The Relation Of the Immune Reaction To Cancer

THE RELATION OF THE IMMUNE REACTION TO CANCER Louis V . Caso* Deportment of Anatomy. College of Medicine. Ohio State University. Columbus. Ohio

I . Introduction . . . . . . . . . . . . . I1. Tumor Tramplantation . . . . . . . . . . A . Transplantation Immunity . . . . . . . . . B. Role of the Adrenal Corticosteroids . . . . . . . 111. Serological, Cytotoxic. and Systemic Effects of Tumor Antisera . A . Serological Changes of Unknown Significance . . . . B . Inhibition of Tumor Growth by Heterologous Sera from Nonimmunized Animals . . . . . . . . C . Passive Immunization . . . . . . . . . D . Change in Electrophoretic Pattrrn of A4ntiscrum . . . . . . . . . . after Immunization E . Immunocheniical Studies of Antibody Distribution . . . F . Some Cytotoxic Effects of Tumor Antisera on Cultured Normal and Malignant Cells . . . . . . . . IV . Inimunochemical Pattern of Tumor-Related Antigrns . . . A . Fractionation of the Tumor Antigen . . . . . . B. Studies with Fluorescent Staining Reactions . . . . . C . Studies with Cells in Tissue Culture: Common Tumor Antigen D . Tumor Lipids and Complement Fixation: Cytolipin H as the Tumor Antigen . . . . . . . . . . . E . Anaphylaxis Studies and the Sclrultz-Dale Technique . . V. iZutoimmunity in Cancer . . . . . . . . . . A . The Concept of Autoimmunity . . . . . . . . B . Occurrence of Autoimmunity to Tumors . . . . . C . The Anemia of Cancer . . . . . . . . . . D . Antigenic Loss in Cancer . . . . . . . . . E . Green’s Theory Involving Antigenic Change . . . . . F . Immunological Enhancement . . . . . . . . G . Burnet’s Theory of Self-Recognit.ion . . . . . . H . Possible Limits to Autoimmunity as It Pertains to the Neoplastic State . . . . . . . . . . . V I . Viral Oncogenesis . . . . . . . . . . . . A . Some Recently Discovered Viral Oncogenic Agents . . . B . Mechanism of Action . . . . . . . . . . C . Relationships among Virus and Host Antigens . . . . D. Possible Role of Immunological Tolerance . . . . . E . Resistance to Tumor and Virus: Possible Separate Mechanisms F . Antigenic Relationship among Viruses . . . . . . G . Relation of Virus Antibody Titer to the Presence of the Tumor

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* Present address : Department of Histology, Temple University School of Dentistry, Philadelphia, Pennsylvania . 47

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H. Cell-Free Induction of Leukemia from Leukemic Murine and Human Brains . . . . . . References . . . . . . . . . . .

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I. Introduction

Although the relevancy of immunology to cancer was noted early in the history of medical research, when bacterial toxins and extracts, such as Coley’s fluid, were studied for their effects on the course of the disease (Stern and Willheim, 1943), rccent years have witnessed a renaissance in the application of immunological methods to the cancer problem. Research in this area now has intensified to such a degree that one cannot hope to assimilate all the data and findings which are accumulating in the areas which immunology encompasses. However, certain trends of thought and method appcar to stand out within the mass of reports and discussions, and this review is an attempt to give them organization and, if possible, meaningful correlation. Bcginning with the very dynamic processes involved in tumor transplantation, moving on to the highly technical fields of serology and immunochemistry, and finally touching on some of the significant virus work, various aspects of immunity during the tumorous state will be considered. Attention is also given to the theoretical implications of the phenomena observed in tumor autoimmunity. For an understanding of the immunological mechanisms encountered, the reader is referred to the general works by Boyd (1947) and Raffel (1961a). II. Tumor Transplantation

A. TRANSPLANTATION IMMUNITY Snell (1957) classifies the main types of tumor transplantation as (1) isotransplantation, in which the graft is made between two members of a highly inbred strain, whose genetic coniponents are believed to be identical or nearly so; and (2) homotransplantation, in which the graft is made bctween members of two different inbred strains of the same species. I n addition, transplantation experiments have included heterotransplantation, between members of different species, which are therefore genetically diverse, and autotransplantation, in which the graft is made within the same individual. As regards tumor transplantation, autotransplantation would require that the tumor which is transplanted arose spontaneously in the same individual, in which case i t would be genetically identical with, or a t least very similar to, the individual in which i t had arisen. It should be mentioned, parenthetically, that the immunologist uses

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these ternis (or rather the terms for the corresponding antibodies) somewhat differently, and care should be taken, in any given context, to keep the distinction in mind. To the immunologist, isoantibodies exist in different individuals of the same species and can react with the specific antigens in another individual. Heteroantibodies are directed against antigens which exist in another species, and autoantibodies are produced in the same individual which possesses the specific antigens. “Homologous” antibody is a general term used to designate the antibody which reacts with the specific antigen. Snell (1957) attempted to organize certain apparent uniformities in the behavior of transplantable tumors by presenting a provisional classification of common experimental tumors. Highly susceptible to humoral antibody are E.L.4 leukemias, Brown-Pearce carcinoma (Kidd subline), Yoshida sarcoma, Olson avian lymphosarcoma (Burmester), Gorer’s strain A tumor, and Bagg rat lymphosarcoma. These frequently give large growths followed by regression and, with the possible exception of the last two, are characterized by rapid growth. The antibody to these tumors can be neutralized by the antigen and is passively transferable. Sarcoma I and lymphosarcoma 6C3HED display low susceptibility to humoral antibody. I n this case the antibody retards rather than prevents growth, and can be demonstrated by neutralization of the antigen. A third group consists of tumors which are probably not sensitive to antisera but fail to grow in most foreign hosts. These include adenosarcoma D1905, fibrosarcoma S620, and carcinoma D22. These are slowly growing tumors which seldom regress once organized growth has begun. As a rule, isotransplants will grow well in the recipient because of genetic similarity. I n the mouse four closely linked histocompatibility genes have been identified, and grafts across divergent types are rejected. Homotransplants are usually rejected, and elicit both a humoral (circulating) and cellular antibody response. The former is frequently manifested by agglutinating or complement-fixing antibodies, while cellular response is seen mainly in lymph nodes and spleen. Rejection of the graft in the homograft reaction is due mainly to the cellular response (Snell, 1957). Immunity to transplanted tumors presumably depends on genetic, and therefore antigenic, differences between the tumor and host. These differences have been investigated by various methods in tissue transplantation, and a brief survey of these follows. 1. I n Vivo Resistance to Tumors

Immunity resulting from comparatively slight antigenic differences is correspondingly difficult to demonstrate, but systems of this kind

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perhaps present a situation which is more comparable to actual changes in the development of neoplasms. Hirsch and co-workers (1958) increased survival time of strain C mice inoculated with a second-generation strain C tumor, mammary adenocarcinoma #10040. A fragment of this tumor was implanted successively in various parts of the same animal, followed by amputation of the parts after tumor growth. Hence, growth of tumor in the right ear was followed by amputation; this was followed by implantation and growth of tumor in left ear with subsequent amputation; then implantation and growth in tail were followed by amputation. Finally, there was a subcutaneous challenge with a 5% homogenate of the tumor. Mean survival time was increased from approximately 91 days in the unimmunized controls to approximately 124 days in the immunized mice. However, there were no differences in the total number of tumors grown or in the time a t which they became palpable, and immunity was concluded to be slight. Martinez et al. (1957) had conducted the same type of experiment in Z (C3H) hybrids using a transplantable adenocarcinoma of the Z (C3H) mouse. He found increased numbers of “takes” at the second implantation in the same animal ( a rise from 67 to 95%), but after the challenge dose, which followed the third immunizing implantation, only 11% of the animals showed tumor growth after 45 days, with no metastases to thc lungs. This tumor had undergone 53 passages prior to the experiment, and it is likely that genetic changes in the tumor were responsible for the marked degree of immunity conferred. Prehn (1960) demonstrated isologous immunity by means of a carcinogen, dibenzanthracene, administered subcutaneously in the backs of C3H/He and BALB/c Am mice. Of 208 mice in which a tumor was transplanted to the subcutaneous tissue of the abdominal wall, only 43% showed tumor growth, compared to 81% of the 204 control animals. Skin from control animals grew well when transplanted into the immunized animal, indicating marked tissue specificity. The eleven tumors used in this study were first generation tumors. I n a later study, Prehn (1962) demonstrated that 3-methylcholanthrene-induced adenoacanthomas elicited tumor-specific immunity when transplanted into isologous mice. Moreover, Old and associates (1962) found tumors induced by 3-methylcholanthrene to be antigenic in isologous hosts. Both workers found that the carcinogen used to induce the tumor affected its subsequent antigenicity, 3,4-9,10-dibenzpyrene producing less antigenic tumors, and urethane producing a majority of tumors which showed no capacity for immunizing subsequent isologous hosts. The work of R6v6sz (1960) with various spontaneous and induced mammary carcinomas, lymphomas, and sarcomas, implanted after

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irradiation into isologous mice, suggests the pertinence of the genetic and antigenic status of the tumor. He found that long-standing, serial transplants, such as two lymphomas of several hundred generations and a carcinoma of 35 to 39 generations, showed incompatibility with their hosts. However, lymphomas and carcinomas of recent origin could produce no such resistance to growth in their isologous hosts. Significantly, inethylcholanthrene-induced sarcomas were capable of immunizing their hosts after one (or in a few cases two) transfers.

2. Resistance to Tumor Metastases There is some indication that resistance to formation of metastases can be developed from primary tumor transplants. Martinez e t al., in the study cited above (1957), used Z(C3H) mice implanted with an isologous adenocarcinoma. This tumor normally metastasizes to the lungs in 12 days. After successive immunization in the right and left ears and tail, there were no metastases to the lung in any of all 11% of the animals, autopsied after 45 days, which developed the tumor in the groin after the final tumor inoculation. Green and Harvey (1960) studied the rate of metastases of a lymphoblastic lymphoma in golden Syrian hamster, presumably a randomly bred strain. This tumor arose spontaneously in the hamstcr, is transplantable a t the third week, and usually will grow in all animals. Transplantation of blood from a tumorous animal to subcutaneous locus on the same animal does not produce a subcutaneous tumor, but, when blood was transplanted t o a tumor-free hamster, a tumor was found to result in 11 of 16 animals. This indication of immunity in the tumor-bearing hamster was tested further by excising the single tumor from a tumorbearing animal. The rate of metastases was found to be 10% among the animals which were allowed to keep the single tumor, whereas the rate increased to 62% among the animals which previously had had the tumor excised. If tumors were simultaneously implanted in both axillae of the hamster, none of the animals showed metastases. Excision of one of the axillary tumors then resulted in the occurrcnce of metastases in 10% of the animals. There is, therefore, in this study, a direct relationship between the presence and amount of tumor and the frequency of metastases. Further discussion of the relationship of immunity to metastases will be taken up in the section on the effects of adrenal corticosteroids (Section II,B,2).

3. I n Vivo Enhancement of Tumors Another finding common in transplantation studies is the phenomenon of enhancement. Enhancement takes place when the time period

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during which the tumor homograft is viable in the homologous host is lengthened, either temporarily or until the death of the host. This is accomplished by pretreating the host with nonliving donor tissue or tissue extracts. Enhancement, therefore, probably depends on the presence of tissue antigens [notably those determined by histoconipatibility-2 (H-2) alleles] which are absent from the host but present in the tumor homograft. Passive enhancement is brought about by inoculating the recipient with an antiserum directed against the homologous tumor which is to be transplanted, or with the serum from anothcr recipient which has already been enhanced for the same tumor genotype (Billingham et al., 1956). The enhanced tumor may or may not retain this growth property indefinitely on transfer to the same strain of recipient (Kaliss, 1957; Snell e t al., 1960), but enhancement cannot be conferred on spontaneous tumors growing in strain of origin, or on tumor homografts which have attained maximum growth adaptation after many generations of transplantation (Green and Wilson, 1958). Billingham e t al. (1956), using skin homografts in CBA- and A-strain mice, showed that enhancement, can be accomplished in normal tissues, but the prolongation of these grafts was slight. Extracts from tumors have been studied for their tumor-enhancing properties. Green and Wilson (1958) obtained full enhancing properties from tumor lipoprotein fraction, and found that soluble protein or phospholipid fraction from normal tissues, or from a tumor of different type, possessed about half the enhancing potency as the extracts from the tumor of the same type used in the graft. Kandutsch (1960) fractionated the ascites form of Sarcoma I (from strain A mice) by centrifugation. These extracts showed enhancement in homologous B10-D2 mice by increasing mortality from Sarcoma I to 50 to 9776, depending on the tumor fraction injected. The soluble part was inactive, but the fluff layer and the microsomal layer were very active, while the nuclear layer was estimated to contain 50% of the total activity of the cell. There was some correlation of activity with hexosamine content, but the microsomes were low in hexosamine content. I n another study Kandutsch and Reinert-Wenck (1957) enhanced Sarcoma I subcutaneously in B10-D2 strain mice with extracts from the tumor and spleen. They found significant activity in the deoxyribonucleoprotein and ribonucleoprotein fractions, but the insoluble residue fraction contained the maximum activity. Chemical analysis indicated the presence of both carbohydrate and protein in the structure of the enhancing substance. Activity was not associated with either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Recently, Kandutsch and Stimpfling (1962) extracted a lipoprotein from the particulate fraction of Sarcoma I which had marked enhancing properties in C57BL/10 mice.

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While enhancement, as defined, requires nonliving tumor tissue in a host of different genotype, it appears that the process may admit of some variation. First of all, genotype can be very nearly identical in both host and tumor. Feldman and Globerson (1960) succeeded in enhancing sarcoma SBLl (carcinogen-induced in strain C56BL mice), so that antiserum to C57BL cells, injected into C57BL recipients, caused formation of SBLl tumors which were larger than those in untreated controls. Moreover, Casey and others (1959) previously had studied a mammary carcinoma which originated in a C57BL/6 mouse. This tumor was grown in the isologous host as (1) a single isograft and (2) a double isograft, the second graft being made in the host 10 to 14 days after the first. The singly grown isograft, when transplanted into homologous mice, showed virtually no growth. When either of the double isografts was transplanted into homologous mice, the tumor was seen to have enhanced status, since it grew in 11 to 23% of the mice. This indicates an immune response by the isologous host which affects each of the double isografts with respect to the mechanism of growth in a subsequent homologous host. 4. In Vitro Treatment of Tumors: Enhancement and Neutralization

Klein and Sjogren (1960) studied several methylcholanthrene-induced neoplasms in both homologous and isologous systems. I n one group of experiments a tumor [such as MSWB from a (A X A.SW) strain mouse] was implanted into a homologous mouse (A x C3H)F,. Antiserum to the tumor, produced in the homologous mouse, and lymph nodes takcn from the homologous mouse, were incubated separately with additional MSWB tumor, which was then reinoculated into the isologous (A x A.SW) mouse. These studies showed that neither immune nor normal serum from the homologous mouse had any effect on the MSWB tumor when it was transplanted into isologous mice. Incubation with homologous lymph nodes, however, did prolong the latency of the tumor growth and reduced the number of “takes” in the isologous animal. I n similar experiments with LNSA lymphoma and MSA sarcoma, both preimrnunieed and unimmunized homologous lymph nodes, when incubated with the tumor, produced inhibition of tumor growth in the isologous animal. Here it is apparent that an isologous antibody of cellular nature was operative in influencing tumor growth. I n another group of experiments the same authors (Klein and Sjogren, 1960) immunized (A x C3H)F, mice against A.SW tissue, and used the immune serum and immunized lymph nodes for separate incubation with MSWB (A x A.SW) tumor. The tumor was then grown in homologous mice, the (A x C3H)F1 strain, Here the antiserum against the tumor brought about enhanced growth in the homologous host, since

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the tumor grew progressively until death. Normally this transplant is incompatible and the tumor regresses. (A X C3H)Fl strain normal serum had no effect when incubated with the MSWB tumor. Lymph nodes from (A x C3H)F, mice immunized against A.SW tissue, when incubated with the MSWB tumor, inactivated the tumor so that no growth was obtained, whereas lymph nodes from unimmunized (A x C3H)F1 mice, when incubated with the MSWB tumor, produced enhancement of tumor growth in the homologous host, but of temporary duration. I n this system, then, there are cellular antibodies which can cause both enhancement and inactivation of tumor, as well as humoral antibody which can cause enhancement of tumor, when grown in the incompatible or homologous host. I n the same study Klein and Sjogren (1960) investigated the effect of antisera, produced in the mouse bearing a primary tumor, and subsequently incubated with two tumors, and found no detectable influence on the growth of the tumors in the homologous host. Winn (1960) studied homotransplantation by grafting lymphosarcoma 6C3HED into the subcutaneous tissue of C57BL/6 mice. He was able to neutralize lymphosarcoma cell suspensions so that, when the antiserum produced by this graft was diluted 1:20 and mixed with 1 x lo5 tumor cells, the suspensions produced no tumors when injected into host mice. More dilute solutions of antisera allowed increasing numbers of mice to develop lethal tumors after injection of the treated tumor. If guinea pig complement was added to the tumor cell-antiserum mixture, the titer of the antiserum necessary to neutralize the same number of tumor cells, and hence prevent tumor growth after injection into mice, was increased to 1:160. This increased potency of the antiserum was also seen when guinea pig complement was injected separately intraperitoneally into the recipient mouse. I n this work the number of tumor cells mixed with the antisera was critical in determining the completeness of neutralization. Incompletely neutralized cells would, of course, produce the lymphsarcoma when injected into mice. It is clear from the work of Klein and Sjogren (1960) and Casey and co-workers (1959) that an immune reaction can enhance the growth of tumor in a homologous host. Martinez et al. (1956) provided evidence that a tumor homologous to the host can initially show a shorter onset of growth, while after 17 days the host will show resistance to growth of additional tumor transplants. This recalls the finding of Klein and Sjogren, already mentioned, that unimmunized, homologous lymph nodes can cause temporary enhancement of tumor growth, while the preimmunized lymph nodes cause complete inactivation of the homologous tumor. It may be possible that there is a stage of antibody production in which the effect of antibody is to enhance the growth of the tumor.

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These enhancements and inhibitions of the growth of tumor cells are common in transplantation studies, and certainly have relevancy to a possible mechanism for the initiation and promotion of spontaneous tumor growth. The mechanism of tumor enhancement will be considered in more detail in a later section (Section II,A,8). 5. Immune Responses to Transplantation Reactions There is some evidence that morphological and immunological changes accompany incompatible grafts of both normal and tumorous tissue. The work of Weinberg et al. (1959) indicated that interference with the activity of the reticuloendothelial system can enhance the growth of homologous grafts. Making the assumption that the rejections of tissue grafts between homologous rabbits was due to an immune response, these authors attempted to “block” reticuloendothelial cells by administration of Evans blue dye a t the time of operation. Pairs of rabbits were joined a t the fascia, muscles, and peritoneum of the abdominal wall. It was found that one member of the untreated parabionts most frequently died in 24 hours. When Evans blue had been administered to the pairs of parabiotic rabbits the union was prolonged for 3 to 21 days. Animals which were rejoined a sccond or third time rejected the grafts more rapidly by necrosing of tissue. Thus there is strong indication that antibody-antigen reactions enter into the homotransplantation of normal tissues. Moreover, Terasaki and othcrs (1959), using White Leghorn and New Hampshire chickens, showed that serum from one breed of chicken which received a skin graft from the other, was capable of agglutinating leucocytes from the donor chicken. It is possible that skin and leucocytes have a transplantation antigen in common, which elicits the production of a n antibody in the recipient of the skin graft. Erdmann and co-workers (1959) found evidence that an immune reaction occurs during heterotransplantation of neoplastic tissue. They observed an inflammatory reaction when mouse leukemic cells (L1210) were implanted as ascites cells into the Sprague-Dawley rat. Infiltration of polymorphonuclears, lymphocytes, plasma cells, and macrophages was seen in the implant of the host rats during the temporary growth. Moreover, these rats showed hyperplastic and swollen reticular cells in spleen and lymph nodes. 6. Use of Immune Tolerance Technique in Tumor Transplantation

Billingham et al. (1953) initiated the technique of establishing the state designated as “immune tolerance.” These workers injected kidney and spleen cells of a strain A mouse into the embryo of a strain CBA mouse. Eight days after birth, they succeeded in grafting skin from a strain A mouse onto the treated CBA mouse. The reaction, which would

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ordinarily cause mice of diverse antigenic make-up to reject the graft in 10 days, had been suppressed, and growth up to 100 days was obtained. Nevertheless, the grafted skin maintained its original strain A antigens, because GBA lymph nodes, previously immunized against strain A skin, when injected into the CBA mouse which carried the grafted skin, caused rejection of the graft. It is probable that the presence of antigen, in the form of the surviving living cells or the foreign protein, must continue in the tolerant animal in order to maintain a fully immunologically nonreactive state (Medawar, 1958, 1961). This technique for immune tolerance was adopted by Pikovski and Schlesinger (1956), who succeeded in growing R I I I mouse mammary carcinoma in an inbred strain of rats. Six- to fourteen-day-old rats were injected with lyophilized R I I I tumor, which rendered them completely tolerant to 120 to 160 mg. R I I I tumor suspension given subsequently in 6 to 8 injections. There were “takes” in 100% of the rats injected. Tolerance could also be induced by injection of lyophilized kidney and spleen of R I I I mouse embryos. Tissues from other mouse strains (C57BL and C3H)-normally resistant to R I I I mammary carcinomagave a weaker tolerance effect. Various human, chicken, and rat carcinomas had no effect a t all in conferring tolerance to the RIII tumor. There is therefore both species and strain specificity operative in actively acquired tolerance. On the other hand, Wallace (1956) had less favorable results in establishing tolerance to homo- and heterotransplantation of tumor grafts. Using Wistar rats which were injected in utero with SpragueDawley liver and kidney cells, he succeeded in establishing a progressively lethal growth of Sprague-Dawley 53FM tumor in only 4 of 18 rats. The 35FM tumor from Sprague-Dawley rats completely regressed in the Hooded rat after 14 days, the latter having been made tolerant to Sprague-Dawley cells. Sarcoma 180 and C3HBA tumor from the C3H mouse completely regressed in 18 days when transplanted into Wistar rats which had been made tolerant to C3H mouse cells. The same was true of the dbrB tumor from the DBA mouse. 7. Effect of Zymosan on Tumor Transplantation

Zymosan has given different results with transplantable and incompatible tumors. Using human colon carcinoma HR132, Herbut and Kraemer (1956a) found that intravenous injection of zymosan could enhance the number of “takes” in Wistar rats, although to a lesser degree than afforded by irradiation of rats before transplantation. Bradner e t al. (1958) studied the effects of zymosan on the growth

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of Sarcoma 180 in Swiss mice. Administered intraperitoneally in carboxyniethylcellulose i t significantly increased the number of mice surviving implantation of the tumor. Since the inhibiting effect is delayed beyond the time of administration, and zymosan is more effective in smaller does levels, these authors believe that zymosan is acting here through the host’s defense mechanisms rather than directly on the tumor. Sarcoma 37, implanted subcutaneously in Swiss mice, was found by Mankowski et al. (1957) to grow until death of the host in about 28 days in all except 6% of the untrcatcd animals. Intraperitoneal injection of zymosan caused complete regression in 67 of 100 mice, while polysaccharide extracted froin Candida guilliemond? caused regression in 62 of 100 mice. The polysaccharides had no effect on Sarcoma 37 grown as ascites tumor. Similar regression of subcutaneous Sarcoma 37 in inice by administration of zymosan was demonstrated by Diller and Mankowski (1960) and Diller et al. (1963), the latter group showing in addition that another yeast polysaccharide, hydroglucan, caused regression in 90 to 9570 of mice with subcutaneous implants of Sarcoma 37 and Sarcoma 180. Zyinosan was found by Martin et al. (1962) to augment the effects of cytoxan and 6-mercaptopurine, and also surgical-chemical conibined therapy, on adenocarcinoma RC in mice. Administration of cortisone was seen to reduce these therapeutic effects significantly. 8. T h e Mechanism of Tumor Enhancement

According to Billingham e t al. (1956), there are three possible ways by which enhancement of tumor growth may be accomplished, assuming that immune responses arc inhibited. First of all, there may be direct inactivation of tumor antigens or prevention of their release from the graft. This is called afferent inhibition. Second, there niay be inhibition of the host’s machinery for antibody production so that a state of tolerance is produced. This is termed central inhibition. Third, the products of the immune response may be prevented from completing their action on the cellular antigens of the graft. This i b known as efferent inhibition. a. Central Inhzbztion. From recent cvidcnce it seems to be unlikely that enhancement of tumor growth is duc to suppression of the host’s antibocly-forming tissues. This is mainly because enhancement does not eliminate certain other immune responses of the host. Feldinan and Globerson (1960)’ using rabbit antiserum, enhanced growth of Lyinphosarcoma 6C3HED in C57BL mice with subcutaneous injections of anti-C3H antiserum. The recipients were shown to produce titers of hemagglutinins for C3H erythrocytes comparable with titers obtained from C57BL mice which received the lymphosarcoma only.

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Consequently, the immune response was shown to be functional during enhancement. However, these authors found that hemagglutinins are not necessarily directly involved in homograft immunity, because rejection of Sarcoma SBLl by C3H recipients was unaccompanied by isohemagglutinin formation. I n addition, i t was found that C3H recipients, after their enhanced SBLl tumors had been extirpated, rejected a subsequent implantation of the tumor. Therefore the host’s immune mechanism remained intact after tumor enhancement. According to Mitchison (1954), passive transfer of transplantation immunity can be accomplished only by transfer of activated lymph nodes. I n mice, serum antibodies do not confer immunity to tumors, except for the leukoses studied by Amos and Day (1957). Feldman and Globerson (1960) inoculated C3H mice with the C57BL sarcoma, SBL1, and subcutaneously injected anti-C57BL antiserum. Enhanced tumor growth was seen. By transferring, by intraperitoneal injection, the lymph nodes from these treated animals to C3H mice, and implanting SBLl sarcoma, they showed that the tumor was rejected. I n untreated controls the tumor grows for about 13 days before regressing. Consequently, the homograft reaction had occurred simultaneously with enhancement in the original host. Moreover, when the second hosts, which had rejected the SBLl sarcoma, were inoculated with a subcutaneous injection of antiserum, growth of implanted tumor was obtained in all animals. From these studies i t is clear that the enhanced tumor homograft does induce immunity, and that the homograft resists the immunity which it produces. b. Efferent Inhibition. While enhancement does not take place if the host has been preimmunized with the tumor (Feldman and Globerson, 1960) ; Moller (1963a) was able to enhance Sarcoma MACD cells, derived from (A X A.CA)F, strain mice, by incubating them with anti-A antiserum before implantation into strain A.CA mice. The A.CA mice had been immunized against strain A normal cells. The MACD tumors grew progressively until the death of the hosts. This was accomplished because tumor antigens had been coated with the antiserum by in vitro incubation. Since mouse antisera were used throughout these and other enhancement studies by Moller (1963a,b,c), the serum of control mice, inoculated with MACD tumor cells, also produced the same type of antiserum (anti-A), but apparently in vivo coating of the tumor cells by antibody was insufficient, and the tumors regressed. Complete covering of the tumor antigens by antibody would render them “unrecognizable” by the antibodies of the preimmunized host. Hence this would indicate efferent inhibition, since the isohemagglutinin titer of all preimmunized mice was 1:8000.

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c. Afferent Inhibition. Moller (1963a) used the isohemagglutinin titer as an indication of secondary immune response in strain A.CA mice. These mice had been immunized previously with strain A lymphoid cells, and after 15 days, when the hemagglutinin titer was zero, they were reinoculated with strain A cells which had been coated with anti-A antiserum. There was no increase in isohemagglutinin titer for the treated mice, while the control mice, receiving uncoated strain A cells, showed a peak antibody rise in 5 days. Therefore, coating of cells with antibody effectively prevented the secondary response. Consequently, it is possible that enhancement might be brought about by blocking of tumor antigens so that they would not initiate the immune (homograft) reaction as either a primary or, in the case of preimmunized recipients, a secondary response. d . Specificity of Ttmor Enhancement. I n another series of experiments Moller (196313) was able to demonstrate that enhancement shows a degree of specificity, and that its induction depends on the presence of antibodies directed against histocompatibility-2 (H-2) antigens of the host. Hence there is strong confirmation for the inimunological basis of the phenomenon. By using various combinations of H-2 antigens contained in tumor and host, a n antiserum to a single H-2 complex was tested for its enhancing properties. I n this way several test systems were set up to determine the relevancy of antigens in tumor and host and antibodies of the injected antiserum. For instance, Sarcoma MACD is derived from strain ( A x A.CA)F, mice. It contains the H-2 antigens G H I from strain A.CA, and antigens A C D H J K M Y from strain A. When this tumor is grown in a strain (A x C571,)F1 recipient, the foreign H-2 antigens are G and I. If the enhancing antiserum is anti-A.CA, this tumor will grow progressively until the death of all the recipients. Here the antiserum, anti-A.CA, is directed against antigens G and I, i.e., against all the H-2 antigens foreign to the host. When the recipient host is changed to the (C3H x 57BL)F, hybrid, the MACD sarcoma contains several antigens foreign to the host, while the anti-A.CA antiserum is directed against only two of them (G and I ) . Here the tumor, when the host is injected with anti-A.CA antiserum, progresses for 12 days, is then inhibited somewhat, but finally kills the host. In another instance, the host was changed to the C57L recipient, a strain which contains no H-2a antigens. The injected anti-A.CA antiserum is therefore not directed against kewn loci of the antigen complement of the MACD sarcoma (i,e., ACDJKMY). I n this graft the tunior regresses, as do the tumors in the untreated (C57L) controls. Moller, in other experiments (1963b), showed t h a t enhancement could

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be re-established in particular combinations of H-2 antigen complexes by supplying antibodies, lacking in a single antiserum, in a second antiserum administered simultaneously. It thcrefore appears t h a t the enhancement of tumor growth is most pronounced when the antiserum is directed against all the H-2 antigens of the tumor which are foreign to the host. When the antiserum is directed against only some of the foreign H-2 antigens, enhancement is diminished or abolished. Conversely, inhibition of the tumor is strongest when the tumor has many H-2 antigens, foreign to the host, which are uncovered by the antiserum. By numerous variations in the H-2 antigen combinations in the experimental design, Moller demonstrates that the enhancing antiserum is insufficient unless directed against the whole antigenic discrepancy (H-2) between tumor and host. Enhancement therefore appears to be related to the number and/or strength of tumor antigens covered by the antibodies. It is also indicated that the antiserum to the tumor does not act by directly stimulating tumor growth or altering of physiology of tumor cells, because in that case its specificity need not be limited to certain host genotypes, but would vary with tumor genotype only. Since the enhancing propcrty of an antiserum, as seen in these studies, does not depend exclusively on tumor genotype, but also on that of the host, there is no compelling reason to assume direct action by the antiserum on tumor growth physiology. Yet there does remain the possibility that host genotype combined with antiserum specificity could have direct action on the tumor cell. Coating of tumor cells, by incubation in vitro with the appropriate antiserum, also enhances their growth in foreign hosts similarly to antisera administered separately by injection (Mollcr, 1 9 6 3 ~ ) .Moller’s studies bring out t h a t inhibition of the immune response, as mcasured by isohemagglutinin formation following inoculation of antibody-coated tumor cells, also depends on the relation of the tumor genotype to that of the host. Inhibition of immune response is related to tumor enhancement because it is specific, depending on the reaction of the antiserum with all the H-2 antigens of the tumor which are foreign to the host (Moller, 1963b). e. Relationship between Tumor Enhancement and Tumor Inhibition. Huinoral antibodies, formed in response to transplantation across the H-2 barrier in mice, are strongly cytotoxic in the presence of complement in vitro. The response of various cell types depends on the surface concentration of cellular antigens. High concentration of surface, antigenic receptors is corrclated with sensitivity to cytotoxicity, while low con-

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centration of receptor sites is correlated with resistance (Moller and Moller, 1962). Moller ( 1 9 6 3 ~ )showed that a tumor which is sensitive to cytotoxic antibodies, such as Lymphoma 6C3HED, when treated in vitro with diluted antiserum against tumor type, showed temporary enhanced growth followed by regression. With undiluted antiserum the tumor showed comparatively rapid regression. FHA sarcoma, an even more susceptible tumor, also showed temporary enhancement of growth, followed by regression, after treatment with the specific antiserum, but dilution of tlie antiserum resulted in decreased enhancement (or increased inhibition) of tumor growth. A tumor quite resistant to cytotoxic antibodies, sarcoma MACD, was enhanced by specific antiserum over a wide range of concentrations with no significant differences in growth. The host genotype was tlie same in all these cases (strain A.CA). The work of Gorer (1962) and Gorer and Kaliss (1959) had indicated possible duality of humoral antibody action in that it could both stimulate and inhibit tumor growth. T o test this concept, Moller ( 1 9 6 3 ~ ) attempted to exclude the effects of cell-hound antibody of homograft immunity by (1) coating tumor cells with the specific antibody and growing them in the isologous host, in which cell-bound antibody would not be expected to be present; and (2) growing tumor cells in cell impermeable chambers, which would prevent passage of cell-bound antibodies, implanted in the preinimunized homologous host. I n each case the humoral antibodies showed only inhibition of tumor growth. The conclusion drawn by this investigator is that enhancement seems to occur in spite of a slight inhibiting rather than stimulating effect of circulating antibody. Moller (19634 was able to show that short-term and long-term treatment of various tumors with specific antibody apparently does not permanently alter the growth properties of the tumors. This indicates that antiserum dose variations, causing both enhancement and inhibition of various tumors, are due to a “walling off” or blocking of cellular antigens by antibodies rather than deep-seated changes in the cells. This can be explained in terms of an afferent mechanism for enhancement. Hence, the action of an antiserum would depend on the susceptibility of the tumor to cytotoxic (humoral) antibodies and the number of antigenic receptor sites on tlie tumor cells. A tumor completely resistant to cytotoxic antibodies, such as Sarcoma MACD, has few antigenic receptor sites. These in turn are easily blocked by specific antibody, and since the tumor is resistant to cytotoxic action, concentration of antiserum is not critical. It is therefore permanently enhanced

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a t both high and low concentrations, A tumor which is moderately sensitive to cytotoxic antibody, such as 6C3HED lymphoma, has more numerous antigenic receptor sites, but low concentrations of antibody are sufficient to coat them completely, so that temporary enhancement results. At higher concentrations of antiserum, cytotoxic antibodies will inhibit the tumor. A tumor which is highly sensitive to cytotoxic antibody, such as FHA sarcoma, has the most numerous antigenic receptor sites, so that low concentrations of antibody, sufficient to enhance the 6C3HED lymphoma, cannot block all the receptor sites on FHA sarcoma cells, and the latter tumor would show more inhibition (and less enhancement) a t lower antiserum concentrations. The ability of transferred, immunized lymphoid cells to neutralize the enhancing effects of specific antiserum on tumor cells also seems to be related to the concentration of antigenic receptor sites on tumor cells and their susceptibility to humoral, cytotoxic antibody (Moller, 1963a). While central inhibition is not ruled out completely [and Brent and Medawar (1961) have presented evidence in favor of i t u s i n g , however, a different test system, i.e., skin grafts], these studies are indicative of enhancement a t the cellular level, as afferent or efferent inhibition of transplantation immunity. The relevancy of host genotype to the enhancement of particular tumors with specific antiserum indicates inhibition of the immune responses of the host rather than direct stimulating action causing permanent changes in tumor cell growth. Moreover, enhancement probably is not restricted to certain antigen systems, because i t has been brought about in a highly sensitive mouse leukemia belonging to a non-H-2 antigen system (Moller, 1 9 6 3 ~ ) . f. Possible Genetic Importance. It is possible to establish some tumors, once enhanced with specific antisera in homologous hosts, as compatible tumors in untreated foreign hosts (Kaliss, 1957). Moller believes that variants have occurred in these tumors after prolonged enhanced growth in foreign hosts. This would represent a genetic change in the tumor rather than a “permanent” enhancement. Hellstrom (1960) showed that lymphomas, derived from F, mice, spont,aneously developed variant sublines, which involved loss of sensitivity to cytotoxic antibodies directed against the H-2 isoantigens derived from one of the parental strains. It is possible that the period of enhancement of tumors, during which the host’s immune mechanism is inhibited over a long series of transplantation generations, allows genetic mutation in the tumor favorable to growth in the foreign host. g. Other Theories of Enhancement. Feldman and Sachs (1957) and Feldman and Globerson (1960) suggest that resistance of the tumor homograft to transplantation immunity may depend on increased pro-

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duction of H-2 antigens, which would neutralize the cytotoxic antibodies from the host. The increased antigen synthesis would depend on the enhancing antibody either by direct or indirect stimulation. Billingham et al. (1956) believe that histocompatibility genes determine antigens of the nucleus, which are responsible for the production of antibodies in transplantation immunity (vs. skin homografts) . Isohemagglutinins would depend on antigens in the cytoplasm for their formation in the homograft reaction. A single allele is supposed to determine both these antigens, i.e., nuclear and cytoplasmic, and therefore they would be very similar structurally. It is possible that humoral isohemagglutinins evolved by response to cytoplasmic antigens could cross-react and combine with the nuclear antigens, thereby blocking them and inhibiting the onset of the homograft reaction. B. ROLEOF

THE

ADRENALCORTICOSTEROIDS

1. Promotion of Growth in Tmnsplanted Tumors

The effective use of cortisone and hydrocortisone in the reduction of strain and species resistance to tumor transplantation is well established in certain cases. Ponieroy (1954) inoculated Swiss mice intravenously with Krebs I1 ascites cells and Sarcoma 37. Those groups receiving 2.5 mg. of cortisone subcutaneously on the day of tumor injection showed increased numbers of animals developing tumor and enhanced metastases to the liver. Toolan (1954) succeeded in growing two human tumors (Sarcoma 1 and Epidermoid carcinoma 3) in the cheek pouch of the hamster after a single, 3-mg. dose of cortisone a t the time of transplantation. These tumors were found to maintain human antigens after having been transplanted, as demonstrated by the agar plate test. The growth rate of the tumors was faster in the hamster than in the human donors, and this may have been a factor in overcoming host resistance. Normally, the tumors will not grow in untreated hamster hosts. Handler et al. (1956) reported successful growth of 28 human tumors, including epidermoid carcinoma of skin and cervix and a lymphosarcoma, after implantation in the hamster cheek pouch. The animals were administered 1 to 3 mg. cortisone acetate two to three times daily. Those tumors which grew best were sarcomas, while the epidermoid carcinomas grew less well, and the adenocarcinomas were least successful. These workers found that all 68 tumors grew briefly in the hamster (5 to 58 days), and that none of the tumors survived multiple transplantation. Metastasizing tumors grew most successfully. If the tumor had been refractory to irradiation or chemotherapy, or was of a recurrent type, growth seemed more likely than for other

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tumors not showing these characteristics. However, irradiation of the hamster host, in addition to cortisone administration, resulted in the best growth of tumors. Boucher et al. (1956) used radioactive-phosphorus and cortisone acetate in transplanting tumors into both susceptible and resistant mice. I n combination these agents accelerated the development of 28352 tumor cells in ZBC mice, and reduced longevity from 21 to 35% of that of the controls. Pnz or cortisone alone produced no enhancement of tumor growth. Combined P3’-cortisone pretreatment of Zb mice, which werc inoculated with 28352 tumor cells, resulted in reduction of longevity by 30 to 40%. With the combined pretreatment C57B46 mice, inoculated with E0771 cells, showed a decreased longevity index of 24%. On the other hand, P3?and cortisone pretreatment temporarily modified the growth of 28352 tumor cells when injected into the genetically diverse C57BL/6 mouse, so that a nodule was produced a t the site of injection and later followed by regression. The strain resistance t o progressive tumor growth could not be overcome in this instance. The authors interpret thcir results to indicate that cortisone and internal irradiation from P S L do not affect immune resistance t o tumor growth, since the strains they used were closely related, but i t should be pointed out that the tumors used, EO771 and 28352, had undergone 22 and 42 passages, respectively, and quite possibly had developed antigenic variations as compared to their original hosts. Herbut and Kraemer (195613) used Toolan’s technique of combining whole body irradiation of the host with cortisone administration, and attempted to implant various human carcinomas, sarcomas, and melanoblastomas into pretreated female Wistar rats. Of 206 tumors transplantcd into the treated rats, 77 survived the first transplantation and only 3 survived to the fourth generation. It was found that only one of these was really growing a t the fourth generation, a n anaplastic carcinoma of the colon; it was of small bulk and slow-growing. 2. Enhancement of Metastases of Transplanted Tumors There is much evidence that systemic injection of cortisone can enhance metastases of an implanted tumor. Baserga and Shubik (1955) studied transplantable bladder epithclial carcinoma of the black mouse, C57(T150), which was carried in the C57 black strain. This tumor metastasizes spontaneously to the lung. These authors found that mice receiving 0.5 mg. cortisone acetate in each of four or five injections showed metastases to the lung in 37 of 40 animals, whereas controls without cortisone treatment showed metastases in 20 of 38 animals. Moreover, the total macroscopic count of metastases in all animals

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showed only 56 for the controls as compared with 196 for the cortisonctreated mice. The average size of the metastases in the treated mice was about twice the diameter of those in the control animals. These workers believe t h a t cortisone acts on host processes after the vascular disseniination of tumor cells. Wood et al. (1956) studied experimental pulmonary metastases in 702 Swiss mice. They found that the number of lung tumors was increased if any one of the following hormones was given prior to intravenous injection of Carcinoma 150: cortisone, corticosterone, ACTH, 9-a-fluorohydrocortisone. Also, prior cold stress or formalin administration has the same effect. Hydrocortisone or growth hormone given prior to intravenous Carcinoma 150 inoculation increased the number of lung tumors, but did not cause an increase if administered simultaneously with, or subsequent to, the tumor suspension injection. Hydrocortisone was found to increase lung metastases from a subcutaneous tumor implant while not affccting the growth rate of the primary tumor. Gasic and Gasic (1957) grew Bladder carcinoma T150, Carcinogen-induced fibrosarconia GL46, and Sarcoma 180 subcutaneously in C57BL/6, DBA/2, and Swiss mice, respectively. The bladder carcinoma nornially metastasizes while the other tumors do not. Free tumor emboli were tested for by cardiac puncture of the tunior-bearing animal, either control or cortisone-treated, and reinoculation of the withdrawn blood intraperitoneally into the susceptible mouse. Donor and recipient mice were then examined for lung metastases. With the T I 5 0 tumor, cortisone caused a slight increase in metastases in thc donor animals, while the recipient mice showed about the same nuinbcr of tumors, whether the blood sample was from a cortisone-treated or control animal. The Sarcoma 180 and GL46 tumors did not metastasize in the donor animals or in the recipient animals, regardless of the presence or absence of cortisone administration. The conclusion is readily drawn t h a t Cortisone had no effect on emboli already present in the donor animal and t h a t i t did not affect the release of emboli from the donor animal. This indicates that cortisone action is mediated through the host in the spread of neoplasms. Cortisone, given 10 days before implantation, was found by Baserga and Shubik (1954) to give only temporary inhibition to subcutaneous implants of DBA mammary adenocarcinoma in DBA mice, aftcr which accelerated growth of the tumor was observed. Metastases to lung, liver, kidney, and other organs occurred in the animals receiving cortisone, but were absent in the untreated controls. I n Swiss mice the DBA mammary adenocarcinoma reached the same size in cortisone-treated animals and in controls. Most of the tumors regressed after 2 weeks.

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However, some studies have indicated little or no enhancement of tumor metastases by cortisone. Cortisone was found not to affect the incidence, time of appearance or distribution of metastases of lyophilized Sarcoma I (from strain A mice) when implanted into C57BL/6 mice, in a study reported by Kaliss et al. (1954). Fisher and Fisher (1960) injected Walker “carcinosarcoma” 256 intraportally into Sprague-Dawley rats and showed equal numbers of tumor “takes” in cortisone-treated animals and pair-fed controls, while ad libitum controls showed increased numbers of metastases. There may be a nutritional factor concerned with liver metastases, although these workers were not able to find any basis for it. The inherent metastasizability of Walker 256 tumor was not evaluated. It is of interest that the American Medical Association Subcommittee on Steroids and Cancer (1951) concluded that cortisone does not alter the course of various types of neoplasia studied, even though i t allays pain and gives the patient an improved appearance and fceling of wellbeing. Despite the subjective response, however, i t was found that in several cases there was evidence of a more rapid spread of the neoplasm after cortisone therapy. Autopsy in these cases showed the spleen to be riddled with metastases.

3. Effects of Corticosteroids on the Action of Chemical Carcinogens I n the experiment of Baserga and Shubik (1954) cited previously, Swiss mice, whose skin had been painted with methylcholanthrene, showed inhibition of tumor development when receiving 0.5 mg. cortisone, administered daily, intraperitoneally. Since the steroid dose was rather toxic, in that i t produced loss of weight in the treated mice, the authors believe that this may have played a part in tumor inhibition. I n mice bearing established skin tumors of gross malignancy, cortisone was found to produce metastases of squamous cells to lung and liver, while the mice receiving no cortisone did not show metastases, as is normally the case with carcinogen-induced squamous cell tumor. When methylcholanthrene was administered subcutaneously to Swiss mice, subcutaneous sarcomas were produced in both cortisone-treated and control animals, with no differences in size of tumor or rate of growth. Findings of inhibition by cortisone of transplantable tumors of epithelial origin havc been negative generally, although there is some disagreement as to the results with carcinogen-induced skin tumors. Zachariae and Asboe-Hansen (1958) induced skin carcinomas in ST/Eh mice by painting with 9,10-dimethyl-1,2-benzanthracene. Hydrocortisone acetate was injected weekly for 16 weeks subcutaneously beneath the tumor. There were fewer deaths and smaller and fewer tumors in a

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24-week period if the animals were injected with hydrocortisone, although the trend reversed in the second half of the period. These authors observed that, when premalignant skin papilloma becomes malignant,, connective tissue mast cells decrease in number. Hydrocortisone also reduces the number of mast cells in connective tissue and causes pronounced morphological changes in them. I n another experiment Zachariae and Asboe-Hansen (1954) injected hydrocortisone subcutaneously beneath precancerous, 10-day-old, benzanthracene-induced papillomas in 65 ST/Eh strain mice, and produced tumor disappearance in 83% of the treated mice. Tumor disappeared in 37% of the 58 untreated control animals. Wolf and Nishimura (1960) applied methylcholanthrene and hydrocortisone topically to the skin of CAF, hybrid mice, and found that the steroid completely inhibited the action of niethylcholanthrene in that epidermal mitoses were relatively rare. With iiiethylcholanthrene alone, there was outstanding hyperplasia of the spinosum and granulosuin layers of the epidermis, with most mitoses occurring in the basal cells. On the other hand, Spain et al. (1956) gave cortisone acetate by subcutaneous injection to BALB/C strain mice bearing methylcholanthrcne-induced skin papillomas. They found that the treated mice had approximately three times as many papillomas after 40 weeks as did the untreated controls. They found no morphological differences in the tumors of the controls and the treated animals and no metastases induced by cortisone. Carcinoma formation roughly paralleled the rate of papilloma development. Sherwin-Weidenreich e t al. (1959) found that 39% of Swiss mice receiving intradermal injections of 9,10-dimethyl-1,2-benzanthracene developed skin tumor when cortisone acetate was administered systematically, compared with 9% among the mice not receiving cortisone. These workers concluded that cortisone contributed to tumor growth by slowing the regrowth of hair follicles and the repair of damage to the skin caused by the carcinogen. Nakai (1961) used various steroids intraperitoneally in an attempt to inhibit the development of subcutaneous sarcoma following the subcutaneous injection of methylcholanthrene. Ninety-seven per cent of the controls developed the tumor, whereas hydrocortisone, iiicthylprcdnisolone, dexamethasone, and triamcinolone produced 75 to 78% tumor development, and cortisone reduced the incidence of tumor devclopmcnt to 93%, this last figure being an insignificant amount compared to the controls. This worker found dosage, route of administration, and frequency and duration of treatment of importance in steroid administration. As Wolf and Nishimura observed, concentration of steroid a t thc tumor site is much greater if it is applied locally to the tumor than when i t is administered systemically. Grccn and Savigear (1951) performed an experiment similar to that of Wolf

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and Nishimura (1960), but administered the cortisone systemically ; they were unable to show decreased mitoses after carcinogen treatment. 4. Inhibitory Effect of Corticosteroids on Lymphoid Tumors

Cortisone has been found to be slightly and temporarily inhibitory to transplantable lymphoid tumors, and corticosteroids have been widely used clinically in recent years (Burchenal, 1958; Burdette, 1958; Haupt e t al., 1960; Zeulzer and Flatz, 1960). According to Forkner, Burchenal, and associates (1961) corticosteroids have a definite effect in decreasing the size of the liver, of the spleen, and of the tumor masses in lymphosarcoma, reticulum cell sarcoma, and in chronic lymphocytic leukemia. Corticosteroids are useful in causing remissions in children with acute leukemia (Burchenal, 1958). Hynian and Sturgeon (1956) used Prednesone, an analog with three to four times the anti-inflammatory activity of cortisone, in treating 21 children with acute lymphatic leukemia. The drug was administered until bone marrow and peripheral blood cell composition returned to normal, followed by additional course of treatment if the signs of disease recurred. Remissions lasted from 19 to 107 days. There were 27 courses of treatment in all, the remissions being shorter after two courses (31 to 56 days). With repeated therapy there was little or no response by bone marrow to treatment. There is quite possibly a relation between the action of certain adrenal corticosteroids on normal hematopoietic and lymphatic tissues and their inhibitory effects on lymphoid tumors and leukemias. The usual response to corticosteroid administration is relative and absolute lymphopcnia, eosinopenia, and neutrophilia (Gordon et al., 1957). The decrease of lymphocytes and eosinophiles is due possibly to direct destructive action, such as reported by Dougherty (1957) for small, mature lymphocytes of lymphatic organs and peripheral blood. On prolonged administration of corticosteroids, impaired production of lyinphocytes and eosinophilcs has been described (Gordon e t al., 1957). Supporting this is the report by Dougherty (1957) that mitoses of lymphocytes are suppressed by these agents. 5. Effect on the Immune Responses of the Host Direct effect of cortisone acetate on the immune reaction was demonstrated by Ward and Johnson (1959), who injected the hormone daily from 1 day before to 10 days after the injection of iodine-labeled antigen (human serum albumin) into rabbits. They found that cortisone inhibited the primary response by early preventing the sensitization of antibody-forming cells. The action was similar to that of X-ray irradiation on rabbits. Ward and Johnson also inhibited the secondary response

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with cortisone injections in 50% of tlie animals, most of which had shown tlie primary response with serum albumin 9 weeks previously. Kaliss e t al. (1956) immunized C57BL/6Ks strain mice against Sarcoma I, S786 fibrosarcoma, and E9514 lymphatic leukemia, and showed the effect of cortisone acetate injections given four days prior to and concurrently with tumor antigen inoculations. These authors reported inhibition of hemagglutinating antibody production in mice given Sarcoma I or S786 tumor during the first course of immunization, and this inhibition persisted in some of the animals during the second course of immunization, without cortisone, using either S786, Sarcoma I, or E9514 antigens. If the animals were immunized with Sarcoma I, without cortisone administration, during the first course of inoculations, cortisone given during the second course of immunization with Sarcoma I or S786 tumor antigens did not prevent the production of hemagglutinins. It is apparent that in niice cortisone can prevent the primary response, but once the antibody-producing mechanism had been established by previous immunization, cortisone cannot prevent the secondary response. The same authors postulated two sites of antibody production, one being insensitive to, tlie other inhibited by, the action of cortisone. Kass and Finland (1953) reviewed the literature concerning the effect of adrenal corticosteroids on immunity up to 1953. It is apparent from their work that resistance to a wide variety of infectious agents is lowered in many species of animals. This includes both natural resistance, as in the rabbit resistant to tuberculosis, and acquired resistance, as in mice which have been inoculated with partially protective doses of antiserum or vaccine. It is believed that adrenocortical hormones inhibit antibody synthesis, since it is known that cortisone inhibits Synthesis of pentose nucleic acids in regional lymph nodes after antigen injection. Of particular interest is the effect of these hormones on the reticuloendothelial system. Infiltration of large mononuclear cells is inhibited, as is local granuloma. I n large macrophages, phagocytosis may be accelerated, but intracellular digestion of particulates, such as foreign erythrocytes or labeled foreign protein, is inhibited. The polymorphonuclear leucocytes may be reduced in number, capillary permeability is diminished, and the inflammatory reaction from burns, trauma, and irritating chemicals is reduced. I l l . Serological, Cytotoxic, and Systemic Effects of Tumor Antisera

A. SEROLOGICAL CHANGES OF UNKNOWN SIGNIFICANCE 1. Effects of Tumor Growth on Erythrocytes The effects of various sera and antisera have long been investigated in the attempt to evaluate the degree of protection, if any, a specific

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antiserum might afford against a particular tumor, and to characterize the changes that occur in the host’s immune reactions in response to the tumor stimulus. Some of these changes are indeed induced by the growth of tumor, but may be manifested by changes in other antibody-antigen reactions not related to the tumor in any meaningful way. It is known, for instance, that certain tumors which grow progressively until death in the rat can alter the host serum, so that the heterophile reactions of rat serum are also changed. I n the study of Bogden and Aptekman (1953) i t was found that isologous tumors, spontaneous or carcinogen-induced, when implanted into P.A. or Lewis inbred strains of rats, cause a lowering of the hemagglutinating titer of the host serum for human erythrocytes. I n strain P.A. there is an initial drop in the titer for AB and 0 erythrocytes until 48 hours after tumor implantation, followed by a rise in titer for from 6 to 9 days, after which the titer again declines until complete disappearance a t 21 days. Serum from the tumorbearing Lewis strain follows the same pattern for the anti-AB titers. The decrease in titer in each case is correlated with the average size or growth of tumor during 21 days. The same workers (Aptekman and Bogden, 1956) also observed that normal serum from P.A. rats has complete reactivity with human red cell A substance. The other reactions for P.A. serum, and all the reactions for normal Lewis serum with AB and 0 red cells, were found to depend on antigens common to the human erythrocyte and not related to human A, B, and 0 substances. It is not known how the tumor affects the natural antibody titer of the rat serum. Differences in erythrocytes from susceptible strains of mice were reported by Adelsberger (1951). Using mouse mammary tumor antigen to immunize rabbits, antitumor antiserum was prepared and complement inactivated. It was shown that erythrocytes from C3H tumor susceptible mice (but not tumor-bearing) were hemolyzed by the antiserum in dilutions of 1:16 to 1:256. Erythrocytes from C57 black mice (tumor-resistant) were not hemolyzed by the antiserum. Erythrocytes from C3H mice bearing spontaneous mammary tumor tended to show decreased hemolysis with the tumor antiserum. Since normal rabbit serum showed a similar effect, the action of the antiserum may be due to a heterophile effect. Moreover, erythrocytes from normal C3H mice were more sensitive to hemolysins in mammary tumor suspensions than erythrocytes from C57 mice, and erythrocytes from tumor-bearing C3H mice showed reduced sensitivity, or no hemolysis a t all, when reacted with the tumor. In another study the same author (Adelsberger and Zimmerman, 1954) tested erythrocytes from C3H mice which bore various transplanted tumors, and found that the incidence of hemolysis with inactivated normal rabbit serum was reduced in mice with malignant glioma, ependy-

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moma, or meningeal fibrosarcoma as compared with the tumor-free mice. It was observed that mice with fast-growing tumors showed limited hemolysis, whereas mice with slow-growing tumors showed hemolysis similar to that of controls (normal C3H mice). Hemolysis tended to be absent or weak in mice during the early and moderately advanced stages of tumor growth (23 to 31 days) and increased in degree during the later stages (40 days after implantation). It is possible that an antibody adhering to these erythrocytes is responsible for inhibiting the action of tumor hcmolysins. For a discussion of the effects of human neoplasms on erythrocytes see Section V,C. 2, Comparison of Strain Susceptibility with Presence of Natural, Heterophile Heinagglutinins Davidsohn and Stern (1949a) studied four strains of mice and found no significant differences in agglutination titers for human erythrocytes with normal sera from the various strains, although the latter were chosen because they differed in susceptibility to mammary tumors. However, the antisheep erythrocyte titer was consistently higher in normal serum from C57 black mice and more frequently present than in the three strains which have a much higher incidence of mammary tumor. Later experiments indicated that this strain gave the highest levels of hemagglutinin and hemolysin titers for sheep and human erythrocytes after immunization against these cells (Davidsohn and Stern, 1949b). The natural antibody against sheep erythrocytes was found not to be of the Forssman type, and the Forssman antigen in normal and neoplastic mouse tissues was found to be independent of strain differences. These authors found no correlation between the incidence of spontaneous tumors and the presence and levels of antisheep agglutinins in eight strains of mice studied (Davidsohn and Stern, 1950).

B. INHIBITION OF TUMOR GROWTHBY HETEROLOGOUS SERA FROM NONIMMUNIZED ANIMALS An unusual effect of a heterologous serum was recorded by Ainis and co-workers (1958). Serum from a single strain of guinea pig, when injected into T;CTistar rats, was found to inhibit the growth of MurphySturm ascites lymphosarcoma in reducing mortality by 50%. Preincubation of tumor cells with guinea pig serum in vitro had no effect on tumor transplantability and growth, although agglutinins which were present in the serum could be removed by tumor cells but not by r a t or sheep erythrocytes. Serum from newborn guinea pigs had no effect on tumor growth when administered in vivo, while serum from normal adults was

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CASO

reduced in in vivo activity by prior absorption with zymosan. I n a separate study Jameson e t al. (1958) had shown the inhibitory effect of guinea pig serum on the solid (subcutaneous) form of Murphy-Sturm lymphosarcoma. It is of interest to compare the work of Winn (1960), already mentioned, in the use of guinea pig complement to enhance the potency of an antiserum to Lymphosarcoma 6C3HED in mice (Section II,A,4). Complement from human serum was found by Landy and others (1960) to inhibit completely the intraperitoneal growth of Sarcoma 37 in CAF, mice after in vitro incubation with tumor cells. Glycolytic activity of the tumor cells was also lost after serum incubation. Preheating the serum, or absorption with Sarcoma 37 cells, prior to incubation of the tumor with the serum, allowed the tumor cells to retain their transplantability and glycolytic activity. The antibody could be eluted from the Sarcoma 37 cells which were used in the absorption, the eluate restoring full antitumor activity when added to the absorbed serum. EDTAl was found to suppress the antitumor activity of the serum, as would the removal of any of the four components of complement. Absorption of the serum with normal mouse erythrocytes could not remove its cytotoxic action. Several other sera were found to have antitumor activity, including that of chimpanzee, cow, pig, dog, rabbit, and chicken. Horse, sheep, guinea pig, and mouse sera were found not to be cytotoxic to Sarcoma 37. Ehrlich carcinoma and Krebs carcinoma were also inactivated by incubation with human serum. If human serum was heat-inactivated prior to incubation of the cells, or absorbed with Sarcoma 37 prior to the incubation, both Krebs and Ehrlich carcinomas were enhanced in their growth properties in mice. This was usually paralleled by increased glycolytic activity of the tumor cells. I n addition, it was demonstrated that Sarcoma 37 cells, when incubated with a noncytotoxic serum, such as t h a t from the horse, caused an earlier appearance of ascites when subsequently inoculated into mice.

C. PASSIVE IMMUNIZATION

A heterologous antiserum was prepared by McCredie et al. (1959) by inoculating rabbits with Walker 256 r a t tumor. The antiserum was absorbed with normal rat tissue, and fractionated for y - and (1.-2 globulins. The latter technique increased the titer of the antiserum 100 times. This antiserum prevented “takes” of Walker 256 tumor in 12 of 20 rats, and delayed the appearance of the tumor in the remaining rats until 22 to 25 days after implantation. Tumor was palpable in the control animals after 3 days. I n rats with palpable tumor, the antiserum caused decrease

’ Ethylenediaminetetraacetate,

for removal of Ca++and Mg” ions.

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in size but not regression of the tumor. Normal rabbit y- or a-2 globulin had no effect on tumor growth. Gorer and Amos (1956) immunized mice by passive inoculation with antiserum to leukosis E.L.4 tumor. The isologous antisera were produced in strains A, BALB/c, and C57BL. As little as 0.1 ml. of antiserum administered to strains incompatible with the tumor (A, BALB/c, C 3 H ) , 24 hours before the animal was challenged with 400,000 to 2 million tumor cells, protected 202 of 211 mice. I n the strain of tumor origin, C57BL, 0.1 ml. antiserum delayed palpable tumor 6 to 17 days and death 5 to 16 days beyond the corresponding occurrences in the controls. Since death occurred 16 days after the appearance of the tumor, i t was believed that there was a progressive growth effect after the single dose of antiserum had “worn off.” These authors showed by absorption of the antiserum for H-2 antibodies and embryonic tissue antibodies, that the anti-E.L.4 antibody is distinct. Antisera to antigens other than those of E.L.4 would not give any protection to the compatible mouse strain against the tumor. Gorer and Kaliss (1959) also showed inhibition of leukosis E.L.4 with isoantiserum, while two sarcomas were found to be enhanced by isoantisera.

D. CHANGEIN ELECTROPHORETIC PATTERN OF ANTISERUM AFTER IMMUNIZATION Hartman and Nungester (1956) studied the change in electrophoretic mobilities of normal C3H mouse serum after absorption with 6C3HED Gardner lymphosarcoma cells, as compared with the change in electrophoretic mobilities of antisera to the tumor after absorption with lymphosarcoma cells. The antisera were produced by C3H mice whose tumors had regressed. The mobility of the antiserum decreased two to four times that of normal serum after absorption by the tumor. Nornial mice inoculated with the lymphosarcoma showed the appearance (or increase) of a substance in their sera which could be removed by absorption with tumor cells. It is not known whether or not this substance is protective, but it was concluded t h a t i t is a n antibody, appearing as a new substance or as one already present but in increased amounts. E. IMMUNOCHEMICAL STUDIESOF ANTIBODYDISTRIBUTION 1. Concentration of Antibody in N o r m a l and Tumorous Tissues

There has been much valuable research which has attempted t o characterize the affinity of tumor antisera for various normal and malignant tissues both by in vivo injection of labeled antisera or by in vitro reactions. Wissler and others (1956) produced antisera to two r a t tumors (Flex-

74

LOUIS V. CASO

ner-Jobling tumor and Jensen sarcoma) and Ehrlich mouse ascites tumor in immunized rabbits. I n vitro precipitation reactions, utilizing 1131labeled antisera, indicated strong specificity for the homologous antisera to the Flexner-Jobling and Ehrlich ascites tumors, with less reactivity for rat spleen, liver, and kidney. The specificity of Jensen sarcoma antiserum was evident but less marked. There was almost complete in vitro cross-reactivity between antisera to Flexner-Jobling and Jensen tumors. Normal rabbit, 1131-labeled globulin was less reactive with the tumors and normal tissues in vitro than were the antisera. I n vivo experiments, in which intravenous injections of anti-Flexner-Jobling tumor antiserum were given to tumor-bearing rats, showed greater localization of immune 7-globulin in spleen, lung, and liver than in the tumor a t 24 hours, while kidney showed poorer localization. However, a t the 24-hour reading, the concentration in tumor had been on the increase, which was contrary to blood and other tissues, in which the concentration was decreasing a t that time. Continued administration of labeled antisera for 16 days did not change this finding, However, intraperitoneal injections of anti-Ehrlich-ascites-tumor, immune, labeled globulin showed marked concentration of 7-globulin in the tumor cells, in contrast t o concentration in liver, spleen, and kidney. The authors point out the importance of route of injection here, and postulate that the capillaries of the Flexner-Jobling tumor are more impermeable to 7-globulin than those in the sinusoidal organs (liver and spleen). Ehrlich ascites tumor localized the immune globulin because there was no capillary-blood barrier. However, another point to keep in mind is the rapidity of elimination of the intravenous injection of the antiserum. With the Flexner-Jobling antiserum this may be accomplished by the lung, spleen, and liver, and lower the effective blood level of antibody before concentration in the tumor is possible. It is interesting to note that these workers found lessened tumor weight and histological evidence of cellular deterioration in FlexnerJobling tumors, after 16 days of daily antiserum administration, even though concentration in the tumor was negligible compared with other organs. Little specific affinity of immune globulin for tumor was obtained by Hiramoto and Nungester (1958) with Gardner lymphoblastoma 6C3HED in mice. These workers labeled the immune, rabbit, anti6C3HED globulin, and also normal rabbit globulin, with P31,and intravenously inoculated each separately into tumor-bearing mice. Labeled immune globulin was concentrated in the subcutaneous tumors in approximately the same amount as the labeled normal globulin. Liver, spleen, and kidney concentrated the P1-labeled immune globulin a t one half to one third this amount, and i t was concluded that relatively little

THE RELATION OF THE IMMUNE REACTION TO CANCER

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globulin penehated the tumor. Use of fluorescein-labeled immune globulin also indicated that there was little selectivity for tumor tissue. Concentration of the dye was found in kidney glomeruli. 2. Concentration of Antibody in Reaction Products of the Host

Because they believed the Murphy-Sturm lymphosarcoma caused enhanced activity by the rat fibrinogen system in the inflammatory area of tumor growth, Spar et al. (1959) immunized rabbits with rat fibrin and produced fibrin antiserum. The antiserum was labeled with 1131,and inj ected intravenously into 11 Wistar rats bearing the implanted lymphosarcoma. The in vivo distribution showed 14.3% of the 1131dose present in 1 gm. of tumor tissue, as compared with 3.85%) 0.3%, and 0.55% per gram in blood, lymphatic, and spleen tissue, respectively. Normal, labeled, rabbit 7-globulin was found in tumor tissue at 2.1% of the dose per gram. Previous studies by the same authors (Bale et al., 1958) indicated strong, preferential concentration of I131-labeled antibody, prepared against lyinphosarcoma tissue, in the tumor when administered in vivo (6.8% dose per gram). However, this antiserum showed much less specificity when compared to other malignant and normal tissues by in vitro immunological tests. Hence, this seems to be a case in which the antitissue component of the antiserum has a low specificity for the tumor, while concentration of the antibody in the tumor is evoked by a normal host substance, fibrin, which is itself concentrated in the tumor as part of the inflammatory reaction of the host. 3. Factors Affecting the in Vivo Distribution of Antiserum

It is apparent that the factors that can affect the distribution of an injected tumor antiserum are numerous and varied. If the antiserum is immunologically closely related to normal tissues, i t may be removed from the circulation, by organs which contain these tissues, to the same degree as by the tumor, or even to a greater extent, thus reducing the effective concentration of antibody a t the tumor site. If i t is immunologically distinct from the normal tissues, but produced by an animal of another species, it still may be removed by the reticuloendothelial system or the kidney, or detoxified in the liver, like any other foreign substance. The permeability of tumor capillaries, and indeed the accessibility of the tumor to the circulatory system, have also been suggested as important factors. There is also the possibility of neutralization or inactivation of the antibody by combination with haptens or other substances in plasma or tissue fluids, or by circulating tumor antigens. The problem here seems to be closely related to that of chemotherapy in

76

LOUIS V. CASO

general, that is, to obtain an effective, final concentration of therapeutic agent a t the site of action.

4. Alteration of the Metabolism of Ascites Tumor Cells by Antisera Flax (1956) reported that in vitro treatment of Ehrlich tumor cells by immune rabbit y-globulin alone reduced viability of the cells when injected intraperitoneally into mice. The animals survived during the 60-day observation period, while control animals died a t the end of the second week. The anti-Ehrlich, immune 7-globulin, when combined with guinea pig complement, caused in vitro cellular damage to Ehrlich ascites cells, as evidenced by staining of tumor cell cytoplasm and nucleic acid after treatment. Similar treatment of Ehrlich ascites cells in vitro caused absence of glucose utilization as determined by the neotetrasolium method and cessation of oxygen consumption (Warburg method), while succinate continued to be utilized. Intraperitoneal injection of the immune 7-globulin and complement into C F 1 mice on the third to fifth day after Ehrlich tumor implantation produced reduced amount of tumor growth and increased the survival time to twice that of saline-treated controls. Utilization of glucose during in vivo immune globulin treatment was reduced in ascites cells, liver, and kidney to about the same extent, and in spleen to a lesser extent. Ellem (1958) described the swelling of Ehrlich ascites tumor cells after in vitro incubation with the antiserum prepared in the rabbit. I n addition, there was increased permeability of the cells to inorganic phosphate, followed by a loss of acid-soluble organic phosphate. Binding of the inorganic phosphate to the acid-soluble organic phosphate, and degradation of the acid-soluble organic phosphate to inorganic phosphate, were also effects of the antiserum, depending on incubation temperature and time sequence. F. SOMECYTOTOXIC EFFECTS OF TUMOR ANTISERAON CULTURED NORMAL AND MALIGNANT CELLS 1. Species-Specific Reactions of Antisera to HeLa, a Tissue Culture Cell Line Derived from H u m a n Carcinoma of the Cervix Mountain (1955) prepared antisera to HeLa cells, grown in tissue culture medium, by freeze-thawing the cell suspension and inoculating the supernatant into rabbits. Applied to HeLa cells growing on glass substrate, the antisera caused cells located in the interior of the sheet to agglutinate in large clumps. At the periphery of the monolayer the normally stellate cells rounded up and became elongate, sending out spicules from the surface. Nuclei became eccentric or terminal. Finally the nonviable cells peeled off the glass and became floating debris.

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When fresh guinea pig serum was added to the medium, the cells became flattened and necrotic on the glass substrate, and lobt the ability to retain the supravital stain, neutral red. There were also cytotoxic effects on human embryo fibroblasts, HEP 1 and HEP 2 (human carcinoma of cervix and larynx, respectively) , and the Flexner-Jobling r a t tunlor. Miller and Hsu (1956) prepared antisera to HeLa by inoculating the washed, ground cells into rabbits. At a dilution of 1: 10, the antiserum caused disruption of the HeLa cell sheet on glass substrate and withdrawal of cell processes. The cytoplasm became vacuolated and granular, and pinocytosis ceased. Cell extrusions (blisters) were observed in many cells. Mitochondria fragniented, contracted, and became spherical. They then remained rigidly fixed in the granular cytoplasm. Nuclei showed increascd density and rounded up or decreased in size. Nucleoli changed refractility and sometimes disappeared. If the nucleus was damaged, cytotoxicity could not be reversed in normal tissue culture medium. Cells in mitosis appeared to he more resistant to damage than interphase cells, but clumping of chromosomes a t metaphase was seen. I n two cases mitoses were not interfered with by the antiserum. Also affected by anti-HeLa antiserum were human leucocytes and the epithelium and fibroblasts from human skin and tonsil. Mouse B3 mammary carcinoma was uninjured by the antiserum. These investigators found that the antiserum lost its cytotoxic activity when absorbed with norinal human skin extract. Cross-reactions of antisera to HcLa and other human tissues were further investigated by Goldstein and Rlyrvik (1958). They found marked cell damage by anti-HeLa antisera (1: 100 dilution) against HeLa cells, H E P 2 carcinoma, leucocytes of inyeloid leukemia, normal leucocytes, fetal liver cells, intestinal epithelial cells, and Q96 nionocytic (human) leukcinia. There was complete reactivity between the antisera to certain of these tissues and the otlicr cell lines enumerated here, but rabbit fibroblasts and leucocytes remained unaffected. Goldstein reported that the presence of complement was necesary for the cytotoxicity of these reactions. Absorption studies with the antisera to these various human tissues also indicate a species rather than a specific cellular antigen in these cytotoxic reactions, but Goldstein does not believe cellular antigens can be ruled out on the basis of these experiments. I n vivo studies or utilization of priniary tissue explants might reveal cellular antigens unapparent in tissue culture. This work does demonstrate, however, the continuity of human antigens in cell lines of human tissue growing in tissue culture. The affinity of anti-HeLa antiserum for other human tissues was investigated further by Hiramoto e t al. (1958). These workers reacted

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LOUIS V. CASO

various human tissues with the rabbit antiserum to HeLa, after which the tissues were stained with fluorescein-labeled antiserum to rabbit y-globulin produced in the horse. This method showed HeLa antibody present in reticular or basement membrane tissue of thyroid and adrenal glands, liver, and kidney. Unstained were thyroid colloid, liver parenchymal cells, and kidney tubule epithelium. For additional reactions of HeLa antisera, see Sections IV,A,2, and IV,C.

2. Two Distinct Antibodies in H e L a Antiserum The work of Goldstein and Myrvik (1960) is of interest in regard to specificity in that these authors were able to distinguish two specific antibodies in anti-HeLa antiserum, prepared by inoculating rabbits with washed, whole-cell HeLa suspension. This antiserum is active in agglutinating A, B, AB, and 0 erythrocytes of the human blood groups as well as inducing cytotoxicity in HeLa cell cultures. By means of absorption of the antiserum with small quantities of HeLa cells, they were able to reduce markedly the cytotoxic action on HeLa cells, while the hemagglutinating action was not lowered to a corresponding degree. They therefore postulated two distinct antigens for HeLa cells, one producing an antibody which is cytotoxic for HeLa, the other producing an antibody causing agglutination of human erythrocytes. The latter would correspond to the H antigen which is common to human erythrocytes regardless of blood group.

3. Histochemical Reactions with Antisera to Ascites Cells The biochemical alterations in Ehrlich ascites tumor cells, induced by the action of the antibody to these cells, were mentioned previously in the study by Flax (1956). This author observed that the immune y-globulin, produced by the rabbit in response to the tumor cells, caused swelling of the ascites tumor cells and increased cellular fragility in vitro. Staining with azure B indicated dissociation of nucleoprotein after treatment with the anti-Ehrlich 7-globulin. It was found that complement was necessary for these reactions, I n vivo studies showed that intraperitoneal injection of the anti-Ehrlich immune globulin produced a loss of basophilia in the cytoplasm, chromatin, and nucleoli of ascites tumor cells. The cells were also seen to lose RNA, followed by a loss of DNA. The action of the antibody to Ehrlich ascites tumor also seems to be species specific for mouse tissue rather than for the tumor cells, and the reactions studied by Flax and others in Ehrlich ascites cells have been observed in normal mouse tissues and in Krebs ascites tumor cells as well.

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However, quantitative differences between normal and tumor cells have been detected in tissues treated with the antiserum to Ehrlich ascites tumor. IV. lmmunochemical Pattern of Tumor-Related Antigens

Central to the concept of tumor diagnosis or therapy by immunological mcthods is the possible existence of specific tumor antigens. Various studies have been carried out to determine whether tumor antigens can be distinguished from corresponding normal tissue antigens or from the antigens of other tumors, or if there is indeed an antigen common to all tumors, or whether tumorous tissue may bc lacking in one or more antigens present in the corresponding normal tissues. Thesc studies are a11 complicated by the fact that normal and malignant tissues contain numerous antigenic components, any of which, because of comparatively low antigenicity, may be obscured in immunological reactions by the others if the latter conipcte more successfully for the production of antibodies in the inoculated animal. Consequently, the siniplc lack of an antibody in a given antiscrum is not proof that the antigen is absent from the tissue. Because of the complex nature of tissue antigens, recent studies have concentrated on the fractionation of components of normal and malignant tissues and their separation into protein and other chemical moieties. Even after separation by biochemical methods, it is still possible that the Components may differ antigenically in some respects and yet be related to each other in certain other respects, so that unless the immunological method is refined enough to show these differences, qualitative discrimination between two given tissue antigens may well be missed. Finally, there is the possibility t h a t antigens in corresponding normal and malignant tissues, while qualitatively the same or similar, may differ quantitatively in the respective tissues. Here again their detection would depend upon the sensitivity of the immunological methods used, there being the possibility that low levels of antigen concentration might be missed completely. The problem of the existence of tumor antigens has been approached from several directions, and the following discussion is an attempt t o introduce some of the more effective methods in current use. A. FRACTIONATION OF THE TUMOR ANTIGEN 1. Chromatography and Electrophoresis

Angeletti and co-workers (19604 studied seven tumors, found in various inbred strains of mice, by fractionation on a cellulose ion-ex-

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LOUIS V. CASO

change column. Previous work had shown that each normal tissue (in rats and mice) has a characteristic enzyme pattern quite distinct from other tissues of the same species of animal. By fractionation of the tumors (which included rhabdomyosarcoma, mammary gland carcinoma, and lymphosarcoma in Swiss mice), it was found that all seven tumors had a similar soluble protein pattern and similar patterns of enzyme activity. The chromatogram peaks for the enzymes were in the same position for each tumor and the proportion of peaks was approximately constant for all the tumors. The authors concluded that the proteins resemble one another closely, regardless of the tumor. This supports the belief that neoplastic tissue tends to approach a common metabolic type. The same researchers (Angeletti e t al., 1960b) compared carcinogeninduced, transplantable rhabdomyosarcorna in C3H mice with normal muscle by means of DEAE-cellulose column chromatography. A large part of muscle protein from normal mice was found not to be bound to the ion-exchange column, representing protein of zero or positive charge and including some glycolytic enzymes and perhaps also myogen and myoglobin. The rhabdomyosarcoma protein differed from the muscle protein from which it was derived originally in that a relatively reduced amount of this basic component of soluble tumor protein was not bound to the column, representing 15 to 20% of the total recovered rhabdomyosarcoma protein. The difference may be due to the fact that some highly specialized proteins of normal muscle are missing in the tumorous tissue. Moreover, elution of much of the rhabdomyosarcoma protein from the column was accomplished a t a higher level of sodium chloride concentration than was most of the normal muscle protein. In the chromatograms some enzymes showed differences in activity and localization. Glucose-6-phosphate dehydrogenase was found to be increased in the tumor as compared with normal muscle and showed two extra peaks in the chromatogram. Alkaline phosphatase was detected in the rhabdoinyosarcoina in several distinct peaks, while i t was not found in measurable amounts in normal muscle. &-Glycerolphosphate dehydrogenase activities, well localized in normal mouse muscle chromatograms, were not detected in various fractions of the rhabdomyosarcorna. This agrees with other reports that 0-glycerolphosphate dehydrogenase has very low levels in various malignant tissues. It is significant that the comparison of rhabdomyosarcoma chromatograms with those of squamous-cell carcinoma of the skin in mice (Angeletti e t al., 1960c) showed a great similarity in protein distribution. Other evidence that different neoplastic tissues tend to develop comrnon metabolic features was contributed by studies of the Walker 256 carcinoma in rats following injection of L-lysine-U-Cl'. Chromatography

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81

of radioactive acid-soluble nucleoprotein of the tumor gave a specific localization in radioactive peak 2 ( R P 2-L). Thirty to thirty-seven per cent of the labeled lysine was found in the cationic nuclear proteins, specific activity being greater in the histone fraction than in any other cellular fraction. Since peak 2 ( R P 2-L) was not found in a number of other tissues, it was postulated that protein synthesis in the tumor was different from t h a t in nontuniorous tissues. T o test this hypothesis, Davis and Buscli (1960) studied various tumorous and noriiial tissues, and found that isolated, fractionated nuclei from tumor in rats with Jensen sarconia and Flexner-,Jobling carcinoma, and in mice with Sarcoma 180 and Ehrlich ascites tumor, after injection with labeled glycine, showed radioactivity a t elutions from a similar chromatographic position [peak 2 (RP 2 - L ) ] . This was true of a human inalignant iiielanoma obtained by surgery. The radioactive peak was not found in normal tissues undergoing rapid growth, i.e., cnibryonic rat tissue and regenerating r a t liver. Hence, these findings suggest that significant differences exist between proteins of tuiiiors and other tissues. The authors mention the possibility that nuclear proteins may undergo genetic change in tumor cells. This change, due either to mutation of nucleic acid or admixture of viral nueleic acid, would take a direction common to neoplastic tissuc. Witebsky and co-workers (1956) in their extensive studies on thyroid glands were able to show hiocheniical differences between the normal gland and inalignant thyroid tissue. Ultracentrifugal analysis revealed that the peak for thyroglobulin in the normal gland contained 58.3% of the components, while the corresponding peak for primary and iiietastatic tumors consisted of only 4.9%. I n addition, electrophoresis showed qualitative differences between the normal gland and the iiietastatic tuinor. Whereas normal thyroid extract showed four main protein peaks (probably thyroglobulin, consisting of 62% of the total components), the metastatic thyroid tissue extract showed nine poorly resolved constituents. Of these, the major peak was a protein other than thyroglobulin, while the thyroglobulin peak made up only 19% of the total protein. Moreover, five of the components of the malignant tissue did not seem to have any counterparts in the normal thyroid gland. Iininunological studics rcviewed by the same authors (Witebsky e t nl., 1956), wlio used the complement fixation reaction carried out by serial dilutions of both the antiserum (antithyroid carcinoma) and the antigens (normal and malignant thyroid tissue), showed t h a t the titer for the homologous (tumor) antigen was 10 tinies t h a t of the normal thyroid antigen. H O W C V Cthe ~ , two antigens eross-reacted, and the common

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antibody could not be removed by absorption without greatly reducing the titer for the thyroid tumor antigen. I n light of the differences disclosed by electrophoresis, i t would be of interest to see these cross-reactions repeated by the more specific double diffusion gel technique of Ouchterlony (1953). 2. Extraction with Fluorocarbon and High-speed Centrifugation

Genetron is a fluorocarbon, trifluorotrichloroethane, which was used originally to separate nonviral protein from virus-tissue extracts. Later this substance was used to separate nonspecific, complement-fixing antigen from specific antigens in tumorous tissue. I n this application the Genetron-treated antigen, present in the tissue supernate, is centrifuged a t 20,000 and 40,000 r.p.m. The resulting pellet represents the purified antigen, which can be used in immunization of rabbits and in the complement-fixation reactions. Taylor et al. (1959), using Genetron extracts, studied a human metastatic ovarian sarcoma, human carcinoma HEP 2 (tissue culture), Rous sarcoma (viral origin in fowl), and transplantable chicken fibrosarcoma. The data in Table I show the results when untreated or crude TABLE I COMPLEMENT-FIXING TITERSOF RABBITANTISERAAGAINST THE OPTIMAL DILUTIONOF THE VARIOUSCRUDEANTIGEN SUSPENSIONS~ Crude antigens

Crude antisera

HEP 2

Human Chicken Rous sarcoma fibrosarcoma sarcoma ~

HEP 2 Human sarcoma Chick tumor (germfree) Rous sarcoma Chick muscle (germfree)

160 80 -

-

40 160 -

-

Germfrce chick muscle ~~

-b

320 320 160

40 320 40

-

40 40 80

From Taylor et al. (1959). The dash indicates titer of less than 20.

antigens are reacted with antisera to these tumors. Note that the human tumors cross-react, as do the three chicken tissues. The two different species do not cross-react, however. From Table I1 it can be seen that Genetron extraction and ultracentrifugation of the purified antigens has resulted in virtual elimination of cross-reactions between these antisera.

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THE RELATIOE O F T H E IMMUNE REACTION TO CAKCER

TABLE I1 COMPLEMENT-FIXING TITERSOF RABBITANTISERA A G S I N S T THE OPTIM.\I, DILUTIONOF THE VARIOUS GENETRON-TREATED ANTIGEN SUSPENSIONS" Genctron-trcated antigens Antisera to Genetron extracts

HEP 2

HEP 2 Human sarcoma Germfree chick tumor Row sarcoma Germfree chick muscle

Human sarcoma

40 -

_c

-

-

-

-

-

-

Chick t,umor

ROUS* sarcoma

so

-

80

-

Germfree chick muscle

40

From Taylor el al. (1959).

* I.D.5, virus titer lo4 in chick embryo tissue culture. c

The dash indicates titer of less than 20.

PlcKenna and colleagues (1962) extracted homogenized suspensions of HeLa and J l l l cells grown in tissue culture, and normal tissues, including uterus removed by surgery. Separation of the Genetron-aqueous antigen mixture was accomplished by four centrifugations a t 30009 a t 10°C. The numerous cross-reactions of the antisera with the crude (untreated) antigens are coniparcd to the reactions with Genetronpurified antigens in Table 111. It can be seen that there has been a marked increase in specificity of the complement-fixation reaction. Note that all antigenicity is lost from uterus by Genetron treatment of the antigens. The data shown in Table I11 for uterus were the same for normal human skin, muscle, and liver. TABLE I11 SPECIFICITY O F CELL EXTRACTS OBTAINED B Y GENETRON TREAT?dENT"* Complement fixation titers with antisera ~~

Antigen

HeLa(C)

HeLa(C)

320 40 40 320

Jlll (C)

Uterus(C) HeLa(G)

J111(G)

Uterris(G)

5 5

Jlll(C) 80

so

80 10 320 5

Uterus(C)

HeLa(G)

Jlll(G)

Uterus(G)

80 40

640

5 80 5 10 640

6

320

5 5 5

5 5 1280

5 5

5

5 6

5 5 5

From McKenna et al. (1962). C, crude antigens and their antisera; G, Genetron antigens and their antisera. (1

* Symbols:

84

LOUIS V. CASO

By means of absorption with crude and Genetron-treated extracts of uterus and HeLa cells, it was found that there is a specific antigen for HeLa, while HeLa also shares a common antigen wit.11 normal uterus. Thus from Table I V it can be seen that antiserum to crude uterus TABLE IV COMPLEMENT-FIXING TITERSOF ANTISERATO CRUDE UTERUS A N D GENETRON HEL.4 EXTRACT, AFTER ABSORPTIONSWITH CRUDE UTERUS EXTRACT (c), GENETRON-TREATED HEL.4 EXTRACT (G), OR CRUI)E HELA EXTRACT (c)' Antigen used for immunization

Absorption of antiserum

Ut,erus (C) Uterus (C) Uterus (C) Uterus (C) HeLa (G) HeLa (G) HeLa (G) HeLa (G)

None Uterus (C) HeLa (C) HeLa (G) None HeLa (G) HeLa (C) Uterus (C)

Complement fixation titers Uterus (C)

HeLa (G)

320 5 10 320

5 5 5 5

5

640 10 40 640

5 5

5

From McKenna et al. (1962).

extract, absorbed with crude HeLa extract, will remove most of the antibody to crude uterus extract. Conversely, the antiserum to Genetron extract of HeLa, absorbed with crude uterus extract, shows no reduction in antibody to the purified HeLa antigen. The same is true of the antiserum to crude uterus, absorbed with the Genetron HeLa extract, i.e., there is no reduction in antibody titer to crude uterus antigen. DeCarvalho (1960), applying the method to precipitating antibodies, used Genetron to separate two main fractions of human leukemia and tumor cells, the protein fraction being examined for specific antigens by the double diffusion gel technique (Ouchterlony, 1953). While several antigens were shown to be associated with normal components, only one antigen each could be found in the highly purified extracts of leukemia and tumor cells. These antigens were different in the leukemia and tumor cell extracts, There were also differences in the antigenicity of the extracts of acute stem cell leukemia and chronic lymphatic leukemia. 3. Dialysis of a Tumor Antigen Component

Bogden and Aptekman studied the effects of tumors on the hemagglutinins to human erythrocytes in the plasma of rats and their results have been discussed (Bogden and Aptekman, 1953; Aptekman and

T H E RELATION O F T H E I M M U N E REACTION TO CAXCER

85

Bogden, 1956). I n these studies i t was found t h a t the snti-A activity of normal r a t scruin was related to erythrocyte A substance, and t h a t the action on erythrocytes of the other blood groups was due to another agglutinin (anti-Xi), which was related to none of the major blood groups. I n a later study, these authors (Bogden and Apteliman, 1957) prepared conccntrated ethanol extracts of the following tumors, occurring spontaneously or carcinogcn-induced, in P.A. strain rats : Sarcoma 6, Careinonia 5 , and Sarcoina 231. These extracts, as well as normal r a t tissue extracts, all inliibited the hemagglutination of liunian A and B erythrocytes by normal rat plasma. After dialysis of the tumor extracts, their ability to neutralize the anti-A and anti-X of r a t serum was still apparent, although neutralization of anti-X activity was found to be reduced. After normal rat muscle extract had been dialyzed, its ability to neutralize anti-A of nornial rat serum was lost, although i t retained its ability to neutralize anti-X. These results indicate t h a t the neutralizer of anti-X is a dialyzable fraction, responsible for the neutralizing effects of norinal rat tissue extracts on the antihuirian erythrocyte hemagglutinins in normal r a t seruni. The neutralizer of anti-A, which is not dialyzable, seeiiis to be peculiar to rat tumor extracts. It is of interest that normal rat tissues lack antigenicity and reactivity in precipitin reactions, perhaps because of the low niolecular weight of anti-X neutralizing substances, which would act like haptens. On the other hand, ethanol extracts of r a t lymphonia and sarcoma are antigenic in rabhits, while r a t muscle extracts are not. 4. Ouchterlony Technique for Diffusible Antigens and Antibodies

Ouchterlony (1953) devised the method whereby precipitating antibodies and antigens will diffuse through agar from adjacent wells and form a precipitate in the region of optimal concentrations. I n 1957 this inethod was reviewed by Korngold (1957), who has done extensive studies on tumor antigens using thc double diffusion gel technique. The valuc of the technique is its ability to separate the reactions of mixed antigens and antibodies, and consequently allow comparisons among different antigens which can establish their identification in extracts of various normal and malignant tissues. The precipitate line, which f o r m in the agar betwecn adjacent wells which contain separately the antigen and the antiserum, will vary in its position relative to the two wells and in its curvature. The position of the line depends on the concentration of the antigen(s) and the curvature of the line depends on their diffusion coefficients. The precipitate forms a t the position of optimal proportions of antigen and antibody a t the

86

LOUIS

V.

CASO

equivalence point. The density of the precipitate is related to the antibody content of the antiserum. It is possible by the Ouchterlony technique to distinguish between several different antigens. If the same antigen is placed in two separate adjacent reservoirs, while their homologous antiserum is placed centrally in a third reservoir (completing a triangle), the precipitate lines will coalesce to form a continuous line between the two antigen wells, showing close relationship or identity between the two antigens (C and D of Fig. 1 ) . This is called the reaction of identity. If the two adjacent reser-

FIG.1. Central well: antibodies vs. serum albumin and y-globulin; wcll A : y-2-globulin; well B: Bence-Jones protein; well C : serum albumin (0.1 mg./ml.) ; well D : serum albumin (0.02 mg./ml.). Sce text for explanation (Korngold, 1957).

voirs contain unrelated antigens, while the antiserum in the central well contains antibodies to each of them (mixed antibodies), the resulting precipitate lines will not coalesce but intersect (A and C of Fig. 1 ) . If one reservoir contains the homologous antigen, and the other a cross-reacting antigen, the two precipitate lines will coalesce only partially, with a “spur” extending beyond the point of juncture. This is the case of B and A in Fig. 1. Most tissue extracts contain several antigens, and antisera made to a given tissue would contain a mixture of antibodies to some or all of these antigens. In the case in which the antigen well contains a mixture

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of antigens, and the antiserum well contains a mixture of antibodies, several precipitate lines will form. These correspond to the different rates of diffusion of the several antibody-antigen systems. The number of lines will depend on the number of different antigens t h a t can react with tlie antibodies present in the antiseruiii. As Korngold points out, a given antiscruin usually does not contain antibodies to all tlie antigens present in the iiiiniunizing mixture. Therefore, the number of precipitate lines represents a niinimum number of antigens present in the mixture in the antigen well. Korngold emphasizes that it is difficult to dcteriiiine that an antigen is lacking in R certain tissue. Varying concentrations of the antigen inust be used in orclcr to ascertain the optimal concentration for producing the precipitate. Moreover, normal and tumorous tissues from different individuals do not contain antigens in the same concentrations. Conseyucntly, in comparing antigen content of a tumor with that of normal tissue, the same individual should be used. I n general, it is easier to show altercd protein structure or abnorinal protein synthesis than coiiiplcte lack of an antigen by the double diffusion gel technique. In Fig. 2 can be seen the precipitate lines of the several antigens

FIG.2. Centrr well: antihuman carcinoma antiserum ; well A : human carcinoma of cervix (40 mg./ml.) ; well B: HEP 3, 13 gcncrations in rats (80 nig./ml.) ; well C: human pla,-ma; wrll D : HEP 3, 7 grnrrations in rats (80 mg./ml.). See text for explanation (Korngold and Lipari, 1955).

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present in human carcinoma of the cervix (upper left well), when reacted with antihuman carcinoma (parotid) antiserum. One of these antigen lines shows the reaction of identity with HEP 3 extract (upper right well), which is a rat-grown transplant of carcinoma of the buccal niucosa. Note the nonrcactivity of human plasma. a. Persistence. of Human Antigens in Rat-Grown Transplantable Tumors. Korngold and Lipari (1955) analyzed the antigens of human tumors, grown by transplantation in rats, to determine whether or not their original characteristics were retained. The sarcoma HS 1 had had 31 transfers in rats; the epidermoid carcinoma HEP 3, ten generations in rats, one generation in the hamster and one in eggs. Using the double diffusion gcl technique, these workers compared the three tumors by reacting the antigen extracts with antiserum to human carcinoma of the parotid gland. HEP 3 showed several precipitin lines, some of which showed identity with antigens of the carcinonia of the human cervix. This tumor lacked several of the antigens (lines) present in the carcinoma of the cervix. HS 1 was also shown in this way to produce human antigens. On the other hand, rat lymphosarcoma, when reacted with the antihuman carcinoma antiserum, showed no human-reacting antigens (Fig. 3, well C ) . When the antiserum to rat lymphosarcoma was rcacted with the human tumors and the homologous lymphosarcoma, the human tumors showed some precipitate lines which were identical with the antigens of the homologous rat tumor. The antiserum did not react with human carcinoma of the cervix and other human tumorous and normal tissues. T o dcterniinc i i the human tumors had had their metabolism shifted by transplantation to the synthesis of rat antigens, thesc researchers grew the H E P 3 tumor in the hamster and finally in eggs, and the HS 1 tumor in eggs also. The tumor transplant thus grown showed no reactivity with the antirat lymphosarcoma antiserum. HEP 3 and HS 1 still showed synthesis of human antigens when reacted with the antihuman carcinoma antiserum. Hence the double diffusion gel technique established t h a t transplantable human tumors continued to retain human antigens, regardless of transplantation generation, and that their reactivity with the antirat antiserum was not due to intrinsic synthesis of rat antigen, but probably to ingestion of rat protein, by the human tumors, The human antigens found in the tumors werc tissue antigens, and no serum albumin, aglobulin, fibrinogen, or ,&lipoprotein was detected. b. Occurrence of Specific Antigens in Various Normal and Malignant H u m a n Tissues and in Transplantable H u m a n Tumors. I n a later study Korngold (1956) found that with each of four antisera to human tissues

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Ii’iG. 3. Ceiitcr well: Antiliunian carcinoma antiseruni; well A : HEP 3, 13 gcncriitions in rats (40 mg./nil.) ; wrll B : HEP 3, 16 grnerations in rats (80 me./ nil.) ; wrll C : rat lymphost~rconia (40 nig./ml.) ; well D : human carcinoma of cervix (40 n ~ g . / n ~ l . )Srcl , t r x t for r>xpliin;ition (Korngold and Lipari, 1955).

a t least one antigcn in eacli tissue rcactcrl strongly with the homologous antiserum, other antigens of the tissue appearing as fainter lines in the agar. Antigen 1 was present in an ovarian cyst; antigen 2 in carcinoma of the ovary; antigen 3 in nornial uterus; and antigcn 4 in carcinoma of the cervix. Screening 46 surgical spceiiiicns of normal and inalignant human tissues by his niodified Ouchterlony method, Korngold found that antigen 1 n.as prcscnt in 50% of the specimens, being absent in five carcinomas of the ovary and present in two of three noriiial ovaries. Antigen 1 was prescnt in all carcinoinas of the cervix and uterus, but absent from inost normal uterine tissues. It was considered a “groupspecific” antigen. None of the tuniors or normal tissues lacked all of the antigens, and only one ( a carcinoiiia of the ovary) lacked three of the antigens. Approximately 50% of the specimens contained all four antigens. Antigen 1 was the antigen found to be absent most frequently. Antigen 2 was present in all surgical specimens, although in low concentration in fibromas, sarcomas, and uterine tissue. Antigen 4 was present in all

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tissues except in three instances,, i.e., in one of five carcinomas of the ovary, in one of seven uterine fibromas, and in one of seven normal uterine tissues. Antigen 3 was also lacking in only three instances, i.e., in one of five carcinomas of the ovary (the same one as the preceding), in one sarcoma of the pelvis, and in one of two reticulum cell sarcomas. I n direct contrast to the above findings on surgical material was the occurrence of the four antigens in human tumors grown as transplants in cortisone-treated rats. The results obtained by double diffusion in agar are shown in Table V. TABLE V DISTRIBUTION OF ANTIGENSIN HUMAN TRANSPLANTED TUMORS IN RATS& ~

No. antigen 1 2 3 4 a

b

HEP 3 (carcinoma buccal mucosa, 13th gen.)

HEP 1 (epidermoid carcinoma of cervix, 74th gen.)

-

-

*b

HS 1 (sarcoma, 32nd gen.) -

+*b

H. Emb. Rb. (embryonic rhabdomyosarcoma, 56th gen.) -

Melanoma 1 (20th gen. in hamster and rat) -

+

From Korngold (1956). Present in low concentrat,ion.

It will be seen that antigen 2 is present in HS 1 only, and antigens 1 and 3 are absent from all five tumors. Antigen 4 was present in the melanoma in usual concentration, but in HS 1 and HEP 3 it was not visible in the 40 mg./ml. concentration. While H. Emb. Rb. and HEP 1 lacked all four reference antigens, the HEP 1 carcinoma did contain another antigen to anti-carcinoma of the cervix antiserum, indicating that i t in fact continued to synthesize human antigen. This study therefore confirms the fact that human tumors have the capacity to produce species specific antigens 3 years after transplantation in a foreign host. Antigen 2 must be considered lacking in 4 of the 5 tumors in the 40 mg./ml. concentration used. Elsewhere (in lyophilized carcinoma) i t can be detected in as little as 1 mg./ml. It is of interest that antigen 3 was absent from HEP 1 (epidermoid carcinoma of the cervix), since i t was present in all the surgical specimens of carcinoma of the cervix. The absence of antigens 1 and 3 from the transplantable tumors may be due to either of two causes. Growth in a foreign host may induce loss of antigen synthesis by the tumor; or the tumor may have con-

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tained antigen-deficient cells originally, which were reproduced selectively when transplanted into the foreign host. The latter possibility seems to be supported by the finding that some surgical specimens were lacking in these antigens, as shown above. I n the case of HEP 3 i t is known that the tumor was already deficient in some antigens after only a few generations in a foreign host. c. Augmentation of Antigenicity of a n Antigen b y Electrophoretic Separation of Its Components. Korngold and Van Leeuwen (1957) used these same four tissue antigens in a more recent study. By double diffusion in agar they also found another tissue antigen reactive with an antiserum to a carcinoma of the ovary, antigen 5 . By fractionation of these antigens by zone electrophoresis on starch, they were able to determine which components reacted with the several antisera. From these antigens five fractions, Corresponding in mobilities to the serum proteins, were obtained. Fraction E had the fastest electrophoretic mobility, corresponding to t h a t of albumin, while fraction A had the slowest mobility, corresponding to that of 7-2-globulin. Most antigenic substances were located in fractions C and D. Fraction A was not reactive with the antisera directed against antigens 4, 2, or 5 . As has been observed before, not all the antigens present in a tissue will stimulate antibody production in any one immunization series. It is believed that the better antigens compete more successfully for antibody-producing sites in the inoculated animal, while the poorer antigens fail to find these reactive cellular areas. This can be compared to competitive inhibition of biologically active chemical agents. With this in mind, Korngold and Van Leeuwen inoculated animals with the separated antigen fractions which had been found to be nonreactive with the antisera. I n this way they hoped to determine whether or not any antibodies could be produced under these new conditions. Fraction A, obtained from carcinoma of the ovary (Ca ov. 11) and unreactive for antigen 2, when inoculated into the rabbit, produced antisera with strong reactivity to fractions A and B a t dilute concentrations. Similar findings were obtained for fraction A derived from normal uterus. These results tend to support the “competitive inhibition” theory of antibody formation. Korngold and Van Lceuwen found that those fractions which did react with the homologous antiserum were identical with the original (unfractionated) antigen and with each other. This is seen to be true, for instance, of antigen 2 in ovarian Carcinoma 111, when the unfractionated carcinoma and its fractions C , D, and E are reacted with antiovarian carcinoma antiserum by the double diffusion gel technique. The precipitate lines of each coalesce, showing identity for antigen 2. The

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indication is that fractionation does not alter the specificity of the antigen. This was also found to be true of the other antigens studied. I n the same study it was found that fractionation can be used to determine whether or not an antigen is really absent from a tissue or simply present in a concentration so low that it cannot be detected by the methods employed. Thus IlEP 3 showed no reactivity for antigen 2 in any of its fractions at a concentration of 30 mg./ml. Comparing this to ovarian cnrcinonia fractions C and D, which reacted for antigen 2 a t a concentration of 0.5 ing./ml., the evidence for the complete lack of antigen No. 2 in HEP 3 is impressive. Similarly, fractionation of HS 1, HEP 3, and reticulum cell sarcoma extracts failed to show the presence of antigen 1 in these rat-transplanted human tumors. Finally, the study of Korngold and Van Lecuwen revealed that, when tissues containing a particular antigen are fractionated, the antigen is found in fractions with the same electrophoretic mobilities, and usually in similar concentrations. It would seem from these studies that the combined use of electrophoresis of antigenic material and the double diffusion gel reaction for antigens and antiscra provides an approach well suited to the complexity of the antigenic constitution of normal and neoplastic tissucs. d. Plasma Changes in Multiple Myeloma and Other h'eoplastic Diseases. (1) Multiple Myelonia Globulins. The Ouchterlony technique was applied to the study of 24 multiple myeloma globulins by Korngold and Lipari (19564. The abnormal globulins were separated electroplioretically a t mobilities ranging from 0.7 to 3.4 cm.2/v./sec. x lo?. When reacted with normal anti-7-globulin antiserum, and compared to Fraction I1 and 7-2 of normal serum, the multiple myeloma (MM) globulins showed partial reactivity (see Fig. 4). This indicated that the disease had altered the globulins so that some antigen or antigens were lacking. When the antinormal y-globulin antiserum was reacted with normal y-2, Fraction I1 or any of its subfractions, only the reaction of identity was seen (see Fig. 4 ) . When reacted with the normal ironbinding fraction (P-1-globulin) , the @-lipoproteins, or the p-globulin fraction, the antinormal y-globulin antiserum showed no reactivity. Therefore, the difference in reactivity of MM globulins and normal y-globulin cannot be due to antigenic difference in Fraction I1 or its subfractions, or possible cross-reactivity with normal p-globulins present as contaminants, or to quantitative elevation of the normal components in the neoplastic plasma. Although one multiple myeloma globulin, MM XIX, had the same mobility as normal y-2, it was still found to be antigenically dissimilar. Using antiscra to three different antigenic MM globulin types, these

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FIG.4. Centor well: anti-y-globulin antiserum; well A : Fraction I1 ( 1 mg./ml.) ; wcll B: y-2-glot)ulin; wrll C 1 M M - S X I glot,nlin (1 mg./nil.) ; wc.11 D: MM-XX globnlin ( 1 nig./rnl.). Sec tcxt for rsl)lanation (ICorngold and Lipari, 1956a).

saiiie authors found that tlie R l R l globulins contain antigenic determinants which are lacking in norinal globulins of Fraction 11. It is therefore clear that altered glohulins of multiple iiiycloma serum are lacking in antigenic determinants present in normal y-globulin, and a t the saiiie tiiiie possess antigenic detcriiiinants which are lacking in normal 7-globulins. Moreover, it was found by using antiscra to each of the three antigenic groups of ?1/IRI globulin (MM I, Alhl 111, and AIM V I I , that thcre is a common antibody in the antinornial Fraction I1 antiseruni and the antiserum to each of tlie three abnormal globulin types. (2) Bence-.Jones Protein and Multiple Myeloma Protein. I n another study, Korngold and 1,ipari (1956b) investigated the structural relationship between normal y-glotdin, niultiplc niyeloina globulin ( M M ), and Bence-Jones (BJ ) urinary protein of itiultiple niyelonia patients. When reacted by double diffusion in agar with antinornial y-globulin antiserum, Bence-.Jones protein, and noriiial 7-globulin showed precipitate lines which indicated a relationship but not identity. A relationship between B J protcin and hlRl globulin n ’ a b also indicatcd. Antiserum to MM globulin, inoreover, reacted with sonic antigenic determinants coni-

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mon to BJ protein and MM globulin, and with other antigenic determinants common to normal 7-globulin and M M globulin. B J protein, therefore, has determinants possessed by M M globulin and not by normal 7-globulin, and consequently i t is closer antigenically to the M M globulin. These authors suggest that Bence-Jones protein is produced by cells which cannot complete the synthesis of M M globulin. I n addition, Morton and Deutsch (1958) found evidence from quantitative precipitin reactions that M M globulin and B J protein show individual variations, the antisera to BJ protein cross-reacting strongly with M M globulin of the same patient, and less strongly with M M globulin of a different patient. This would seem likely, according to these authors, if BJ protein is a product of incomplete protein synthesis. Experiments such as these formed the basis of the immunological methods for diagnosis of multiple myeloma and macroglobulineniia described more recently by Korngold et al. (1962). (3) Comparison of Immunological and Physicochemical Analyses of Proteins in Multiple Myeloma. The physicochemical data supplied by Putnani and others (1955) on abnormal serum proteins in multiple myeloma and related obscure diseases tend to confirm the immunological findings. The abnormal globulins have been found to have characteristic physical-chemical constants and N-terminal &-amino acids. These globulins are homogeneous when compared to normal globulins electrophoretically. Most are of the 7-type in mobility range and have a similar shape and molecular weight. Specific abnormal globulins are synthesized by individual patients, They can be divided into two groups: those closely related to normal 7-globulin; and the P-type, which is distantly related to normal 7-globulin. Moreover, these abnormal myeloma globulins are not identical with one another but have different N-terminal residues. It has been found that N-terminal aspartic acid and glutamic acid have a different distribution from that of normal 7-globulin and its common subfractions. Other myeloma globulins are practically devoid of aspartic or glutamic acid, and they contain other amino acids not found in normal globulin fractions. Cryoglobulins (which solidify in the cold), from M M serum, also differ from normal pooled serum in the N-aspartic and N-glutamic acid group distribution, as well as in two or more of the following characteristics: crystal form, solubility, isoelectric point, electrophoretic mobility, sedimentation constant, and homogeneity. Putnam reports that physicochemically Bence-Jones protein is essentially an incomplete protein. The molecular weight is one quarter that of normal serum globulins. BJ proteins are of different types, apparently individual in character, and show unusual heat coagulation properties.

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Cl4-1abelcd glutaniic acid and lysine injections in vivo have indicated that it is not a clcavage product of abnormal serum proteins. Putnam suggests that BJ protein may result from a n abortive attempt to synthesize normal globulins by a deranged mechanism of protein synthesis. (4) Serum Protein Changes in Experimental Animals with Spontaneous and Transplanted Tumors. I n addition to the globulin changes seen in multiple iiiyeloina, there have been increasing reports of serum changes in experimental animals with other neoplastic diseases. Clausen and associates (1960) studied two lines of spontaneous hcpatomas from (CBA x DBA/2)F, hybrids by transplanting the tumors into mice of the same genetic constitution. They reported an increase in the p-fraction been in paper electrophoresis. Inimunoelectrophoresis showed t h a t this incrcabc was clue to a strong increase in the p-2 I globulin fraction. This fraction was identificd by autoradiography as the iron-binding protein corresponding to transferrin (siderophilin) of human serum. The rise in the p-2 I fraction was observed in all mice examined. I n all the hepatomatous sera, moreover, in the y-area, liypogammaglobulinemia was seen to occur. I n inice with two lines of plasma cell leukemia there was observed a moderate or slight increase in the p-fraction, and this was not as constant as that seen in hepatoma. The p (transferrin)-level was seen to be normal or decrease in other transplantable leukemias and tumors of various typcs. Other fractions which were found to be changed were p-3 I ; a-2 I ; CU-211. The p-2 I11 change in mice with all types of spontaneous and transplanted tumors was probably due to the production of immunoglobulin directed against the tumor. Increased amounts of a-globulin fractions of plasma have been reported in human patients and in animals with neoplastic diseases, including C57BL/6 inice bearing Sarcoma 180. Miller and Bernfeld (1960) analyzcd the globulins of C3H mice with spontaneous mammary carcinoma. Fractionating serum by zone elcctrophoresis, they produced antisera to various fractions of plasma from tumorous animals. Antiserum to the c~-1and (Y-2globulins, when in double diffusion gel with plasma from tunior-bearing mice, showed a strong extra precipitate line. This was in addition to the strong, single line seen with the plasma from normal C3H mice. Moreover, a fainter line was produced with anti(1-2 globulin antiserum and plasma from tumorous mice, and this was in addition to the two or three normal precipitate lines formed with normal C3H plasmas. These researchers found that they could identify accurately plasma from mice bearing the mammary carcinoma by the appearance of these extra lines in the plasma fractions. By this technique it was therefore possible to identify an abnormal plasma component

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which could not be found by electrophoresis. There is a strong implication that such procedures may have a diagnostic value for human cancer. e. Antigens of H u m a n Leucocytes. By means of double diffusion gel studies Korngold et al. (1961a,b) detected three antigens in human leucocytes. Antisera to high-speed leucocyte sediments were prepared in rabbits. Most of the antisera contained an antibody against the same granulocytic antigen. Antigen 1 was found by reacting an antiserum to chronic myelocytic leukemia cells. The antiserum showed high potency toward the homologous leucocytes. Antigen 2 was detected with an antiserum to normal leucocytes and was seen in relatively high concentration in granulocytes. Antigen 3, described as relatively specific, was detected by using an antiserum to cells of acute leukemia. It produced a distinct precipitate line with the homologous cells and certain other acute leukemic cells. Antigen 3 was not specific for the latter, however, since it also reacted with extracts from normal lymph node, liver and spleen. According to the relative amounts of these three antigens, three antigenic groups of leucocytes were described. Group I, which includes granulocytes, normal leucocytes, and chronic myelogenous leukemic cells, contains much antigen 1 and antigen 2 and little antigen 3. Group 11, found in relatively immature cells and in leucocytes from certain acute leukemia patients (although also in normal spleen and liver), is rather deficient in antigen 1 and contains considerable amounts of antigen 3. Group 111, made up of all cells from chronic lymphocytic leukemia and certain cells from acute leukemia, as well as Osgood J l l l and J96, is deficient in most of the antigens which are found in high concentration in Groups I and 11. Presence of the leucocyte antigens in other tissues and organs is difficult to determine because of leucocyte contamination of the extracts. However, cells grown in cortisonized rats or in tissue culture should be free from contaminating leucocytes. Four such cell types contain antigens which react with antileucocytic antisera: Osgood J l l l and Osgood J96 (isolated from the buffy coats of acute monocytic leukemia serum); HeLa cells; and HEP 2. However, some antigens present in granulocytes are lacking in HeLa cells and HEP 2. HEP 2 grown in tissue culture, has only one antigen in common with granulocytes. Among the surgical tissue specimens tested with antichronic myelocytic leukemia antisera, and showing antigenic differences compared to leucocytes, human reticulum cell sarcoma and normal uterus were found to be lacking in at least one antigen found in the homologous myelocytic leukemia extract.

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The authors of this study conclude that i t is evident that human granulocytes contain large amounts of several antigens not produced in other cells, or produced in quantities too small to be detected. No specific antigen for lymphocytes could be demonstrated.

B. STUDIES WITH FLUORESCENT STAINING REACTIONS There are two ways in which fluorescein is used in cytological studies of neoplastic tissue. One method is dependent on a reaction unrelated to the usual antibody-antigen reactions. Weiler (1956a) demonstrated that binding of tissues by carcinogens could be shown by means of fluorescein-globulin stain. Normal liver cells were seen to combine with fluorescein, while hepatoma cells did not. Others found that specific carcinogens were bound to skin and other tissues, there being a correlation between carcinogen-binding by tissues and carcinogenesis. Differential centrifugation and electrophoresis of carcinogen-bound tissues showed that the carcinogen-dye was bound to the soluble proteins of cytoplasm. This formed a complex of low electrophoretic mobility a t alkaline pH. Corresponding components were found to be absent from neoplastic tissues. It is believed, therefore, that neoplastic tissue undergoes a deletion of protein or a change in protein structure, viz., deletion of a growth-controlling component (protein or protein complex) of the soluble cytoplasm. The fluorescein-globulin staining reaction depends on the presence of this cytoplasmic protein for its combination with the protein of the cell. It is this dye-binding basic protein complex which is thought to be lacking in the neoplastic cell. This type of fluorescein reaction has been reviewed by King e t al. (1958, 1959), who have reported on the use of the dye in detecting neoplastic tissue. These workers found that the specificity of the globulin used in the fluorescein-globulin stain has no effect on its ability to discriminate between normal and neoplastic tissues. Several mammalian globulins, including that from monkey and quinea pig, and fowl globulin, were found to give the same nonspecific staining properties when combined with fluorescein. They also demonstrated that the tissue which was stained was not specific for the dye, since normal mammalian, fish, amphibian, and reptilian tissues all showed staining with fluoresceinglobulin or rhodamine-globulin stains. I n these reactions the cytoplasm stains, but the nucleus does not stain. These researchers also found that cancerous tissue from man and experimental animals does not stain with fluorescein. Leucocytes in most leukemias of the mouse do not stain, but leucocytes in most human leukemias do stain. Moreover, normal tissue of the central

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nervous system, normal erythrocytes, and intercellular substance of normal connective tissue, in man and experimental animals, do not stain with fluorescein. The above reactions, then, are not immunological but physicochemical in nature. They depend upon the alteration of the charges on globulin end groups by the dye, which in turn results in a change in the isoelectric point of globulin. The fluorescein-globulin combination is then able to combine with normal cellular protein. The other fluorescein technique consists of coupling the fluorescein dye with the globulin of the specific antibody, so that its binding to cells is truly immunological, being the union of antigen with the homologous antibody. This method was described by Coons and Kaplan (1950). In this technique the nonspecific staining of normal tissue, which was little understood a t the time, is eliminated by absorbing the antiserum containing the fluorescein-globulin conjugate with ground liver. The fluorescent particles are then all removed from the antiserum by filtration or centrifugation. By means of prescribed controls of normal tissue, and tissue treated with unstained antibody (antibody inhibition) , this method has been used with reliable specificity. Fluorescence of structures stained with the antibody-fluorescein complex can be visualized in ultraviolet light in the fluorescent microscope. 1. Comparison of Myosin and Connective Tissue Antigens in Normal

Muscle and Rhabdomyosarcoma Hiramoto and associates (1961a) applied the immunological fluorescein technique in an effort to explain the derivation of human rhabdomyosarcoma. They proceeded on the premise that a substance present in a tumor, and found only in the tissue of origin, is certain evidence that the tumor is derived from the normal tissue. On the other hand, the lack of a substance in a tumor is no evidence that the tumor was not derived from a particular tissue, because the substance may have been deleted from the tumor during the process of neoplasia. Myosin is the substance characteristic of all types of muscular tissue. These workers prepared antimyosin antisera in rabbits and antirabbit globulin antiserum in the horse. The latter antiserum was coupled with fluorescein dye. The antimyosin antiserum was reacted with sections of normal and tumorous tissues obtained a t autopsy, It was seen that normal skeletal and cardiac muscle stained most intensely, usually a t the sarcolemma and along the cross-striations: the antirabbit globulin antiserum (fluorescein-coupled) combined with the antibody to myosin, which was held fast by the myosincontaining parts of the muscle fiber. Hence a green fluorescence “tagged” these parts of the cell. Applied to the rhabdomyosarcomas of six in-

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dividuals, these antisera gave two kinds of reaction. Three embryonal tumors showed intense cytoplasmic staining for myosin, especially at the periphery of round, carcinomatous cells. Three other pleomorphic rhabdomyosarcomas were all negative with the antimyosin antiserum. While anticonnective tissue antiserum did not stain normal muscle or the embryonal rhabdomyosarcomas, after treatment with the antirabbit globulin-fluorescein antiserum, i t did show reactivity with the tumors of two individuals with adult rhabdomyosarcoma and one area of the tumor of a third individual with the adult rhabdomyosarcoma. The cells which showed reactivity with the anticonnective tissue antiserum appeared to be fibroblastic rather than carcinomatous. I n a later study Hiramoto et al. (1961b) applied the fluorescent staining technique to determine whether or not the tumor cells of the pleomorphic rhabdomyosarcoma had changed their antigen-synthesizing properties. They used both frozen sections obtained from autopsy material and tissue derived from the same source by 4 weeks’ growth in tissue culture. These researchers found that normal muscle in tissue culture possessed cells which were positive for myosin, as shown by the indirect fluorescein staining technique which was used in the previous study. The muscle cultures also contained cells, growing intimately with the myosin-containing cells, which were positive for the anticonnective tissue antiserum. The same authors (Hiramoto e t al., 1961b), in a direct staining double-label study, coupled the antimyosin antiserum with fluorescein, and anticonnective tissue antiserum with tetramethylrhodamine, an orange-colored fluorescent dye. Applied to muscle cells in tissue culture, it was found that the fluorescein-labeled antibody stained one type of cell which contained myosin, while the rhodamine stained another type of cell which contained connective tissue antigens. Furthermore, the myosincontaining cells showed perinuclear staining with the rhodamine-labeled (connective tissue) antibody. Therefore, muscle cells, while growing in intimate contact with connective tissue cells in vitro, possess connective tissue antigens. On the other hand, frozen autopsy sections of adult and fetal skeletal muscle were found to be positive only for myosin by the direct fluorescein staining method. The anticonnective tissue antiserum stained only the endomysium. Applied to embryonal rhabdomyosarcoma tissue, the direct staining fluorescein and rhodamine technique revealed a change in antigenic properties of the tumor after i t had been grown in tissue culture. One tumor from a 14-year-old child showed positive staining to antimyosin and negative to anticonnective tissue when the original frozen sections were

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reacted with the antisera. After 4 weeks’ growth in tissue culture, the roundish, carcinomatous cells disappeared and fibroblasts became evident. All the cells stained positive for connective tissue, while there was no convincing staining for myosin in any of the cells. I n another rhabdomyosarcoma there were originally both fibroblastic and roundish cells in the solid tumor, all of which stained only with the antimyosin antiserum. After 2 weeks’ growth in tissue culture, the myosin became negative to staining while the connective tissue became positive. After an additional 2 weeks’ culture in vitro, there were only sheets of fibroblast-like cells which did not stain with antimyosin antiserum. As these authors conclude, it is impossible to show from these studies that the pleomorphic rhabdomyosarcoma undergoes similar transformations in vivo. However, it appears that in adapting cell lines of normal muscle in tissue culture, there are a t first two antigens in the cells, as shown by the direct staining double-label technique. It may be that the transformation of mesenchyme into muscle in the embryo involved the (partial) loss of connective tissue antigen synthesis and the retention of myosin synthesis. I n tissue culture the connective tissue antigen would be produced again in detectable quantities in later generations of cells. As the authors suggest, the transformation of muscle into neoplastic tissue may involve the presence of myosin with minimal synthesis of connective tissue antigen in the original muscle cells, myosin being synthesized in embryonal rhabdomyosarcoma and connective tissue antigen in pleomorphic rhabdomyosarcoma, each to the exclusion of the other. I n either tumor type, it is possible that there are cells which would synthesize both antigens (as in normal muscle in tissue culture) or even neither antigen. The indicated loss of myosin synthesis in some human rhabdomyosarcomas recalls Weiler’s observation about the loss of antigen in carcinogen-induced rat hepatoma, as shown by the inability of r a t hepatoma to bind fluorescein-globulin in the nonspecific reaction described earlier (Weiler, 1956a). 2. Discrimination between Normal Fibrin and the Antigens of

Murphy-Sturm Rat Lymphosarcoma

I n a series of papers Day, Hiramoto, Yagi, and colleagues (Day

et al., 1959; Korngold and Pressman, 1954a; Yagi and Pressman, 1961; Hiramoto e t al., 1959) investigated the antigens responsible for the

localization of antisera to Murphy-Sturm rat lymphosarcoma. Distribution of the antibody possibly depends on the distribution of fibrinogen in vivo. As in the work of Spar, Goodland, and Bale, already cited (Spar et al., 1959; Bale et al., 1958), it was found that in vivo localizations of antibodies to fibrin and ascites lymphosarcoma, as visualized

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by use of fluorescein-labeled antirabbit globulin, were similar. I n the case of each antibody there was staining in the periphery of tumor masses in loose stromal areas and in necrotic foci. Actual tumor cells remained unstained. Intensity of staining was much less with the antitumor antiserum, since it contained less antifibrin antibody. I n vitro staining, following exposure of sectioned tumor to antiascites lymphosarcoma antiserum, showed intracellular staining of tumor cells as well as fibrin areas. Sectioning of the tissue therefore exposed an additional antigen to the tumor antiserum. The antifibrin antiserum did not show this intracellular staining. Moreover, Novikoff and Miller hepatomas and Walker tumor did not show this antigen. The ascites lymphosarcoma cells were agglutinated in higher titer by the antitumor antiserum than by the antifibrin, and stained a t the surface only, unless sectioning of the cells exposed intracellular antigens, in which case intracellular staining was observed. In vitro treatment of normal tissues with antifibrin antiserum, followed by exposure to antirabbit fluorescein-labeled antibody, showed staining of stromal and vascular elements, and in the case of lymph node and thymus, staining of reticular fibers and reticular cells particularly. Parenchymal or epithelial cells remained unstained. Similar results were seen after treatment of normal tissues with antiascites lymphosarcoma antiserum, except that, in addition to stromal elements, lymphoid cell cytoplasm stained in spleen, lymph node, and thymus. Lymphoid cell nuclei did not stain. Since normal spleen sediment was found to absorb the antibody in the tumor antiserum directed against both lymphosarcoma and spleen, no antigen specific for ascites lymphosarcoma cells could be demonstrated. Nevertheless, absorption and elution studies, using I131-labeled antilymphosarconia and antilymph node antisera, and ascites lymphosarcomn and normal lymph node sediments, indicated multiple components in the tumor and the lymph node. Insoluble components of ascites lymphosarcoma were of two kinds: one component common to both ascites lymphosarcoma and normal lymph node, and one specific for ascites lymphosarcoma. Insoluble components of lymph node showed similar results: one component common to ascites lymphosarcoma and lymph node, and one component specific for lymph node. On the other hand, Korngold and Pressman (1954b), using gel diffusion techniques, which would involve soluble components, found that lymph node shared all five lymphosarcoma antigens detected by antilymphosarcoma antiserum.

C. STUDIES WITH CELLSIN TISSUE CULTURE: COMMON TUMOR ANTIGEN Bjorklund and co-workers used pooled, human, malignant, epithelial tumors obtained a t autopsy to make a lyophilized lipoprotein extract.

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This extract was presumed to contain concentrated “cancer antigen.” It was used to immunize the horse (Bjorklund and Bjorklund, 1957), and the resultant antiserum reacted with HeLa cells in stationary tube cultures. The 1:20 dilution of the antiserum caused retraction of cell ectoplasm and cessation of surface activity (pinocytosis) . Fresh culture medium failed to provide regrowth after the HeLa cells had been treated with 1:20 dilution of antiserum for 24 hours, while cells treated with the 1 :40 solution showed slight regrowth in fresh medium. It was also noted that the HeLa cells had absorbed the cytotoxic antibody from the antiserum, so that reuse of the antiserum was found to be nontoxic to HeLa cells. It was also found that the pooled tumor antigen could absorb cytotoxicity from the antiserum, but a pool of 14 normal human tissues failed to absorb cytotoxicity. The antibody-absorbing capacity of the tumor antigen was found to be inactivated completely at 70°C. after 60 minutes. The same researchers (BjGrklund et al., 1958) characterized this tumor antigen by physicochemical analysis. The results indicated that the active groups =were not bound to soluble protein, not nucleoprotein, nor associated with sulfhydryl groups. Denaturation with either urea, or a quaternary ammonium compound, or phenol destroyed antibodyabsorbing capacity, indicating that the antigen is dependent on protein for its activity. Preliminary reports indicated (Bjorklund and Bj orklund, 1957 ; Bjorklund et al., 1957) that vaginal, endometrial, and other normal tissues were unaffected by the antiserum to this common tumor antigen, whereas the malignant cell lines, HeLa and a cervical cancer, showed cytotoxicity. Bjorklund et al. (1961) showed this antiserum to be cytotoxic to three atypical cell lines, Detroit-6 (Berman and Stulberg), conjunctiva (Chang) and heart (Girardi), as well as HeLa, in stationary tube culture. Cytotoxicity to these strains could be absorbed with extracts from 60 human malignant tumors, including ovary, breast, colon, lung, liver, and several other carcinomas. It was concluded, therefore, that the antigen was diffused widely in different types of human malignancies. Since the three atypical cell lines could absorb from the antiserum the cytotoxicity for the three cell lines, common antigenic receptors on these cells was indicated. Goldstein and Hiramoto (1961) re-evaluated the Bjorklunds’ studies on the common tumor antigen, using cells in tissue culture and the same horse antitumor antiserum. They found that the antiserum in the 1: 10 dilution was strongly cytotoxic to HeLa and lymphoblasts from an acute leukemia patient, as well as to normal muscle fibroblasts, leucocytes, and human amnion cells. Heating the antiserum reduced its cytotoxicity,

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which could be returned to the normal level by the addition of complement. Higher dilutions (1:20) of antiserum produced retraction of cytoplasm and clumping in HeLa cells and normal tissues (fetal fibroblasts, adult muscle fibroblasts, and foreskin fibroblasts). Observations were made up to 48 hours. The antibody could be absorbed from the antitumor antiserum by normal human liver sediments, pooled lyophilized tumor tissue (Bjorklund’s), and normal human umbilical cord. It became apparent, therefore, that there is no specific tumor antibody in Bjorklund’s antiserum, but one which shares reactive sites on both normal and malignant human cells. It is a species-specific antibody, and does not react with rat liver sediments or hamster or rabbit cells. However, a t higher dilution ranges, in which HeLa cells were not severely damaged by thc antitumor antiserum, the capacity for regrowth in fresh medium was not reported on by these investigators. Goldstein and Hiramoto believe that Bjorklund and his group did not find cytotoxicity toward normal tissues with this antiserum because these were grown by them in a plasma clot, and therefore insufficiently exposed to the antiserum in the medium (Bjorklund and Bjorklund, 1957). On the other hand, the HeLa cells had been grown as monolayers on glass and had maximum exposure. Moreover, Bjorklund (1956) reported that gel diffusion studies had indicated that the tumor extract contained four components in common with normal human tissues, a finding which would support the results reported by Goldstein and Hiramoto (1961). Of these components, antigens 2, 3, and 4 were generally distributed in 16 normal tissues, while antigen 1 was present in brain, liver, lung, spleen, and kidney, and absent in the others. I n addition, antigen 1 was demonstrable in the tumor extract a t the 1:400 dilution, whereas normal tissue gave a weak precipitate a t a dilution of 1:80. The significance of antigen 1 remains to be investigated, but BjGrklund believed that antigens 2, 3, and 4 were contaminants in the tumor pool from normal tissues.

D. TUMOR LIPIDSAND COMPLEMENT FIXATION: CYTOLIPIN H AS THE TUMOR ANTIGEN 1. Hapten from Rat Lymphosarcoma

Rapport and colleagues have carried out extensive studies which have contributed to refining the complement-fixation reaction applied to tumor antigens. The reaction is valuable because i t has ten times the capacity of the precipitin technique for measuring antibody (Rapport and Graf, 1957a). Furthermore, i t can be used for measuring antigenicity of cellular and subcellular units (insoluble antigens) and lipids. Sensitivity

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LOUIS V. CASO

can be increased by reducing the number of red cells used in the test, by adding Ca++and Mg++,by decreasing the excess quantity of guinea pig complement used in the primary reaction, and by raising the per cent hemolysis taken as the end point of reaction. Because absorption of the antiserum causes anticomplementary reactions in complement fixation, Rapport and Graf used lipid extracts and washed mitochondrial fractions of tumors, a technique which removes completely the anticomplementary reactions (1955, 1957a). Another precaution taken by these authors (Rapport and Graf, 1955, 1957b) is the use of three dimensional titrations, in which both the antiserum and the antigen are serially diluted in order to determine the optimal concentration for the reaction. Use of a single concentration of either antigen or antiserum is capable of introducing serious errors in complement-fixation reactions. Using an antiserum prepared by inoculating rabbits with the mitochondrial fraction (Fraction M ) of rat lymphosarcoma (Rapport and Graf, 1955)) and extracting lipid from tumors and normal tissues with chloroform-methanol, Rapport e t al. (1955) found that the antiserum was nonreactive with tissue extracts from man and several species of experimental animals. Also unreactive were whole tissue extracts of rat adrenals, brain, kidney, and other tissues, although the mitochondria1 fraction of rat kidney, liver, and testis showed positive reactions. This antiserum to Fraction M is therefore species, and to some extent tissue, specific, although r a t lung, spleen, skin, stomach, and intestine were also found to give positive reactions. Among tumors in various strains of rats, Jensen sarcoma, Flexner-Jobling carcinoma, Lewis carcoma 10, Fibrosarcoma 4, and Walker 256 tumor were found to be reactive. The cross-reactivity which Witebsky (1929) found between anti-Jensen sarcoma antiserum and alcoholic extracts of normal r a t brain, heart, kidney, and liver was eliminated by the use of the well-defined cell fraction in immunization and by the extraction of the tissue lipids with chloroformmethanol. Rapport e t al. (1958a) studied the chromatograph fractions of the rat lymphosarcoma hapten obtained in the chloroform-methanol extract. The active fraction was found to require the addition of lecithin or cholesterol for specific complement fixation. It was found not to be anticomplementary. Antigenic activity roughly paralleled the ninhydrinpositive, phospholipid fraction. 2. Hapten from Transplantable Human Tumor and Lung Carcinoma In chromatograph fractionation experiments with human lung carcinoma, these authors found an active hapten which was eluted in the fraction preceding the main (amino nitrogen) phospholipid peak. The

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lipid hapten was therefore chemically different from the rat lymphosarcoma hapten. Since many human tumor extracts react more strongly to tumor antisera than do extracts from normal tissues (but with variable results, however), Rapport et al. (1958b) extracted human HEP 3 carcinoma, which is grown as a transplant in rats, in order to obtain a purified lipid hapten. This extract reacted with both antihuman tumor antisera and with antirat lymphosarcoma antiserum. Korngold and Lipari (1955) had shown that this human tumor probably had absorbed the rat antigen from its rat host. Rapport et al. (1958b) were able to separate the two haptens, the human lipid being eluted in a fraction similar to the active fraction obtained from human lung carcinoma. The two fractions (from human HEP 3 and rat tumor) did not cross-react with antihuman tumor antisera and antirat lymphosarcoma antisera. Antiserum prepared to HEP 3 carcinoma reacted with HEP 3 extracts, but not with human tumors obtained by surgery or autopsy. The fraction was assayed for complement fixation by using antihuman reticulum cell carcinoma antiserum. Lecithin-cholesterol mixture was found to enhance this reaction greatly. The authors named this human lipid hapten cytolipin H. I n a later study Rapport et al. (1959) concluded that the cytolipin complement-fixing reaction was markedly dependent on auxiliary lipid mixtures, a lecithin-cholesterol 5 : 100 mixture increasing the sensitivity of the reaction 7 times. Since tissue extracts differ appreciably in their lipid content, and since mutual lipid interactions cannot be controlled, quantitative complement fixation data are unreliable. Nevertheless, these authors were able to report that cytolipin H reacts strongly to the antisera to most human carcinomas, including those of lung, breast, and kidney, myelogenous leukemia, and malignant melanoma. Most normal tissue extracts have little capacity for such reactions. I n addition, it was found that lipid interactions were important for the ability of cytolipin H to combine with the specific antibody and fix complement. Pure cytolipid H did not elicit antibody formation on inoculation, even when auxiliary lipids or serum were added to the inoculum (Rapport e t al., 1959). The protein moiety is apparently necessary, since crude lipids from human lymphosarcoma did elicit antibodies to cytolipin H. Cytolipin H has been shown to contain four molecular residues: fatty acid, lipid base (sphingosine) , glucose, and galactose. Graf and Rapport (1960) reacted 17 antihuman tumor antisera with cytolipid H and found 14 positive and 3 nonreactive. Four of the five most reactive antisera showed rather weak reactions with cardiolipin (Wassermann hapten) and 3 were lacking in Forssmann antibody. It is therefore indicated that cytolipin H is distinct from cardiolipin and

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Forssniann antigens. The weak reactivity of normal tissues (such as lung or colon) for cytolipin H activity is believed to indicate a lower cytolipin H content relative to most tumor tissue. Glycolipid hapten extracted from ox spleen was found by Rapport et al. (1960) to give the complement-fixation reaction with antihuman carcinoma of the cervix antiserum, the reaction being indistinguishable from that of cytolipin H. The carbohydrate portions of the haptens are believed to be reactive sites, because these are the same chemically, whereas the fatty acid residues differ chemically in the ox and human haptens. It is apparent that loss of species specificity has resulted from purification of this glycolipid hapten of human tumors. The significance of quantitative differences discernible in tumorous and normal tissues remains to be investigated, as does the lack of or weakness of cytolipin H activity seen in some antisera to carcinomas of rectum, breast, and stomach (Graf and Rapport, 1960).

E. ANAPHYLAXIS STUDIES AND

THE

SCHULTZ-DALE TECHNIQUE

1. Indication of Specific Tumor Antigens

Zilber (1957) summarized his studies on anaphylaxis with tumor antigens in 1957. The antigen consisted of nucleoprotein, the fraction being precipitated a t pH 4.5. Human tumors were extracted and the nucleoprotein fraction injected into guinea pigs. After 25 to 30 days, the animals were desensitized by small doses of corresponding normal tissues during two or three successive periods of inoculation, until desensitieation t o normal tissue was established. Injection of the sensitizing tumor extract was then found to produce anaphylactic shock in the guinea pigs. Zilber reported that these results occur invariably. The conclusion is that the tumor possesses, in addition to the antigens of normal tissue, an antigen specific for the tumor. The reaction was seen with a wide variety of human tumors, including carcinoma of liver, stomach, mammary gland, uterus, bladder, several sarcomas, and leukemic spleen. Zilber and co-workers confirmed these conclusions by demonstrating the converse reaction. This consisted of immunizing the guinea pigs to normal tissues followed by desensitizing with tumor tissues. On subsequent challenge with the corresponding normal tissue no anaphylactic shock was seen. Zilber stated that the tissues and tumors used in these studies were of the same blood groups, but unfortunately he does not give details in his review. Experiments on laboratory animals also indicate the existence of

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additional antigens in tumors. Results with a hepatoma from an inbred (C3HA) strain of mice showed anaphylaxis with the tumor extract, after inoculation of guinea pigs with the hepatoma antigen and subsequent desensitization with normal C3HA liver. The tumor in this case was carcinogen-induced. Zilber’s (1962) limited studies with antistomach cancer antisera, by the double diffusion gel technique, did not reveal such clear-cut results, although there was high specificity for the stomach cancer extract. The antisera, suitably absorbed, were unreactive with extracts of various other cancerous tissues, stomach, spleen, and other normal tissues. However, i t was concluded that normal spleen contains an antigen present in stomach cancer tissue, because the latter gives a positive precipitin reaction with antispleen antiserum. I n normal spleen extract, according to Zilber, the antigen may be present in too small a concentration to react with the antistomach cancer antiserum. Hence the necessity of carefully controlling antigen concentrations in these reactions, and of testing antinormal tissue antisera with the various tumor extracts. 2. Existence of Tumor Antigens in Sera: Use of the Schultz-Dale Test

Because of its high degree of sensitivity and specificity, the anaphylactic reaction and the related reaction in the Schultz-Dale technique seem to commend themselves to the detection of specific tumor antigens in complex mixtures of tissue antigens. Provided adequate measures are taken to control the action of blood group antigens, whether in the tissues, blood plasma, or other fluids, these methods would appear to be very promising for the detection of tumor antigen in patients’ body fluids as a general screening test. Makari and Huck (1955) used the Schultz-Dale test to examine 707 serum samples from patients and sera from 111 healthy individuals. The healthy controls showed only 6 positive reactions t o sensitized guinea pig uterus (5%). All 6 were shown to have pressor substances and therefore may not have been false positives in the usual sense. The guinea pig uteri were sensitized actively and passively with antigens from HeLa cells (Gey) (antigen B ) , from tumor suspensions (antigen A ) , and from supernatant from tumor suspension (antigen C) , obtained by centrifugation between 3200 and 10,OOO r.p.m. Some of the results are seen in Table VI. It can be seen that the results with antigen C are in good accord with the criteria set down by Dunn and Greenhouse (1950) for false positive and false negative reactions. Of interest also are 9 cases of questionable clinical diagnosis, which were followed up for 1 year. The results show early diagnosis of the

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V. CASO

carcinomas, elimination of other tumors, such as sarcomas, and elimination of other conditions, such as lupus and fat. necrosis. These cases, too few for generalization, are summarized in Table VII. The author states that the antigen is a common carcinoma antigen, since it is present in carcinomas of tissues and bIood from untreated carcinoma patients, regardless of site of origin or type of carcinoma. I n patients with diseases other than cancer there were 6 positives out of 123 (4.8%),when active immunization of guinea pig uterus with TABLE VI DISTRIBUTION OF SITES OF ORIGININ 134 UNTREATED CASESOF CARCINOMA TESTED WITH SCHULTZ-DALE TESTBY ACTIVE SENSITIZATION' Antigens A and B (H.S. and HeLa) Source of carcinoma Abdominal cavity Bladder Bone Brain Breast Bronchi Cervix Colon Generalized (metastasis) Larynx Lung Mediastinum Neck Esophagus Ovary Pancreas Penis Pharnyx Prostate Recurrent Rectum Site unrecorded Skin Stomach Testis Tongue Thyroid Uterus a

Total

Correct diagnosis

6 1

6 1

-

-

1

1

7 1 6

7

1 2 1 1 1 15 1 15 1 3 4

2 1

From Makari and Huck (1955).

Antigen C (C.H. and M.J.) Total 4 1

2

4 2 1

Correct diagnosis 4 1 2

4 2

1 9

4

1 1 9 4

0

4

4

2

1 1 2

1 1 2

2 2

2 0 1 1 1

1 3

1 1 1

-

10 1

14 1 3 4 1 1

1 1 1 16 2 1

16

2

1

-

-

2

2

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THE RELATION OF THE IMMUNE REACTION TO CANCER

TABLE V I I FOLLOW-UPOF CASESWITH QUESTIONABLE CLINICAL

DIAGNOSIS OF CARCINOMA^

Case

Diagnosis

Result of test

+ + + +

Carcinoma of bladder (?) Carcinoma of pancreas (1) Carcinoma of lung (?) Carcinoma of gums (?) (recurrent) Papilloma (?), carcinoma of bladder Carcinoma of breast (?) Carcinoma of lymph node (1) Carcinoma of stomach (?) Carcinoma of stomach (?)

+

a

Final diagnosis by biopsy or necropsy

Interval between test and definite pathological diagnosis (day4

Carcinoma of bladder

28

Carcinoma (head of pancreas) Undifferentiated carcinoma of lung Extensive carcinoma (recurrent)

10

Papilloma sqnamous cell tumor with invasion of bladder wall Fat necrosis (no carcinoma) Lupus erythematosis Hemangiosarcoma (tail of pancreas) Spindle cell lipofibrosarcoma

4

151

I 14 20 56 1

From Makari and Huck (1955).

antigen C was carried out. With antigens A and B, 19 of 23 cases were positive in which there was non-neoplastic disease. Makari has presented additional results with the Schultz-Dale technique and discussed their implications concerning cancer screening and diagnostic testing elsewhere (1962). The findings of Burrows (1958, 1962) in over 500 individuals, using the Schultz-Dale technique of Makari, gave substantially the same results: 96.7% of the carcinoma patients were positive, while 96.7% of the patients without carcinoma negative. There were 10 false negatives in the first group and 7 false positives in the second group. However, accuracy was reduced to only 77% positive in cases showing small, early lesions. McEwen (1959) was unable to reproduce the results of Makari and Burrows, and notes the problems in technique which are inherent in the Makari modification of the Schultz-Dale test. He failed to demonstrate

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CASO

carcinoma antigen. Hackett and Gardonyi (1960) were unable to achieve the accuracy in diagnosing carcinoma which was reported by Makari, but did find an antigenic difference between normal and carcinomatous extracts when used to immunize guinea pigs in preparation for the Schultz-Dale test. V. Autoimmunity in Cancer

A. THECONCEPTOF AUTOIMMUNITY Under ordinary conditions, the body is not believed to produce antibodies to substances normally present in its own circulatory system. Hence, A substance of human erythrocytes, injected into individuals belonging to blood group A, will not elicit detectable antibody formation, whereas B substance, injected into blood group A individuals, will cause production of anti-B antibodies. It is conceivable, if injury should occur by which a substance not normally present is introduced into the circulation, viz., injury to the lens of the eye, that the body would respond by producing antibodies to that substance. I n the case of optic lens injuries (from cataract operation) , inflammation can sometimes occur even in the unoperated lens, but antibodies are not detectable in the patient’s serum (Raffel, 1961b; Waksman, 1959). The disease, phacoanaphylactic endophthalmitis, can be produced experimentally in rabbits by needling the lens after the animal has been sensitized to lens extract and Freund’s adjuvants (Raffel, 1961b). Precipitating antibodies are produced in these animals, and autologous lens can be used in sensitization (Raffel, 1961b; Muller, 1952). Such production of antibodies to an animal’s own tissues is the result of an autoimmune reaction. The autoimmune response has been observed to occur in patients with certain diseases. Paroxysmal hemoglobinurea, acquired hemolytic anemia, and certain other hematological conditions have been linked to the production of autoantibodies directed against the formed blood elements (Witebsky and Rose, 1956). Antibodies to human heart have been reported in the sera of patients with active rheumatic fever (Cavelti, 1945). Experimentally, extracts of certain organs, such as the lens of the eye and the brain, elicit antisera which are organ specific, ie., will react in vitro with the organ extract regardless of species. Lipid extracts of rabbit brain, when injected into the rabbit, do not elicit autoantibodies, perhaps because the antigen is a hapten requiring the presence of a foreign protein for the immune response. However, when rabbit brain lipid is injected with Freund’s adjuvants (bacterial suspension) into the rabbit, a disease causing damage to the nervous system (allergic enceph-

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alomyelitis) is seen to occur (Witebsky e t al., 1956; Waksman, 1959). Witesby and co-workers used this technique of inoculation with Freund’s adjuvants to produce autoantibodies to rabbit thyroid extract (1956). Autoantibodies were indicated by their agglutination of thyroid-coated, tanned erythrocytes, which is the sensitive detection method devised by Boyden (1951). Beutner et al. (1958) succeeded in showing localization of thyroid autoantibodies in rabbit thyroid tissue (colloid and rim of epithelium) by the fluorescein-conjugate method, when high concentrations of antisera were used. Aspermatogenesis in the rat and the guinea pig was induced by Freund et al. (1953, 1954) by immunizing the animals to their own testicular tissue in the presence of adjuvants.

B. OCCURRENCE OF AUTOIMMUNITY TO TUMORS There is some clinical evidence that autoimmunity occurs in certain cases of human cancer. Graham and Graham (1955) reported complement-fixing antibodies in the sera of 12 of 48 gynecological cancer patients, with titers ranging from 1: 16 to 1 : 128. Sera were tested with antigen extracts from the patients’ own tumors. Later these authors (Graham and Graham, 1959) inoculated cancer patients with preparations from their own tumors mixed with Freund’s adjuvants, and observed them up to 3 years after inoculation. They believe that the inoculations may have exerted a potentiating effect on subsequent irradiation of the tumors, and report an increase in numbers of SR (sensitivity response) cells and histiocytes in cancer of the cervix, the increase being associated with a somewhat more favorable prognosis after radiotherapy. Ishibashi and co-workers (1961) performed intracutaneous transplants of patients’ own tumors (lung, pancreas, colon, etc.) and found some indication that complement fixation, agglutinin, and hemolysin titers were increased. These transplants were observed not to infiltrate the skin and muscles. Finney e t al. (1960) found that 3 of 5 patients showed increased precipitin titers (range 1:64 to 1:640) to their tumor biopsy tissue after irradiation of the tumors. When homogenized tumors were injected intramuscularly with Freund’s adjuvants, patients in another group showed a much greater increase in antibody titers to their own tumors, as tested with Boyden’s hemagglutination method (Boyden, 1951), using tanned, antigen-coated erythrocytes. Moreover, injection of Cohn fractions I, 11, and I11 of serum from patients of this group who had malignant melanoma, reticulum cell sarcoma, or Hodgkin’s sarcoma into the area of their respective lesions, produced a significant reduction of tumor sizes. Corresponding fractions of normal serum did not produce these regressive changes. These authors mention an important point in evaluating these and similar autoimmune experiments: that the extent

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of change in tumor tissue brought about in excision and homogenization (or extraction) is unknown, and antigens possibly are changed even by relatively mild t.reatnient, a fact which would affect antibody production when reinoculated into the host. Furthermore, as Osler (1961) points out in his interesting review of methods of immunological detection of cancer antigens, normal tissue mixed with homogenates from the patients’ tumors may have contributed to the immune response, as also the tubercle bacilli of Freund’s adjuvants. The production of a positive, standardized, immediate skin response when tumor polysaccharide substances (TPS) are injected intradermally, has been found by Makari (1960) to occur in carcinoma patients and certain others in much higher proportion than in healthy volunteers, and he believes the basis for this reaction is an autoimmune hypersensitivity to the polysaccharide antigens of tumorous tissue. The transplantation studies of Prehn (1962) and Old and associates ( 1962) showed the isoantigenicity of certain carcinogen-induced tumors in mice, and this has been discussed in an earlier section (Section 11,A). C. THEANEMIAOF CANCER 1. Evidence of Autoimmunity in Clinical Disease

Green (1957) has expressed the belief that the anemia of cancer may be due to tumor hemolysins. He observes that the life span of the erythrocyte is diminished often by one-half or more in human cancer, and that the degree of anemia is not correlated with replacement or destruction of hematopoietic tissue. I n certain types of disease (lymphosarcoma and chronic lymphatic leukemia) the erythrocytes show a positive Coombs reaction, indicating coating of the cells with human globulin due to an autohemolytic type of anemia. This author (Green, 1957) used Coombs’ reagent (in greater concentrations than used for the standard globulin test) and found that 53% of the general cancer cases tested (excluding leukemias and reticuloses) gave positive erythrocyte agglutination, as compared with 16% for other diseases and 3% for normal individuals. He concludes that the cancer patient’s erythrocyte is more susceptible to antihuman globulin antibody, and finds that this susceptibility increases proportionately with tumor mass. I n addition, Green (1957) found that erythrocytes from myeloma patients gave strong direct and indirect standard Coombs’ reactions. This antibody could be eluted from the red cells, as could the antibody in many cases showing positive for the modified Coombs’ test mentioned previously. However, the antibody eluted from the cells was not anti7-globulin, since absorption with the y-globulin fraction did not remove its potency. Potency was removed by absorption with whole serum, and

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therefore it may be anti-a-globulin or anti-/3-globulin. As such i t would be akin to the antibody present in acquired hemolytic anemia. Accordingly, Green believes the evidence indicates that the anemia of cancer is due to an immunological attack on host cells, there being true hemolysins present in host serum. He poses two alternatives for the origin of these antibodies: (1) production of antibody by host cells in response to tumor, which would be very close antigenically to erythrocytes, causing antibody to cross-react with them; or (2) the tumor itself may be deficient in an antigen, and therefore actively produce antibody directed against host cells. 2. Animal Experiments

Green (1957) studied mice and rats bearing sarcomas and carcinomas and found that all the tumors contained hemolysins in amounts usually exceeding those present in spleen, lung, kidney, or liver. Hemolytic activity varies with the same tumor type and depends on whether the erythrocytes used are homologous, isologous, or autologous. The tumor extracts show hemolytic activity (usually heat stable) and hemagglutinating activity (often increased after heating), the latter most usually induced after inoculation of homologous erythrocytes or the autologous erythrocytes of the tumor-bearing animal. Since these two activities are often greatest in extracts of the spleen and lymph nodes, a possible immune relationship was indicated and therefore investigated in the tumors. Accordingly, Green found that direct injection of erythrocytes into tumor tissue, or even remote injection of erythrocytes into the tumorous animal, resulted in quantitative increase of both the hemolytic and hemagglutinating properties of the tumor extracts. This occurred with homologous and also autologous erythrocyte injections. Hemolytic and hemagglutinating activities accumulated in the tumors more than in the spleen, were found to be species specific, and were associated (in rat sarcoma and carcinoma) with tumor lipoproteins and phospholipids. Both transplanted and spontaneous tumors reacted in the same way to erythrocyte challenge. Green reported that the lipoprotein fraction gave strong hemolytic activity, while the phospholipid showed strong hemagglutinating activity and sometimes mild hemolytic activity. Strong hemolytic activity was found in the serine and ethanolamine phosphatides fraction, and in the sphingomyelin and lecithin fraction of these tumors. The cephalin fraction showed high hemagglutinating activity and species specificity (the latter distinguishing i t from egg cephalin, which shows no specificity). I n contrast to the 10 different mouse and rat tumors investigated above, fresh human tumor extracts were found by Green to be slightly active or not active a t all. These inhibited other tissue hemolysins and

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spontaneous hemolysis. The cephalin fraction showed high hemagglutinating activity and was inhibited by lecithin. It is probable, therefore, that a strong inhibiting factor in human tumor extracts masks the effects of the hemagglutinating and hemolytic agents. It is noteworthy that lipoproteins in carcinoma of the stomach did show high hemolytic activity. I n connection with these tumor extracts reported by Green, reference should be made to the complement-fixation reactions investigated by Rapport and associates, who extracted the lipid hapten, cytolipin H, from the human carcinoma, HEP 3 (see Section IV,D).

3. Nature of the Hemolytic and Hemagglutinating Agents Present in Tumor Extracts Green (1957) has concluded that the evidence points to the antibody character of tumor hemolysins and hemagglutinins. The erythrocytes of the tumorous animal usually show greater sensitivity to tumor (hemolytic) extracts. These erythrocytes have been shown to be coated with rat antibody globulin in about half the rats bearing the Rd/3 sarcoma. Moreover, erythrocyte destruction in animals bearing transplanted tumors is not due to nutritional deficiency, infection, or hemorrhage, but rather is related directly to tumor mass and increases proportionately. Regardless of the nature of the tumor, destruction of normal erythrocytes takes place. It is possible that antierythrocyte agents are formed elsewhere and absorbed or fixed by tumor tissue. However, after challenge with homologous erythrocytes, rat tumors show a significant rise in hemolysin and hemagglutinin activity, while the spleen shows only a slight rise. Nevertheless, the other lymphoid organs have not been eliminated as producers of antierythrocyte agents. Green would explain the antierythrocyte activity of tumor extracts as arising in the tumor parenchymal cells or in primitive, mesenchymal tissue of the tumor. He postulates the lack in the tumor of antigen or antigens which are present in the host, and consequently an active production by the tumor of antibodies directed against cytoplasmic lipoproteins. Work in Green’s laboratory showed that Rd/3 rat sarcoma extracts have a rather specific action on splenic plasma cells. He therefore envisages a two-way flow of antibody production: from tumor to plasma cells (as tumor progresses) and from splenic plasma cells to tumor (as tumor regresses).

D. ANTIGENIC Loss IN CANCER Central to Green’s hypothesis that tumors can produce antibodies which react unfavorably with normal host cells is the concept of the

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loss of one or more antigens during tumor initiation and progression. Such a loss might also make the neoplastic cell unrecognizable by normal control mechanisms, so that processes like phagocytosis or growth inhibition would be inoperative. The difficulties in proving the lack of an antigen in any tissue have been mentioned in the discussion of Korngold’s work using the double diffusion gel technique (Section IV,A,4). The differences in antigen content of leukemia cells, normal leucocytes, and other normal human tissues have been considered previously (Section IV,A,4,e). That biochemical changes do take place in tumor-bearing animals has long been appreciated, viz., decreased activities of certain liver enzymes, and decreased quantities of fatty acids, steroids, and phospholipids in the adrenal gland of tumor-bearing rats (Greenstein, 1947). According to the studies of Weiler (1956a) with fluorescein dye, and those of King e t al. (1958, 1959) on the lack of species specificity of fluorescein, it is apparent that tumor tissue lacks a dye-binding, basic protein complex of the soluble cytoplasm of the cell, and does not take the fluorescein stain. Most normal tissues stain with fluorescein, although there are notable exceptions in man (i.e., tissue of the central nervous system, see Section IV,B). However, to demonstrate specific antigen absence in tumor tissue is another matter, and the evidence appears to be meager (Hieger, 1961). Weiler (1956b,c) produced an antiserum to cytoplasmic particulates of hamster kidney, which, after absorption with normal tissues, was found by complement fixation to bc specific for kidney microsomes and mitochondria. The absorbed antiserum was inactive with particulates from stilbestrol-induced kidney carcinoma, and it appeared that the organspecific antigen was absent or deficient in the tumor. Using the fluorescein-antibody method of Coons and Kaplan (1950) (see Section IV,B) for visual localization of antibody in a specific tissue, normal kidney tubules were stained when the antiserum was applied to cryostat sections, whereas tumor cells, even in early stages of formation, remained unstained. Weiler’s work on kidney and liver neoplasms was confirmed by the studies of Nairn et al. (1960). I n addition to fluorescein-antibody studies, they used the double diffusion gel technique of Ouchterlony (1953) and obtained an organ-specific precipitin line for normal hamster kidney and rat liver. Normal homogenates from these organs completely absorbed the antisera so that no precipitin line was produced with the appropriate antigen, but liver tumor and kidney tumor preparations could not absorb the antibody completely. Parallel results were obtained with fluorescein-antibody to the normal organs. It was concluded that the

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tumors were lacking in the organ-specific antigen. These authors also obtained different staining qualities in normal human epidermis and benign tumors of the skin, on the one hand, and 12 of 13 carcinomas of the skin, on the other, when the fluorescein-antibody technique was used. Hiramoto et al. (1961b) showed that muscle cells, growing under the highly modified conditions of tissue culture, can produce both myosin and connective tissue antigens, whereas frozen muscle sections demonstrate only myosin antigens. Embryonyl rhabdomyosarcoma was seen to give a positive reaction for antimyosin antibodies when the frozen sections were treated, but after growth in tissue culture the myosin reaction became negative and the reaction with connective tissue antiserum positive. Moreover, certain pleomorphic rhabdomyosarcomas were found to be negative to antimyosin antiserum (Hiramoto e t al., 1961a) (see Section IV,B,l). Using antisera to four different antigens, Korngold (1956) demonstrated by the double-diffusion gel technique the variability of the presence of these antigens in different tumors and normal tissues. Approximately 50% of the normal and tumorous tissues obtained a t surgery contained all four of these antigens (see Section IV,A,4,b). The conclusions to be drawn from these studies await more extensive investigation, but the techniques employed indicate an experimental design suited for obtaining an understanding of possible antigenic changes in neoplastic or abnormal tissues. I n all of these serological techniques the relative concentrations of antigen and antiserum are critical in the interpretation of results.

E. GREEN’STHEORY INVOLVING ANTIGENIC CHANGE Green (1958, 1961) believes that the tumor inhibitory effects of certain polycyclic hydrocarbons, and certain carcinogenic hydrocarbons in small doses, are due to alteration of the tumor antigenic complex so as to render i t susceptible to the host’s immune responses. Spontaneous tumors and highly strain specific tumors are relatively unaffected, but about 50% of transplantable tumors are seen to regress completely when compounds such as napth-2’: 1’-1: 2-anthracene are injected into host animals. During this reaction plasma cell proliferation is also indicated. A change in the antigenic structure of these tumors would introduce foreign antigenic sites, which would in turn elicit antibody inhibitory to tumor growth. When Green treated strain specific tumors with either carcinogenic or tumor inhibitory substances in vitro, prior to implantation in the compatible mouse strain, the tumor behaved like one transplanted into a foreign host. Growth was either delayed or repressed completely. The implication is that polycyclic hydrocarbons, both car-

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cinogenic and noncarcinogenic, can bind tumor substances, change the antigenic structure of the tumor, and therefore render i t susceptible to an autoimmune reaction. It should be added, however, that specific antibody in carcinogen-treated animals has not been demonstrated. While Green (1958) envisages tumor antigens as being changed by the various treatments with carcinogens or tumor inhibitory hydrocarbons, carcinogens in full dosage are seen by him to initiate antigenic change in normal tissues. Once changed, an autoimmune reaction to the tissue involved causes promotion of tumor growth. He points out some facts suggestive of an autoimmune reaction in animals undergoing chemical carcinogenesis : the spleen of these animals contains a tumor inhibitory factor ; trypan blue blockade of the reticuloendothelial system of animals fed azo dye resulted in fewer liver tumors which showed less malignant growth ; small doses of superficial irradiation throughout carcinogenesis may suppress liver tumor development, while general exposure of the animal to irradiation will accelerate carcinogenesis. It is not clear, however, how these opposite radiation effects would derive from action on the same reticuloendothelial elements. Neither is the action of cortisone in inhibiting carcinogen-induced skin papilloma understood. Besides suppressing the immune response, there is the possible direct inhibitory action on epithelial cell mitoses (Green, 1957) (see Section II,B,3). Green postulates the loss of an “identity” tissue protein resulting from the formation of a carcinogen-protein complex during chemical carcinogenesis. Antibody would act on cells containing this complex, and “adaptation” would produce a race of cells finally lacking the affected protein. Granting that the reticuloendothelial system responds by an immune reaction to these changes in tissue antigens, the mechanism by which these cells, even if self-replicating, would fail to be “recognized” by growth regulating mechanisms, and so become neoplastic, is utterly unknown.

F. IMMUNOLOGICAL ENHANCEMENT The method of enhancement of tumor growth was devised by growing tumors in homologous hosts and has been discussed in detail previously (see Sections II,A,3 and II,A,S). It was found that lyophilized tissue corresponding to the tumor genotype, inoculated previously into the recipient, homologous animal, would enable the transplanted tumor to grow progressively until the death of the host (Kaliss, 1958). Conversely, previous implantation of living tumor cells would cause speedy rejection of the tumor. Antiserum to the tumor, produced either in rabbits or in

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mice, and inoculated into the recipient, homologous animal, can also induce enhancement to tumors of the same genotype. It should be noted that not all tumors can be enhanced, and that this work has been developed in connection with the H-2 antigens in mice (Snell, 1954; Gorer, 1958; Kaliss and Bryant, 1958). Supernatants and extracts of tumors, previously inoculated into mice, can also enhance tumor growth. Kandutsch and Stimpfling (1962) extracted a Triton-soluble lipoprotein from a particulate fraction of Sarcoma I, which could enhance the growth of Sarcoma I in C57BL/10 mice. Green (1958) had also found that tumor lipoprotein fraction would induce enhancement, and believes that this substance sensitizes the host to the tumor tissue, and that the latter, when implanted into the homologous host, produces the secondary response which brings about enhancement. Green further conjectures that antibodies to this lipoprotein complex (which is probably a cytoplasmic antigen related structurally to nucleoprotein), when combined with the antigenic complex in the cell, produce a permanent antigenic change. This finally results in the emergence of a new cell strain which has lost the antigenic complex concerned. However, the studies of Moller ( 1 9 6 3 ~ )indicate that short-term growth of tumors in enhanced hosts does not confer increased homotransplantability in successive recipients. Moreover, by removing antibody from coated tumor cells (by trypsinization) he was able to show that the tumor cells are not permanently enhanced (altered physiologically) by in vitro incubation with enhancing antiserum. The enhancement of tumor cells was abolished by trypsinization, and could be reestablished by a second coating with antiserum. The work of Snell e t al. (1960) supports the evidence for lack of permanent change in tumors growing in enhanced hosts, while the findings of Green (1958) and those of Kaliss (1958) with Sarcoma I and Carcinoma E0771 would indicate permanent alteration of the tumor in the direction of enhancement. However, during many successive transplants and with long growth in foreign hosts, there is always the possibility of the development of nonspecific tumors, or of isoantigenic variants of genetic or “epigenetic” origin of the type demonstrated by Hellstrom (1960). This investigator showed that H-2 isoantigens of F, lymphomas were indeed lost, so that tumor variants could finally be grown in one or the other strain of parental origin. Comparable findings were reported by Klein and Moller (1963) for MCAG sarcoma (A X A.CA) F, grown in parental strains, and these authors suggest that the lower concentration of H-2” antigens in the (A X A.CA)F, tumor might have led to immunological enhancement of growth in the parental A.CA strain mice. There is a question, of course, whether or not immunological enhance-

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ment is involved in the process of tumor autoimmunity. The fact that various workers (Ishibashi e t al., 1961; Finney e t al., 1960) have succeeded in showing increased antibody after implantation of the patient’s own tumor tissue, in effect a secondary response, would indicate the existence of part of the mechanism, i.e., inimunological competence on the part of the host. Whether the antigens of the tumors correspond exactly to the antigens in normal tissue is of some importance, because the evidence would indicate that a rather sharp genetic difference between tumor and host is necessary for immunological enhancement. The findings of Casey and co-workers (1959) and Feldnian and Globerson (1960) would modify this conclusion somewhat, since both groups showed enhancement of tumor in strain of origin, the former with carcinogeninduced sarcoma, the latter with a transplantable tumor of inany generations. Slight antigenic differences, therefore, were possible in either case (see Section II,A,3). Assuming that antigenic differences do exist between tumor and host, i t would seem t h a t immunological enhancement is a possible explanation of the promotion phase of tumor growth. Once initiation of the tumor has begun, which would involve antigenic change essentially, whether due to chemical or physical carcinogens, mechanical cellular injury, viral invasion of nucleic acids, spontaneous genic mutation, or delayed genic expressivity, the immune response of the host could either destroy thc neoplastic cells or enhance their growth. When the developing tumor produces one or more antigenic sites not present in the corresponding normal tissue of the host, i t is possible that the host would respond by producing two kinds of antibody: cellbound antibody, which tends to inhibit tumor growth as if it were a new “transplant’, in normal tissue; and humoral or circulating antibody, which also inhibits growth by its cytotoxic effect on tumor cells. Cytotoxicity, however, has been found by Moller and Mollcr (1962) to vary in effectiveness with tumor type. The tumor is more susceptible to humoral antibody when i t has a greater surface concentration of antigenic sites, and less susceptible when it has a less concentrated disposition of these sites. This would imply, in the present view of tumor promotion, that the specific antigenic changes in the previously normal tissue would govern the subsequent interaction between tumor and host. Humoral antibody, in addition to its cytotoxic activity on tumor cells, has been demonstrated by Moller (1963b) to induce enhancement by “coating” on the tumor cells antigenic sites which are foreign to the host. It is further indicated t h a t the concentration of antibody, in relation to the concentration of antigenic sites on the tumor cells, also has a direct bearing on the degree of enhancement. Some tumors, which have

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a very high concentration of antigenic sites, are enhanced only within certain ranges of antibody concentration (Moller, 1 9 6 3 ~ )Moreover, . the best enhancement of tumors occurs when all the antigenic sites foreign to the host are covered by the antibodies, i.e., the effectiveness of the enhancing qualities of the antiserum is related to host genotype relative to tumor genotype. Covering of the antigenic sites on tumor cells in some manner “blocks” the action of tumor inhibitory antibodies, either by preventing the formation of tumor antibody by “walling off” the antigenic stimulus (afferent inhibition), or by preventing the direct action of the inhibitory antibodies by blocking receptor sites on the tumor cells (efferent inhibition). Therefore, given a tumor which is less susceptible or unsusceptible to the cytotoxic effects of humoral antibodies, the latter in proper concentration can sufficiently coat the receptor sites on tumor cells so as to render them unaffected by inhibitory, cell-bound antibodies. These effects have been indicated by the studies of mouse tumors (Moller, 1963b), and there is no reason to believe that they would not be operative in human tumors, granting sufficient antigenic differences between tumor and host. The fact that the enhancing effects of tumor antisera can be neutralized by the inoculation of preimmunized lymph nodes demonstrates that cell-bound antibody is also involved (Moller, 1963a) and of possible function in suppressing tumor growth in certain circumstances. Cellbound antibodies have been indicated in the homograft reaction by the action of lymphocytes in the destruction of cellular transplants in diffusion chambers (Weaver et al., 1955). With this in mind, care should be taken that the use of cytotoxic, antimetabolic drugs such as 6-mercaptopurine (Schwartz and Dameshek, 1959; Schwartz et al., 1959; Genghof and Battisto, 1961) does not depress the natural antibody defense mechanisms. In this regard, it may be mentioned that Ishibashi and his associates (1961) believed that the use of mitomycin-C was more effective in causing regression of certain carcinomas after the patients had been autoimmunized by intradermal transplants of their own tumors. Both cell-bound antibodies and humoral antibodies have relevance to the postulated immune response by the host to its tumor, and quite plausibly this response could involve enhancement of tumor growth. As long as the host could produce humoral or circulating antibodies, presumably in low concentration, new tumorous cells would continue to be coated and therefore insulated from the effects of more lethal antibodies or prevented from eliciting their production by the host. No additional mutations or new races of cells need be predicated to keep the process advancing, once the initial, self-replicating changes in cellular

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antigens have occurred. An immunological “chain reaction” due to constantly modified cytoplasm or antigen-antibody complexes, such as envisaged by Caj ano (1960), would seem unnecessary. Conceivably, the presence of antibody on cellular antigenic sites might also isolate the cells from mechanisms other than those associated with immune responses. Reactive sites of hormones might be affected, for example, as might the permeability of mctabolites affecting intracellular enzymatic reactions. The coated cells would exist in a changed environment, resulting in possible changes in cell growth, mitosis, and such little understood processes as contact inhibition.

G. BURNET’STHEORY OF SELF-RECOGNITION 1. Self-Markers and Recognition Units

Directly bearing on any concept of the development of neoplasms by antigenic changes which induce an autoimmune reaction is the manner in which the body’s immune mechanism distinguishes between its own “normal” and foreign antigens. Since an immune response can be elicited by a potentially unlimited number of antigenic groupings, Burnet (1956) postulates the existence in cells of a relatively few chemical configurations which are complementary to configurations in other cells of the same organism. The relationship between these configurations serves to prevent the induction of the immune response. One set of these configurations comprises the means of self-recognition, and the chemical groups are termed self-markers. Immunologically competent cells, such as macrophages, would carry protein configurations which match the self-markers, and by this means would be able to discriminate living cells of the body from heterologous or foreign antigens. Burnet calls these groups recognition units, and pictures them, in the case of macrophages, etc., as enzymes which can destroy the self-markers (antigens), thus rendering them nonantigenic. The stability of cells in a particular tissue would depend on the interchange of molecules between self-markers and recognition units, which would also be present in the nonimmunological cells of the various tissues. There is therefore an equilibrium between neighboring, adjacent cells. If this equilibrium is disturbed, viz., by injury, the recognition units would react to the loss of self-markers in adjacent cells by inducing hypertrophy or mitosis, the cells proliferating until self-markers were again encountered, i.e., when healing has been completed. It is the loss of self-markers, in Green’s theory of carcinogenesis, which causes cells to escape the control imposed by the inhibition of cell division. Both Green and Burnet present the evidence of rats which

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have been fed carcinogenic azo dye. The liver cells of these animals first bind the carcinogen, but the ensuing hepatomas are found to be incapable of combining with the carcinogen (Miller and Miller, 1952). This is interpreted as a loss of protein (self-marker) from the hepatic cells. 2. Recognition Units and the Immune Response

If a n immunologically competent cell encounters an antigenic configuration not comparable to t,he self-markers of host cells (or, in other words, its own recognition units), i t will synthesize a primary template for that configuration. These primary templates are termed “antibody units” and are analogous to recognition units, but in contact with the appropriate foreign antigen they will synthesize antibody. Both selfmarkers and antibody units are conceived of as producing a genocopy, which is the means of impressing on other cells the protein-synthetic mechanism for producing the complementary recognition units and antibody units. Therefore, the mechanism for self-recognition or response to foreign antigen is passed from the original stem cells to successive generations of cells derived from them. As regards the primary and secondary responses of the immune reaction, Burnet (1961) believes that there are three grades of immunologically competent cells derived from a single affected clone. Grade 0 receives inhibition of part or all of its cellular activity by contact with the antigenic determinants. It is present in the embryo mainly but also occurs in the adult, and explains why immunological tolerance and immunological paralysis require the continued presence of antigen. After birth, grade 0 cells develop, either directly or by cell division, into grade 1 cells. These are believed to produce the primary response when in contact with antigen, and also reactions of the delayed hypersensitivity type. Antigenic stimulation of grade 1 cells would produce grade 2 cells, which are capable of antibody production. Burnet’s immunological concepts depend on reproducible molecular configurations in successive generations of cells. Recent investigations of the thymus would tend to support a cellular theory of immunization (Miller, 1961, 1962; Marshall and White, 1961).

H. POSSIBLE LIMITS TO AUTOIMMUNITY AS IT PERTAINS TO THE NEOPLASTIC STATE While Burnet’s hypothesis is essentially a n immunological conception, i t would not seem necessary to reduce all the processes responsible for controlled growth and differentiation to immunological terms. There are now voluminous findings from experimental embryology indicative of the importance of various cells or tissues which exhibit local effects

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on the differentiation of many organs and regions. These “inductors” are not only limited to certain tissues a t a definite stage of development, but even to certain parts of the same general tissue. To postulate immune reactions for all of these stimuli to differentiation and regulation of growth would be too complex and would hardly serve to clarify the processes involved. It is possible that potentially antigenic substances, such as enzymes or hormones, are acting through other than immune mechanisms. It is also possible that nonantigenic reactive groups are involved. There is good evidence that diffusible substances mediate the action between the inductor and developing tissue. These may consist of large molecular structure because their action is prevented, possibly, by intervening, experimentally placed, semipermeable membranes (Grobstein and Dalton, 1957; Grobstein, 1957; McKeehan, 1958). However, the necessity for direct contact between cell processes of the two tissues has not always been ruled out (Grobstein, 1956). Studies from tissue culture indicate the importance of tissue or organ architecture for the maintenance of normal growth. A tissue explant placed in liquid culture medium undergoes steady disorganization, during which reticuloendothelial cells, fibroblasts, and epithelial cells migrate in succession from the explant (Paul, 1961). If some means of physical restraint is used, such as in the cellophane strip method of Rose and associates (1958) or the cellulose sponge matrix technique of Leighton (1951), something of the normal cellular organization of tissue can be preserved. This would seem remarkable in media which, although it may contain normal human serum, almost certainly is deficient in a t least some of the metabolites normally present in the intact animal. Certainly it is difficult to imagine immunological processes, in the usual sense, as controlling such growth patterns. It is well known, however, that normal tissues can develop specific “cell lines” in tissue culture. These established strains of cells usually emerge as a rapidly growing strain from a slowly growing primary explant. The strain, once established, will usually grow indefinitely in subsequent subcultures. Very often, but not always, the established cell strain has an unusual chromosome complement, and this is taken to be a selective advantage in cells growing in an artificial environment. It is debatable whether or not the chromosomal changes are necessarily associated with the malignancy of certain cell lines. It is true, however, that cell lines derived from normal tissue can become malignant in tissue culture when inoculated into the strain of animal from which i t was derived. Such instances are reported by Gey and associates (1954) and Sanford and associates (1954, 1956). Loss of malignancy in cultured tumors has also been observed. From phenomena such as these the

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parallel between “normal” and malignant cells in tissue culture becomes quite evident. If mutations are involved, i t would seem a fruitful line of investigation to determine whether or not these are initiated by a “release” from normal control mechanisms (such as immune responses) or by abnormal conditions of the culture medium (such as low oxygen tension, etc.). I n addition, i t would be useful to know if reinoculated tissue culture cells, derived from normal tissue but showing neoplastic growth in the isologous animal, have changed in antigenicity with respect to the strain of origin. More recent investigations have revealed interesting surface phenomena relating to both malignant and tissue culture cells. Luibel e t al. (1960) used the electron microscope techniques t o demonstrate differences between normal, human, uterine cervix epithelium and carcinoma of the cervix. Attachment plates of the cell membrane were present in the in situ and invasive carcinoma cells, but were less numerous than in normal, stratified, squamous epithelium, and in some regions were lacking entirely. I n addition, villuslike cytoplasmic projections from the surface of carcinoma cells were observed. Coman (1947, 1953), by measuring the force exerted in micromanipulation, found that significantly less force was needed to separate cancerous, squamous, epithelial cells from each other than to separate a pair of adjacent, normal, epithelial cells. This decreased adhesiveness of cancerous cells is related in some way to local calcium deficiency in the cancerous tissue. Abercrombie and Heaysman (1953, 1954) showed that the movement of chick fibroblasts in tissue culture is influenced in velocity and distance of migration by the number of contacts between adjacent cells. Two migrating, monolayer sheets of fibroblasts were observed to stop their movement toward each other when the cells of the leading edges of each sheet met and touched. There was a minimum amount of overlapping of the adjacent cells, although the fibroblasts freely adhered to one another. This prohibition of movement of sheets of cells in tissue culture was called contact inhibition, and represents some restrictive influence on the surface on the cells. Abercrombie e t al. (1957) demonstrated contact inhibition between fibroblasts from two different vertebrate classes (mouse and chick), yet two mouse sarcomas showed no cessation of movement with normal mouse fibroblasts. The sarcoma cell sheets were seen to move over the mouse fibroblast sheets, while the latter moved underneath the sarcoma sheets. The sarcoma cells seemed to be lacking in some element necessary for the contact inhibition normally shown by fibroblasts. If antigenic differences are involved, i t is difficult to understand why the same unresponsiveness is not shown between fibroblasts of mouse and chick origin.

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Finally, there are the tissue culture experiments of Leighton and Kline (1954), Leighton and associates (1956) which demonstrate that HeLa cells (derived from human carcinoma of the cervix) can infiltrate and replace “normal” human fibroblast culture with apparent dissolution of the connective tissue. HeLa cells also show invasiveness to rapidly growing chick embryonic tissues such as connective tissue outgrowths from heart and bone, osseous tissue, liver, and brain. After invasion by HeLa cells, no residue of the displaced normal cells could be discovered by these investigators. Moreover, embryonic chick lung and intestinal epithelium were observed to invade HeLa cells in tissue culture. It is tempting to speculate on the possible intercellular immune responses among these cells, in the light of Burnet’s self-markers and recognition units, but in fact the mechanisms concerned remain quite unknown. VI. Viral Oncogenesis

A. SOMERECENTLY DISCOVERED VIRALONCOGENIC AGENTS Recent studies in virology have produced an increasing knowledge of agents which are associated with malignant growths in experimental animals. While Ellerman and Bang (1908) described avian leukosis as being of viral etiology in 1908, the years from 1951 to 1959 saw the characterization of several new viral leukemia agents. Among these have been the viruses described by Gross (1951) in AK strain mice, and later as “passage A” strain in Bittner C3H mice (Gross, 1957). Lymphocytic leukemia was found by Schoolman e t al. (1957) to arise in Swiss mice which had been inoculated with cell-free brain filtrate from leukemic Swiss mice. Friend (1957) demonstrated leukemia production in Swiss and DBA/2 mice after inoculation of cell-free spleen filtrate from mice harboring Ehrlich ascites tumor cells. This disease was described by Furth and Metcalf (1958) as reticulum cell disease. Also Graffi (1957) summarized his studies on a myeloid leukemia virus, which is recoverable from five transplantable mouse tumors, and which produces granulocytic leukemia when inoculated into Agnes-Bluhm sg and db strains of mice. Lieberman and Kaplan (1959) demonstrated a leukemia-inducing agent in lymphoid tumors arising in X-ray-irradiated C57BL/ka mice. Newborn normal mice are susceptible to infection by this virus. Moloney (1960) extracted a virus from a connective tissue neoplasm, Sarcoma 37, which elicited leukemia in susceptible hosts. Unlike the other murine leukemic viruses, this last shows a lack of strict strain or species specificity as regards susceptible hosts, and it is not limited in effectiveness by the age a t which the animal is inoculated. Also showing a lack of strict strain or species specificity is the virus

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isolated by Rauscher (1962). This agent induces erythrocytopoiesis and lymphoid leukemia in various mouse strains, and leukemia in OsborneMendel rats. In addition to the murine leukemia-inducing agents, viruses have also beer. associated with the induction of solid tumors in experimental animals. Tlie Gross leukemia virus (Gross, 1956) was reported to produce parotid gland tumors in newborn C3H mice. Stewart and associates (1957) isolated polyoma virus from a tissue culture which, through repeated passages in. vitro, had supported growth of a leukemia virus from mice. Polyoma virus produces a variety of solid tumors when inoculated iato mice. Bittner (1936, 1945) had earlier described an infective agent in the milk of nursing C3H mice, and the isolated, particulate substance was characterized as mammary tumor-inducing by Moore and co-workers (1959). Rous described a sarcoma-inducing virus in fowl as early as 1911, and Shope and Hurst (1933) a papillomainducing virus in rabbits. The Shope papilloma can become malignant on successive transplantation in rabbits. Munroe and Windle (1963) reported growth of subcutaneous tumors from inoculation of newborn monkeys with Rous sarcoma virus. Adult monkeys did not develop the tumors. This was the first reported instance of the development of sarcoma in primates after injection of virus. Possibly, immune mechanisms may have protected the adults from development of the tumors, but additional research is indicated here. An interesting, if unusual, aspect of the virus work is the ability of certain viruses to infect tumor cells and cause destruction of the tumor. Results vary with virus strain and tumor type, but many viruses are seen to produce cytopathogenicity in tumor cell lines in vitro (Hammon et al., 1963). B. MECHANISM OF ACTION The application of immunology to the virus oncogenic field will depend on the precise nature of the method by which the tumor is induced and propagated, processes which are receiving much attention a t the present time. The recent work of Martin and associates (1961), showing that simultancous inoculation of a virus and a carcinogen into Swiss mice can result in lymphomas, myeloid leukemias, and sarcomas, which normally do not develop in these animals, indicates a “carrier” role for viruses. Here vaccinia virus was used, and was thought to enhance the subthreshold dose of 9,10-dimethylbenzanthracene-1,2,producing tumors in animals susceptible to vaccinia. Mice which had been immunized against vaccinia did not develop tumors. Hence, an entirely new concept of tumorigenesis has emerged in which common viruses

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might induce tumors in the presence of minute amounts of chemical carcinogens by enhancing the toxicity of the latter. However, these workers were unable to show in vitro absorption of carcinogen on the vaccinia virus. Along similar lines Duran-Reynals and Stanley (1961) used vaccinia virus to enhance the action of methylcholanthrene on noninbred male albino mice. They reported skin tumors and lymphomas in 88% of the experimental animals and in 38% of the control mice, which included mice immunized against vaccinia. These authors concluded that the chance of malignancy was increased significantly by the exposure to methylcholanthrene during vaccinia infection. When a virus siich as polyoma or Rous sarconia is grown in tissue culture, initiation of tumorigenesis is indicated by a characteristic change in cell morphology which is called conversion. It is important to note that polyoma virus, grown in mouse or hamster tissue culture cells, need not continue in the cells of succeeding generations to perpetuate this change. According to Dulbecco (1960, 1961), the “converted” cells continue to produce virus for a time, but clones from succeeding generations of these cells cease to produce virus, although the converted cell morphology remains. Under these circumstances, i t has been supposed that viral DNA remains in these cells in an incorporated state as part of the cell genome (and this is indeed the case with the Escherichia coli bacteriophage cell types). However, Dulbecco has reported that it has not been possible to detect any viral DNA in the polyoma-converted cells, although i t could be extracted from primary converted cells having virus in the vegetative state. The work of Mellors (1960) with Shope papilloma virus would seem to indicate much the same phenomenon in viral tumor induction. This investigator used fluorescent antibody to show intracellular localization of Shope papilloma virus in the rabbit papilloma and in VX7 carcinoma, which is derived from the papilloma by transplantation in New Zealand rabbits. Fluorescent antibody to papilloma virus was found to have identical intracellular distribution in both papilloma and VX7 carcinoma, but the latter showed one third the intensity of staining, indicating some loss of the original viral antigens. The VX2 carcinoma, surviving 120 generations, was found to display no staining with papilloma fluorescent antibody. This loss of, or change in, viral antigenicity correlates with the inability of VX2 grafts to immunize rabbits against papilloma virus. Therefore, this may be another case of the apparent disappearance of the virus from an established tumor after the oncogenic process had been successfully promoted. The findings of Gerber (1963) with long-standing in vitro growth of

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hamster ependymomas, indicate that the original infecting simian virus 40 (SV40) was not detectable in cell lysates and cell-free fluids, although the cells retain their neoplastic potential when inoculated into newborn hamsters. I n contact with sensitive monkey kidney indicator cells, they bring about characteristic cytopathogenic effects in the adjacent indicator cells, a t which time virus 40 (SV40) is detectable by neutralization tests with the specific antiserum. Possibly, viral nucleic acid persists in some tumor cells, and under proper conditions these can introduce genetic information to indicator cells, thereby initiating viral synthesis. The tumor cells themselves seem unable to synthesize virus, and there is no evidence to indicate whether or not the viral nucleic acid is in a provirus or incorporated state.

C. RELATIONSHIPS AMONG VIRUS AND HOST ANTIGENS The studies of Eckert and associates (1955a,b) on avian myeloblastic leukosis virus revealed that a common antigen was shared by virus, chicken tissue, and Forssman antigen. Antiserum was produced in the rabbit by inoculation of virus which had been extracted from chicken serum. It was then absorbed with sheep red cells and chicken tissue homogenate. The absorbed virus antiserum showed a decrease of 90% in the complement fixation reaction for both virus and chicken cell antigens. Also reduced by a similar magnitude was the capacity of the antiserum to neutralize viral infectivity. Moreover, virus obtained from chicken serum showed sedimentation constants, ATPase (adenosine triphosphatase) activity, and electrophoretic mobilities which were identical to those of chicken antigen and Forssman antigen. Thus there is much physical and immunological evidence that chicken antigen and Forssman antigen are integral parts of the virus of avian myeloblastic leukosis. However, the chicken can produce antibodies to the virus when infected with this agent. Eckert presents this as evidence that the virus must possess an antigen distinct from chicken tissue. Working with Rous sarcoma virus, Rubin (1955, 1956) demonstrated that antichicken antiserum and antivirus antiserum neutralized infectivity by different mechanisms. The antiserum to chicken cells is bound reversibly to the virus and will reduce infectivity by 90%. But if the neutralized virus-antichicken-antiserum complex is diluted before inoculation, infectivity of virus is restored. This is not true if antivirus antiserum is used. The union of antiserum with virus is then irreversible. Nor is complement required for the neutralization of virus with antivirus antiserum, whereas it is required for neutralization with antichicken antiserum. The effect of the antichicken antiserum was found to be on chicken cells, because it could be injected 2 days after the inoculation

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with Rous sarcoma virus and still prevent infection. The antivirus antiserum was ineffective if injected 4 hours after the virus, because the virus rapidly penetrates the chicken host cells and loses direct contact with the virus antiserum. The antichicken antiserum prevented cell multiplication. However, i t is possible that the chicken cells continue to release virus (Rubin, 1957). These and other experiments utilizing absorptions with Forssman, chicken, and viral antigens are believed to indicate that in Rous sarcoma the host antigen is not an integral part of the viral antigen (Rubin, 1957).

D. POSSIBLE ROLEOF IMMUNOLOGICAL TOLERANCE As already noted, the age of the animal is an important factor in susceptibility to oncogenic viral infection, young animals usually being more susceptible. Syverton (1960) has suggested that this is due to the fact that immune mechanisms are poorly developed in newborn animals. At the same time, for this very reason, immunological tolerance is possible if the newborn animal receives a small dose of virus congenitally. The virus might then be harbored indefinitely without producing disease symptoms. Thus Gross (1953) was able to extract parotid tumor virus from normal C3H mice. When this virus was injected into newborn C3H mice, the increased virus dose was in excess of the immune responses of the newborns, and the parotid tumor developed. Similarly, Syverton (1960) suggests that irradiation of normal C57BL/Ka mice causes a breakdown of immune mechanisms and allows latent leukemia virus to manifest itself in thymic leukemia. He notes that the disease does not occur if the bone marrow is shielded in the irradiated mice. Kaplan’s own interpretation of this phenomenon is, however, quite different (Kaplan, 1960, 1961). He believes that immature thymic cells are essential for the oncogenic process. These would be present in both newborn mice and in the irradiated thymus of mature mice, since new cells are generated in thymic repair, which follows irradiation. Bone marrow would promote rapid regeneration and maturation of thymic cells, thus inhibiting the leukemia process. This would also be true for normal thymic implants into irradiated mice, because here also the earliest morphological changes resemble arrested maturation of lymphoid cells.

E. RESISTANCE TO TUMOR AND VIRUS: POSSIBLE SEPARATE MECHANISMS Chambers and co-workers (1960) studied oral papilloma virus in dogs to determine mechanism of resistance. These tumors always regress and never become malignant in the dog. Inoculation with papilloma tumor suspension rendered dogs resistant to superinfection to virus 2 to 16

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weeks after recovery. However, tumors can be transplanted into the immune dogs and grow normally. It is evident that the humoral antibody versus virus is ineffective against the tumor, therefore, but effective against the viral agent. Immune serum gave some evidence, in fact, of enhancing tumor growth when injected into tumorous dogs. On the other hand, suspensions of lymph nodes or spleen cells from recuperated dogs, when injected intravenously or intraperitoneally into dogs still bearing tumors, showed some indication that the tumors regressed. There may be a cellular antibody involved, therefore, in tumor regression, although the authors were not able to eliminate genetic differences in donor and recipient dogs as an additional factor to be taken into account in their transplantation studies. Nevertheless, humoral antibody to either virus or tumor is of little effect in causing regression of the established tumor. I n addition, there is the possibility that, once a tumor has been promoted by the virus, a different immunological mechanism becomes operative, because of antigenic change in the tumor. It is therefore not clear whether a cellular antibody would be directed against the virus or the tumorous cells.

F. ANTIGENICRELATIONSHIP AMONG VIRUSES Rowe and co-workers (1958) showed that three strains of polyoma virus, derived from mouse neoplasms and grown in tissue culture, would produce in mice antisera which were detectable by viral hemagglutination inhibition, complement fixation, and neutralization of tumor infectivity. Certain of these strains did not react with antisera produced by such viruses as ECHO types 1 to 20 and poliomyelitis types 1 to 3. Positive reactions were obtained with antisera from mice with filtrateinduced parotid tumors or leukemia (Gross virus), while reactions were negative with the antisera from mice carrying the Bittner milk agent, Friend’s hemopoietic tumor-inducing virus, and generally from infected strains of mice showing high rates of spontaneous leukemia. Therefore, the strains of polyoma virus tested may be distinct from these nonreactive viruses. Also negative in reaction to polyoma virus by complement fixation were 162 human sera, including 72 from patients with solid tumors or leukemia. On the other hand, the SE polyoma strain showed reactivity with the antisera from CSH/P mice infected with the cell-free filtrate leukemia virus described by Schwartz and associates (1959a). G. RELATION OF VIRUSANTIBODY TITERTO THE PRESENCE OF THE TUMOR Fogel and Sachs (1959) studied susceptibility and resistance to polyoma virus which was grown in tissue culture, being derived from

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mouse parotid tumor cells and mammary adenoma. The potency of antisera, which were produced by various species of animals, was measured by its inhibition of viral hemagglutination of erythrocytes. It was found that in susceptible species such as hamsters and rats there was correlation between high antibody titers and tumor progression. In the rabbit, in which the virus-induced fibrosarcomas developed and then regressed after 10 weeks, high antibody titers also depended on the presence of tumors, and dropped after the tumors had regressed. Among resistant animals, the dog and guinea pig, no tumors developed and antibody titers were low, reaching a maximum of 1:320 in the guinea pig in 5 weeks, and dropping to 1:20 in 10 weeks. On the other hand, chickens, which are also resistant to polyoma virus infection, showed antibody titers up to 1:2560 in 4 weeks, which are as high as those found in the susceptible hamsters. These findings are of interest in that they indicate unknown areas in antibody action, especially the inhibiting or enhancing of the growth of tumors in the presence of a viral causative agent.

CELL-FREE INDUCTION OF LEUKEMIA FROM LEUKEMIC MURINEAND HUMANBRAINS Schwartz, Schoolman, and associates (Schoolman et al., 1957; Schwartz e t al., 1956) have studied the effects of cell-free filtrates from mice with leukemia and found a heat-labile agent from leukemic AKR mouse brain which produced lymphoblastoma when injected into 6- to 12-week-old AKR mice. Intracerebral injection of the filtrate produced the disease in 47 of 99 animals, and intraperitoneal injection in 40 of 76 animals, a t 22 weeks of age. This is a high leukemia strain of mouse, and ordinarily 90% of the animals develop leukemia, but not until 6 to 12 months of age. Using low-leukemic Swiss and DBA strains, which have a rate of spontaneous leukemia of 1 and 2%, respectively, these authors were also able to produce lyniphoblastoma by injection of leukemia cell-free filtrates. Those filtrates from Swiss or DBA leukemic brains induced lymphoblastoma in 48 of 106 adult Swiss mice inoculated intracerebrally and in 39 of 55 inoculated intraperitoneally. Those cell-free filtrates inoculated into DBA mice intracerebrally produced lymphoblastoma in 8 of 19 mice. Recipient AKR mice, a total of 20 animals, did not develop the disease from inoculation of leukemic Swiss or DBA cell-free brain filtrates. Cell-free extracts from the tumor had no effect when inoculated into DBA or Swiss mice. Other studies (Schwartz et al., 1957) indicated that the AKR mice showed accelerated development of leukemia when inoculated with cell-free filtrates from H.

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the brains of patients dying from various leukemias. Of 326 mice, 71 showed the disease a t 22 weeks. Cell-free extracts from 8 nonleukemic human brains failed to produce leukemia in a total of 110 mice. Most effective in producing leukemia were the brains from patients with myeloblastic leukemia who had had the disease for a short duration. Leukemia was not produced with heat-inactivated filtrates, or with filtrates from acute lymphoblastic leukemia patients (4brains) or from acute monoblastic leukemia patients (2 brains) in a total of 111 mice. 1. Passive Immunization with Rabbit and Mouse Antisera Schwartz et al. (1959a) were able to induce leukemia in C3H mice after inoculation with cell-free brain filtrate from a C3Heb mouse which had developed spontaneous leukemia. These authors (Schwartz et al., 1959b) also conducted immunological studies by producing in rabbits antisera to the cell-free leukemic brain filtrate. C3H mice were passively immunized by intraperitoneal injection with the antisera on three successive days. The animals were then challenged with an intracerebral inoculation of cell-free brain filtrate from leukemic C3H mice. No leukemia developed in 40 immunized animals, while leukemia did develop in 33 of 40 control animals, which had been immunized with antisera to nonleukemic, cell-free brain filtrate from C3H mice. Challenge with tumor cell suspension, however, produced leukemia in 23 of 40 animals which had been immunized with the antisera to the leukemic, cell-free brain filtrate. Comparable results were obtained with Swiss mice which had been passively immunized with antisera to Swiss cell-free, leukemic brain filtrate and subsequently challenged with the homologous infective agent. Absorption studies showed that normal brain homogenate did not remove the antibody from the antisera to the cell-free, leukemic brain filtrate, whereas absorption with leukemic brain homogenate removed the antibody almost completely. These investigators (Schwartz et al., 1959b) also studied the protection of C3H mice with heterologous antisera. They reported that passive immunization of C3H mice with antisera to Swiss leukemic cell-free brain filtrate, or to human leukemic, cell-free brain filtrate, gave significant protection to the leukemic C3H filtrate, but to a lesser degree than did the homologous antiserum. It is not clear why a species difference should occur, unless different strains of virus are involved. Challenge with tumor cells always produced leukemia in some of the immunized animals. This could be due to a larger “dose” of virus when tumor cells are given, or to changed antigenicity of virus in “converted” tumor cells, or simply to the inaccessibility of the antiserum to the virus as it exists in tumor cells.

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2. Active Immunization of Mice

Maduros et al. (1958) studied active immunity to leukemic, cell-free brain filtrate in C3H X 100 strain mice. Cell-free, leukemic brain filtrate from mice of this strain was inactivated by irradiation with ultraviolet light and inoculated into mice of the same strain. Nonleukemic, cell-free brain filtrate was also irradiated and used in immunizing controls. On challenge with leukemic, cell-free brain filtrate, 3 of 19 mice developed leukemia if they had been immunized previously with the inactivated leukemic filtrate, whereas 17 of 20 developed leukemia if they had been immunized with the nonleukeniic brain filtrate, this being the number expected in unimmunized mice. Those mice developing leukemia developed it in 2 to 4 weeks, and a 20-week period was considered negative in the experiment. Active immunization was demonstrated, therefore, and apparently a specific antigen (virus) is responsible for the immunizing effect, being present in leukemic but not in nonleukemic C3H brain.

3. Immunization of Mice with Human Antileukemic Antisera Logothetetis et al. (1960) applied immunization experiments t o human volunteers, using the high leukemic AKR strain mouse as the test animal. Cell-free filtrates were extracted and pooled from four brains from patients with acute leukemias (lymphoblastic, myeloblastic, and monoblastic). Pooled antiserum was obtained from 14 normal males after having received five injections each (both subcutaneous and intradermal) of the cell-free human brain filtrate. The filtrate used in the challenge dose was from patients with myeloblastic, monoblastic, and stem cell leukemia (4 brains in all). Table VIII shows TABLE VIII IMMUNIZED AND CONTROL ANIMALSDEVELOPING LEUKEMIA AFTER INOCULATION WITH VARIED CONCENTRATIONS OF LEUKEMIC HUMAN CELL-FREEBRAINFILTR.ATE (CFBF)a Conc. of leukemic CFBF

Protection with antiserum to CFBFb

Protection with normal human serumb

Untreated controlsb

0.25 0.025 0.0025

7/15 10/25 3/15

15/15 19/25 9/15

10/10 7/10 5/10

From Logothetetis et al. (1960).

* Number developing leukemia/number

injected.

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about 20 to 50% protection of the mice after intraperitoneal inoculation of the challenge dose. It is not clear how long a time the protected mice were observed during this experiment. The AKR mice were inoculated with the leukemic, cell-free brain filtrate at 10 to 12 weeks of age. This strain normally develops leukemia in 6 to 12 months to the extent of 90%. I n the experiment already cited (Schwartz et al., 1957), cell-free brain filtrate from human leukemic brain accelerated the development of leukemia in AKR mice to 22 weeks of age. The viral studies, especially those relating to human neoplastic disease, are as yet a t the preliminary stage. Nevertheless, i t can be seen that the immunological mechanisms observed earlier in regard to tumor growth and transplantation, and tumor antigen extraction and analysis, are much in evidence in the current investigations in the virus field. The virus represents an additional antigenic factor. Whether or not i t remains distinct from host antigen in causing neoplastic disease, or whether it can combine with and in some way change the quality of host antigen (such as DNA or RNA), are certainly aspects of the problem which will remain crucial in elucidating the immune responses of the host t o its tumor.

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