Electron microscopic localization of viral antigens in mouse mammary tumors by ferritin-labeled antibody

VIROLOGY

33,

Electron

197-214

(1967)

Microscopic Mammary

Localization Tumors

by

of Viral Ferritin-Labeled

I. The Homologous HARUTAKA The Rockefeller

Antigens

TANAKA2 University, Accepted

AND

New June

in Mouse

Antibody’

Systems DAN York,

New

H. MOORE3 York,

10011

3, 1967

Using purified viruses and antibodies against them, the ferritin-labeled antibody technique was applied to mouse mammary tumors to localize viral antigens at the electron microscopic level, and the results were discussed from virological and technical aspects. The following conclusions were reached: (1) only B particles of virus were specifically tagged with antivirus conjugate; (2) intracyt,oplasmic A particles were not correlated antigenically to B particles even wit,h conjugated antibody to ether-treated virus; (3) coat proteins of highly oncogenic &ITT- particles and of far less oncogenic, morphologically indistinguishable particles called NIT were antigenitally indistinguishable; (4) tumor-bearing mice failed to show any antidody formation against the causative virus.

et al., 1967). It is surprising that so few fluorescent antibody studies on these tumors have been made (Lasfargues et al., 1959; Brown and Bittner, 1961a,b). It should be pointed out,, however, that the mouse mammary tumor is a very complicated system, containing various particles and antigens of virus or of presumed viral nature. Tumor extracts usually used as antigens in experiments in this area contain such elements as A and B particles recognized in electron microscopy (Bernhard, 1958), three active or interfering particles proposed by Moore et al. (1959), a highly oncogenic virus (MTV), and a far less active one, named nodule-inducing virus (NIV) by Pit,elka et al. (1964) (Sandi and DeOme, 1965). Newly formed tumor antigens and transplant’ation antigens may also be present, in addition to strain-specific antigens of the host mouse. Under t’hese circumstances even the most careful immunological study can fail to establish an immunizing part,icle to tissue particle correspondence. Successful purification of the virus (B particle) from mouse milk report’ed by Lyons

INTRODUCTION

More and more evidence has accumulated indicating that immunological studies are among the most successful and indispensable approaches to the problem of cancer in general and to cancer of viral etiology in particular. In the area of spontaneous mouse mammary tumors Andervont and Bryan described neutralizing antibody to the causative agent (MTV) as early as 1944. But it was not until 1961 that the gel diffusion technique was first introduced into this field by Lehzneva (1961), although the technique is now employed by many authors (Blair, 1965; Blair and Weiss, 1966; Blair et al., 1966; Cryan et al., 1966; Nowinski 1 This investigation was supported in part by research grant CA-04573 from the National Cancer Institute, National Institutes of Health, Public Health Service, and by a grant from The Lillia Babbitt Hyde Foundation. f Present address: Department of Pathology, Institrlte for Virus Research, Kyoto University, Kyoto, Japan. 3 Present address: Institute for Medical Research, Camden, New Jersey. 08103. 197

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and Moore (1965) greatly simplified the matter, providing us with a highly specific antibody against B particles of MTV. Using this specific antibody we attempted to localize viral antigens in mouse mammary tumors at the electron microscopic level by the ferritin-labeled antibody method proposed by Singer (1959) and applied to tissues by Morgan and his co-workers (1961 a-c). The aims of our studies are to ascertain whether: (1) the ferritin-labeled antibody method is applicable to MTV, since no tumor virus has been systematically examined by this method; (2) it is possible to correlat*e antigenically intracytoplasmic A particles with mature B particles; (3) biologically weak NIV cross-reacts immunologically wit’h biologically active MTV; (4) mice can immunologically distinguish MTV from their own tissues under natural conditions; (5) any immunological cross-reactions can be detected between MTV from different, strains of mice; and finally (6) MTV shares any antigens with murine leukemia viruses and ot’her oncogenic viruses. This paper will be concerned with the first four problems, utilizing the homologous systems which consist mainly of t’umors and viruses of RI11 as well as RIIIf st’rain mice and rabbit antibodies against these viruses. The other problems will be treated in a separate paper (Tanaka and Moore, 1967). Because of the scarcity of fluorescent antibody studies on mouse mammary tumors, explanation of the findings in immune electron microscopy was expected to be difficult. Fortunately, however, the pictures were interpretable with confidence, and we believe these findings will lead to a better understanding of the immunology and oncogenicity of the mouse mammary t,umor virus. MATERIALS

Mice. used in by Dr. versity. develop females, whereas

AND

METHODS

Mice of RI11 and C57BL strains, all our work, have been maintained C. D. Haagensen of Columbia UniSpontaneous mammary tumors in more than 90 % of breeding RI11 usually at 6-12 months of age, not a single tumor has developed

AND

MOORE

among C57BL mice. C57BL mice are free of Bittner agent by all known tests. The RIIIf strain was established in 1963 by fosternursing RI11 newborns on C57BL mothers. Spontaneous tumors develop in only 6 % of them at an average age of 14 months. A high cancer strain IBA mice was given us through the courtesy of Dr. Anna Goldfeder, Cancer and Radiobiological Research Laboratory, New York University and Delafield Hospital. Viral adigens. Virus was purified from milk4 of RI11 and RIIIf strain females by the method of Lyons and Moore (1965), using a 40-10% layered Ficol15 density gradient column. Usually 4 bands and a pellet were formed; band 4, a major viral fraction,6 sometimes combined with band 3, which also contained an abundance of virus, was resuspended in PBS, filtered through Millipore filters (0.45 p), and centrifuged at 100,000 g for 1 hour. The final pellet was resuspended in a small amount of PBS and used for inoculating rabbits. In one series of experiments a suspension of purified RI11 virus was mixed with an equal volume of ethyl ether and magnetically stirred at room temperature for 1 hour in an attempt to destroy the envelope and thus expose the internal antigens of viral particles. Ether was removed by centrifugation and nitrogen bubbling, and the aqueous phase was used as antigen. Antisera. New Zealand rabbits were given 4 weekly intramuscular injections of either one of the above virus preparations, each equivalent to 5-10 ml of milk with complete Freund’s adjuvant. An aliquot of the viral 4 Milk was diluted 4- to &fold with PBS during milking process by moistening the nipples. 5 The Ficoll (Pharmacia, Uppsala, Sweden) was purified by dialyzing a 20y0 aqueous solution against several changes of distilled water over a period of 3 days, followed by centrifugation for 3 hours at 100,000 g to remove insoluble impurities. This was t,hen lyophilized and extensively desiccated over a molecular sieve (Linde Products, Union Carbide Co.). 6 Band 4 in the present paper is substantially identical with band 3 of Nowinski et al. (1967)) who used a mechanical gradient maker inst,ead of manually layering Ficoll solutions of decreasing concentrations. the

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IN

preparation was pelleted in the ultracentrifuge, fixed with OsOd and prepared for electron microscopy. Rabbits were bled one week after the final injection, and globulin was prepared by the method of Strauss et al. (1960). This was filtered through a Millipore filter and kept in a refrigerator until used. In order to test for antibody in tumorbearing mice, globulin of IBA strain mice wit.h spontaneous tumors and C57BL strain mice with experimental tumors was pooled and used for conjugation. Experimental tumors in C57BL mice were induced by RI11 virus inoculated at 2-4 weeks of age. A globulin fraction from normal rabbits was also prepared as control. Conjugation of globulin with horse ferritin. The conjugation was done by Singer’s method (1959) as modified by Rifkind et al. (1964), using meta-xylylene diisocyanate as conjugant The final concentration of conjugates was 10 mgiml with respect to ferritin. Absorption of conjubates. After repeated uhracentrifugations to remove uncoupled globulin, conjugates were absorbed with and combined tissue rat liver powder, powder and milk powder of agent-free C57BL mice in this order, according to the method of Coons et al. (1955). The conjugates were then cleared by centrifugation at 18,000 rpm for 15 minutes. Although we did not employ such purifying procedures of conjugate as continuous flow electrophoresis, recommended by Borek and Silverstein (1961), this exhaustive absorption procedure was found to be very effective in eliminating not only immunologically specific, undesired reactions but also the nonspecific staining of conjugate encountered so often. For the absorption test conjugates were further absorbed with fresh RI11 or RIIIf virus; milk was mixed with EDTA and centrifuged at 18,000 rpm for 15 minutes to remove gross insolubles, followed by another centrifugation at 27,000 rpm for 1 hour. The pellet was resuspended in PBS and filtered through a Millipore filter. One milliliter of conjugate was mixed with fresh crude virus preparation, equivalent to 5 ml of diluted milk, at

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room temperature for 1 hour, and then kept overnight in a refrigerator. It was cleared next morning by a centrifugation at 18,000 rpm for 1 hour. Repeated absorption and subsequent centrifugations inevitably resulted in a dilution of conjugate solution (originally 10 mg/ml with respect to ferritin). However, no adjustment of its concentration was made except to condense it by centrifugation to the original volume. Experimental procedures. Spontaneous RI11 mammary tumors were mainly used in the present study; however, spontaneous tumors in IBA females and experimental tumors in C57BL females (see above) were also used when necessary. Tumors in RIIIf strain could not be used, simply because the incidence of tumors was so low that not a single tumor was available during the experimental period. Tumors, removed and cleared from necrotic materials, were minced with a razor blade in cold 5 % formalin in PBS placed on a wax plate, and if necessary, ground in a mortar to make a tissue paste. This was suspended in formalin-PBS, and differentially centrifuged at low speed to get a fraction of single cells and small cell groups; usually pellets of centrifugation at 1000 rpm for 2 minutes after removing gross pieces by prior centrifugation at 500 rpm for 30 seconds were used. This step was repeated if necessary. The pellet was resuspended in a small amount of formalinPBS and subjected to 3-5 cycles of freezing in dry ice-acetone and thawing in a 37” water bath. The suspension was washed several times with PBS, divided into tubes and incubated in 0.5 ml of appropriate conjugate solution for 20-60 minutes at room temperature with occasional shaking. Then the suspension was vigorously washed, fixed with 0~04, dehydrated with alcohol, and embedded in Epon. IJltrathin sections of conjugate-incubated materials were stained with uranyl acetate only; lead was not used because of a possible mistaking of fine lead precipitate for ferritin. For blocking tests the cell suspension was treated with appropriate unconjugated globulin for 20-

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TANAKA

60 minutes at’ room temperature incubation in conjugate solution.

prior

AND

to

RESULTS

Fine Structure

of Nontreated

RIII

Tumors

Electron microscopic structural det’ails of mouse mammary tumors have been repeatedly reported by many authors (see Moore’s review, 1962), and here only bhose are briefly described which will be specifically related to understanding the pictures revealed by the ferritin-labeled antibody method. The principal constituent of tumor tissue is an acinus surrounded by connec-

MOORE

tive tissue. An acinus consists of a lumen surrounded by an epithelial wall. Viral particles are found in connection with epithelia directly facing the acinal lumen. Bernhard (1958) distinguished two kinds of viral particles usually associated with mouse mammary t’umors: A and B particles. A particles are found only in the cytoplasm, often in clusters or lining vacuoles and secretion droplets (Fig. 1, A). They are thick-walled (20 rnp) spheres whose profiles in thin section are often described as doughnut-shaped, the diameters of the outer and inner membrane being 70 rnp and 50 rnp, respectively. Free B particles are found in the acinar lumens (Fig. 1, MB),

FIG. 1. A part of spontaneous mammary tumor in RI11 mouse. In the cytoplasm of a tumor cell occupying the left two-thirds of the picture are various organelles including vacuoles, obliquely sectioned centriole (C) and some secretion droplets (D). In association with secretion droplets are clusters of doughnut-shaped A particles (A). The cell surface lining an acinar lumen (L) is modified by many processes, and at the tip of some of them are budding B particles (BR). Several mature B particles (MB) are seen in the lumen filled with amorphorm, dense materials.

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IN

a.nd, in smaller numbers, within the intracyt,oplasmic vacuoles. They have a saclike envelope covered with protruding spikes, 95 A long, spaced at 70 A (Fig. 4a,b), and an eccentrically located dense nucleoid. A second membrane closely covering the nucleoid is sometimes well preserved and sometimes not. These particles are rather variable in size, ranging from 100 to 120 rnp in diameter. They are referred to here as mature B particles. They are formed t’hrough a budding process at the cell surface facing the acinar lumen, oft’en at the bip of microvilli (Fig. 1, BB), and, less frequently, at the membrane limiting t,he intracytoplasmic vacuoles. The outermost layer of budding B part’icles is an extension of the surface membrane of host cells, and the int,ernal structure is morphologically similar to A particles. Imai et al. (1966) found by serial sectioning method that all doughnutshaped particles were still connected with host cells, alt’hough some of them appeared free in a single section, whereas mature B particles were in realit’y free. Thin

Sections oj Viral as Antigens

Preparations

Used

Major components found in sections of RI11 (Figs. 2a, 3, and 4a) and RIIIf (Figs. 2b and 4b) virus preparations were identical. They cont,ained abundant mature B particles, a few secretion droplets (D in Fig. 2a,b), and only occasional budding B particles (Fig. 3), but no A particles. A number of small, round, dense struct.ures seen in bet,ween B particles are tangentially sectioned profiles of the latter. The fine structure of B particles is described above. General Appearance qf Conjugate-Incubated Tissues and Some Associated Technical Problems Tumor tissues were subjected to drastic treatments before fixation with OsO1, such as mincing, grinding, freezing and thawing, and repeated washing. This inevitably caused some damage to and morphological alteration of tissue components. Damage to cells was sometimes very heavy, but usually not’ of a degree sufficient to prevent)

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detailed cytologic or even histologic examinations. Acinar lumens sometimes remained completely surrounded by lining epithelia and sometimes only (“closed” lumen) partially surrounded (“open” lumen). Mature B part8icles were not found associated with such an “open” lumen because they had been washed away during the experimental procedures, while budding B particles in abundance remained attached to cells. These findings confirm the report by Imai et al. cited above. The conjugate particles are too big to penetrat,e the cell membrane under natural conditions; tissues were therefore frozen and thawed, a,s proposed by Morgan et al. (1961a,c), to cause small holes to form in the cell membrane so that the conjugate could gain access to the cell interior. Our experience confirmed that this procedure would permit the conjugate particles to penetrate the surface membrane and to reach almost every part. of the cell. However, successive penetration of membranes was increasingly difficult. For inst,ance, B particles within the cytoplasmic vacuoles and those in the “closed” acinar lumens were only rarely reached and it was difficult to wash away non-reacting conjugate particles, resulting thus in a high degree of local nonspecific staining. For this reason a small cell group lining an “open” lumen provided the most favorable situation for ferritin-labeled antibody studies. One important problem was the occurrence of undesired tagging by the conjugate particles of tissue components other than virus. Although we believe that our viral antigens were highly pure, there still sometimes occurred an undesirable t#agging of tumor cell surface, seemingly based on specific immunological reaction by antibody evoked by contaminant host components in the antigen preparations. This could be eliminated rather easily by absorbing the cor1jugat.e with tissue powder of C57BL mice. More hazardous was the nonspecific t’agging. Intracytoplasmic A particles and intranuclear chromatin structures were the two preferential sites for this, although

FIG. 2. Section of virus purified from milk of (a) RI11 and (b) RIIIf mice. Mature B particles are abundant and a few secretion droplets (D) are seen. Those smaller, roughly round, dense structures are tangentially sectioned B particles. Note that there is no morphological difference between HI11 and RIIIf virus. FIG. 3. Similar section to Fig. 2a. Structures identical with budding B particles (BB) are occasionally found, but no A particles. FIG. 4. Higher magnification of purified virus from (a) RI11 and (b) RIIIf mouse milk. Spikelike protein units are regularly arranged on the outer slu-face of the envelope (arrows). 202

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IN

it could also occur as diffuse st,aining throughout t’he cytoplasm. The situation was serious because soluble antigen of MTV was expected to reside in these structures. Nonspecific tagging of chromatin structures was sometimes so heavy that it was comparable with pictures of cell nuclei presentted by Morgan et al. (1961c, 1962b) in which the conjugate was said to be localizing soluble antigen of influenza virus. An example of nonspecific tagging

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of A part,icles is shown in Fig. 5. At the beginning of our study we thought this represented specific reaction and so reported (Tanaka and Moore, 1966a). This was corrected in subsequent reports (Tanaka and Moore, 1966b,c) when it turned out that this was nonspecific staining because (1) it was eliminated by vigorous absorption with rat liver powder followed by complete clearing of the conjugate by centrifugation; and (2) ferritin-labeled, normal rabbit

FIG. 5. RI11 tumor incubated in anti-RI11 virus conjugate. The conjugate was not adequately absorbed with tissue powder in this case, and many structures are tagged with the conjugate particles specifically and nonspecifically as well, including A (‘4) and B particles, cell membrane, and cytoplasm. .4fter adequate absorption, only budding B particles remain specifically tagged.

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AND MOORE

globulin gave the samereaction if absorption was inadequate. Now most of these hazards are eliminated by procedures described in Materials and Methods. However, occurrence of such nonspecific staining of intracellular structures shows that the conjugate particles can readily penet,rate the cell. A number of factors may be involved in nonspecific tagging, but it is certain that substances responsible for it were produced during conjugating procedures, since unconjugated free ferritin did not cause it. Preexistence of natural ferritin which may interfere with interpretation of experimental results does occur in mammary tissue (Miyawaki, 1965), and an accumulation was expected in RI11 tumors because of their strong tendency to hemorrhagic necrosis. Fortunately, it occurred only infrequently and was confined within lysosomes in a large mass or freely distributed throughout the cytoplasm, and soon became recognizable. Misinterpretation caused by mechanical displacement of ferritin molecules by the microtome knife during sectioning (Casley-Smith, 1962) could also be avoided by careful examination of pictures.

findings, therefore, confirm the supposition that budding B particles are really immature B particles. It should be emphasized that the surface of budding B particles has already acquired viral antigens different from those of the host cell even in the very early stages of the budding process, as is the case in myxovirus formation (Morgan et al.: 1961a,b, 196210;Due-Nguyen et al., 1966). RIII

Tumors Incubated in Conjugated Antiserum against Ether-Treated RIII Virus

It was hoped that ether would destroy the envelope of B particles and that the antibody against their internal components would react with intracytoplasmic A particles if the latter were the real precursors of the former. We employed a similar method of ether-treatment to that used for disrupting myxoviruses (Choppin and Stoeckenius 196.5; Leif and Henle, 1956), which produces a soluble antigen from B particles that readily reacts with rabbit antiserum in immunodiff usion technique (Nowinski et al., 1967). However, this new conjugate again failed to tag A particles, although it still reacted with budding B particles (Fig. 8a,b). A few ferritin particles were sometimes observed around an aggreRIII Tumors Incubated in Anti-RIII gation of A particles (Fig. 8b); but their Virus Conjugate appearance was inconstant and they were As stated above, many structures could too few in number to be called positively be tagged by the conjugate particles specific, because similar pictures were specifically and nonspecifically as well, obtained in the control experiments with including A and B particles, the nuclear ferritin-labeled, normal rabbit globulin. chromatin, cytoplasmic ground substance Reasons for this failure will be discussed and surface membranes of host tumor later. cells (Fig. 5). After the appropriate treatment of t,he conjugate, only the budding B RIII Tumors Incubated in An.ti-RIIIf particles remained specifically tagged (Figs. Virus Conjugate 6 and 7). A particles did not react with the conjugate. Reaction of A particles in Using RI11 tumors as substrates, exactly the same results were obtained with antitumors prefixed wit’h a lower concentration of formalin (2%) or even without pre- RIIIf virus conjugate as with anti-RI11 fixation was also negat,ive, excluding the virus conjugate (Fig. 9), the surface of possibility that prefixation with 5% for- budding B particles being tagged while malin destroyed the antigenicity of A the other structures, including A particles, particles. Antibody produced in rabbits was were not. In the extreme left of Fig. 9, a primarily against, mature B particles be- single mature B particle is present’ and cause few budding B particles were con- tagged, a rare phenomenon. The results t#ained in the antigen preparations. These indicate that biologically inactive RIIIf

FIG. 6. filled with conjugate FIG. 7a specifically

Part of an acinar lumen of RI11 tumor incubated many microvilli and budding B particles at their particles. and b. Part of RI11 tumor incubated in anti-RI11 tagged while A particles (A) are not. 205

in anti-RI11 tips which virus

virus conjugate. The space is are specifically tagged with the

conjugate.

Blldding

B particles

are

FIG. 8a and gate again fails BB in Fig. 8b). reaction. FIG. 9. RI11 this conjugate. tagged.

b. Part of RI11 tumor incubated in anti-ether-treated RI11 virus conjugate. The conjuto react with A particles (A in Fig. 8b) although it can still tag B particles (Fig. 8a, and Few ferritin particles around an aggregation of A particles do not represent specific tumor incubated A single mature

in anti-RIIIf virus B particle is present

conjugate. All in the extreme 206

budding B particles left of the picture

are tagged with (MB) and is also

VIRAL

ANTIGENS

virus shares some surface antigens biologically active RI11 virus. RIII

IN

with

Tumors Incubated in Ferritin-Labeled, Normal Rabbit Globulin

Reaction of budding B particles was completely negative in this control experiment, even when some nonspecific staining by the conjugate took place throughout the cytoplasm (Fig. 10). Blocking

Tests

To further confirm the specificity of reaction of budding B particles and also to see to what extent RIIIf virus was antigenically related to RI11 virus, four kinds of blocking tests were performed with RI11 tumors: combinations of pretreatment with unconjugated anti-RI11 or-RIIIf virus globulin and subsequent incubation in anti-RI11 or -RIIIf virus

FIG. 10. RI11 tumor incubated in ferritin-labeled, seen associated with budding B particles although plasm of tumor cell.

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conjugate. In all of t,hese four combinations, the final pictures were essentially the same, therefore only two pictures are presented (Fig. lla,b). In the entirely homologous sysbem, in which RI11 tumors were pretreated with unconjugated anti-RI11 virus globulin and then incubated in anti-RI11 virus conjugate, reaction of budding B particles was substantially reduced, but was rarely completely negative (Fig. lla). On the other hand, there appeared a number of nonspecific aggregations of conjugate particles adherent to tumor cell surfaces and even to budding B particles themselves. Some of them seemed to be tangential sections of specifically tagged B particles. This phenomenon is compatible with a well known fact that in the fluorescent antibody technique a completely negative result is rarely obtained in blocking experiments (Nairn, 1964). Existence of free anti-

normal rabbit some nonspecific

globulin. staining

No conjugate is observed

particles in the

are cyto-

FIGS. 11-12 208

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body which reacted wit,h antigens on budding B particles and cell surface (the antiserum was not absorbed) seems to facilitat’e aggregation of conjugate particles, a phenomenon compatible with the “ant’i-antibody” theory recently revived by Robinson (1966) in electron microscopy. A similar reduction of reactivity of budding B particles and a concomitant nonspecific aggregation of the conjugate particles were also found when pretreatment with anti-RI11 virus globulin was replaced by pretreatment with anti-RIIIf virus globulin (Fig. llb), and when RI11 tumors were treated with anti-RIIIf virus conjugate following pretreat’ment with unconjugated anti-RI11 or -RIIIf virus globulin. All this suggests that RI11 and RIIIf virus are antigenically identical. Absorption Tests Another and perhaps more reliable approach to this interesting problem is the absorption test,. Reaction of budding B particles of RI11 tumors with anti-RI11 virus conjugate was completely prevented by previously absorbing the conjugate with homologous, fresh RI11 virus (Fig. 12a), as well as with RIIIf virus (Fig. 12b). r\‘o nonspecific aggregation of the conjugate particles took place. Although we could not examine RIIIf tumors, these results together with those of blocking tests and simple incubation experiments without any pretreatment again confirm that the coat protein of RI11 and RIIIf virus are antigenically identical. CG’BL Tumors Incubated in Anti-RIII Virus Conjugate Budding B particles in tumors of C57BL mice experimentally induced by inoculating RI11 virus in early life reacted with anti-

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RI11 virus conjugate in the same manner as those in RI11 tumors did (Fig. 13). This is important t’heoretically; the conjugate against RI11 virus generally used in t,he present study was absorbed mit’h tissue powders, including those of agentfree C57BL mice, but powders of isologous RI11 mice were not used simply because virus was not successfully removed from this strain. It was, therefore, possible that t)he reaction of budding B particles to the conjugate in case of RI11 tumors might represent t,he strain-specific antigens to RI11 mice not, necessarily relat’ed t,o virus. This was, however, not likely because such a reaction was found to be restricted to budding viruses, but the specific tagging of virions in C57BL tumors negated this possibility since no RI11 strainspecific ant,igens should reside on C57BL particles. This, in turn, shows that reactions observed in RI11 tumors are also mostly virus-specific. Tumors Incubated in Fey&in-Labeled Isoglobulin from Tumor-Bearing Mice These experiments were carried out t’o determine whether tumor-bearing mice could respond to mammary tumor virus and/or any newly-formed antigens, as shown by antibody formation against these antigens. No reaction was found on budding B particles or anywhere else, either in spontaneous IBA tumors or in experimental C57BL tumors incubated in ferritin-labeled, pooled globulin of tumorbearing mice of the respective strain. I>ISCIJSSION

It was found that only the surface of budding B part,icles and of mature B particles under favorable conditions were specifically tagged with antivirus con-

Fro. 11. Blocking tests. RI11 tumor incltbated in anti-RI11 virus conjugate following pretreat,ment with unconjugated, anti-RI11 virus globulin (Fig. lla) or anti-RIIIf virus globulin (Fig. llb). The findings in these two cases are very similar; tagging of budding B particles and mature B particles (MR in Fig. llb) is reduced, but a number of nonspecific aggregat,es of the conjugate particles appear. FIG. 12. Absorption tests. RI11 tumor incubated in anti-RI11 virus conjugate which has been absorbed with fresh RI11 virus (Fig. 12a) or RIIIf virus (Fig. 12b). In both cases, react,ion of bltdding B particles completely disappears, which means that RI11 and RITIf virus are antigenically identical.

FIG. 13. Experimental tumor in C57BL mouse incubated in anti-RI11 virus conjugate. Budding B particles of C57BL tumor are also react,ing with anti-RI11 virus conjugate rejecting t,he possibility that the reaction of B particles in RI11 tumors is due to strain-specific antigen of RI11 mice. FIG. 14. Experimental tumor of C57BL mouse incllbated in ferritin-labeled isoglobulin of tumorbearing C57BL mice. Ko reaction is found. 210

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jugate. The criteria for the specificity of reactions were: (1) tagging was restricted to virions and found nowhere else; (2) such tagging took place only with antivirus conjugate and not with ferritin-conjugated, normal rabbit globulin; (3) the reaction was blocked by pretreating tumors with unconjugated antivirus globulin; and (4) the reaction was eliminated by absorbing the conjugate with fresh virus. In addition, Gross leukemia virus was not tagged by anti-RI11 virus conjugate (Tanaka and Moore, 1967). Participation of strainspecific antigens in such reactions was not probable because B particles in C57BL tumors also reacted similarly with anti-RI11 virus conjugate. It has also been found that B particles of mammary tumors of C3H, A, and DBA/212 strain mice were antigenically very close to those of RI11 strain mice (Tanaka and Moore, 1967). Two kinds of antigens specific to mouse mammary tumors have been reported by gel diffusion technique; one is as large as B particles (Blair, 1965; Blair et al., 1966; Blair and Weiss, 1966), and the other is much smaller and soluble (Lehzneva, 1961; Nowinski et al., 1967). Antigens reported by Cryan et al. (1966) may belong to the latter category, judging from the relative position in agar plate of the precipitin line. A decision has not been made as to the nature of this smaller antigen; it could be a fragment’ of the envelope or the internal antigens of B particles, or it could represent the smaller active agent originally proposed by Moore et al. (1959) and more recently confirmed in the circulating red blood cells by Nandi et al. (1966a,b). However, our study failed to localize specific viral antigens anywhere other than on the out’er surface of B particles. Appearance of viral antigens on budding B particles still connected with host cell surface is compatible with the similar findings in myxovirus infection (Morgan et al., 1961a,b, 196213; Due-Nguyen et al., 1966). In the cytoplasm, just beneath the host cell surface where budding is taking place, specific structures are usually found, such as aggregat,ion of amorphous material in influenza virus (Morgan et al.,

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1961 c), nucleocapsids in parainfluenza virus (Compans et al., 1966), and A particlelike structures in mouse mammary tumor virus. It is likely that reconstruction of host cell membrane has been put’ in motion by information released from viral RNA contained in these structures. Myxoviruses in general differ from MTV in that with myxoviruses viral antigens tend to spread to nonbudding parts of the host cell surface (Morgan et al., 1962a; Matsumoto, 1966; Due-Nguyen et al., 1966). Mature B particles were contained in purified pellets from milk but were almost completely absent from minced and repeatedly washed tumor tissue, while exactly the reverse situation was observed with respect to budding B particles, an indirect confirmation of the report by Imai et al. (1966) that mature B particles are free but immature ones are not. Correlation of intracytoplasmic A particles with extracellular B part,icles was not successful. Some authors believe that A particles migrate to the cell surface or cytoplasmic vacuoles to mature there into free B particles through the budding process (Bernhard, 1958; Imai et al., 1966). Fact’s supporting this idea are these: (1) A particles are morphologically identical to internal structures of budding B particles; (2) both A and B particles are universally associated with spontaneous mammary tumors; (3) large numbers of A particles and occasional budding B particles are frequently found around vacuoles containing mature B particles. Against that idea are observations that: (1) steps of the assembly of immature B particles can occasionally be seen at the cell membrane starting with a small crescent, in profile, lying against a protrusion of t’he membrane, followed by a filling in of the proximal side of the crescent to form a circle, which then looks like an A particle; (2) no or few migrating A particles are usually observed in areas between the cell surface and accumulations of A particles locat)ed deep within the cytoplasm; (3) in cultures of tumor cells no A particles are observed and the synthesis of B particles at the cytoplasmic membrane is evident (Lasfargues

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et al. 1959); and (4) intravacuolar B particles are usually too few in number, as compared with A particles surrounding the vacuoles, leaving a possibility that those B particles have been phagocytized by cells. Other authors are, therefore, inclined to regard A particles as not’ necessarily precursors to B particles (Lasfargues et al., 1959; Amano and Ichikawa, 1959). It seems to us, however, that the association of A particles with B particles is more t’han accidental. It should be taken into account that even budding B particles can remain as such without further maturation into free particles for a long time in generations of subcutaneous transplantation of tumors (Feldman, 1963). In addition, it is not a rare observation that in some acini of spontaneous tumors only one or the other kind of particles is seen. This would seem to indicate that mat,uration of MTV does not proceed uniformly, but is affected by many factors resulting in an accumulation of one or the other kind of particles in a population of tumor cells. Then, the real question is nob whet)her A particles represent an indispensable stage of MTV, but what are the factors facilitating an accumulation of one or the other particles. Kobayashi et al. (1966) showed that in Friend tumor cells inoculated into mice immunized with formalin-treated virus vaccine, mature leukemia C particles disappeared and only A part,icles remained in clusters. This, together with Feldman’s report’ cited above, seems to indicate a relationship between A and B particles. Nevertheless, our present study failed to correlate A particles antigenitally with B particles even when ether-treated virus was used as antigen. One can argue t’hat the failure in immune electron microscopy is simply due to the fact that conjugate particles did not gain access to the cell interior, but this premise cannot be accepted since so many conjugate particles in a form of nonspecific staining were often found within the cell. On the other hand, one can also argue that A particles and the internal part of B particles are not, antigenic in rabbits. Another possibility is that ant,ibodies to the internal

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structure of B particles do not react with A particles because of different exposed antigens. REFERENCES S., and ICHIKAWA, Y. (1959). Electron microscopical aspects of developing modes of the cancer virus and the problem of “pseudovirus particles.” ilcta Pathol. Japan. 9, 445479. ANDERVONT, H. B., and BRYAN, W. R. (1944). Properties of the mouse mammary tumor agent. J. Nutl. Cancer Inst. 5. 143-149. BERNHARD, W. (1958). Electron microscopy of tumor cells and tumor viruses. Cancer Ren. 18, 491-509. BLAIR, P. B. (1965). Immunology of the mouse mammary tumor virus (IMTV) : A qualitative in vitro assay for MT\.. Nature 208, 165-168. BLAIR, P. B., and WEISS, I>. W. (1966). Immunology of the mouse mammary tumor virus: Comparison of mammary tumor virus with the agent found in C3Hf/Crgl mice. J. Xatl. Cancer Inst. 36, 423-429. BLAIR, P. B., LAVRIN, 1). H., DEZFULIAN, M., and WEISS, D. W. (1966). Immunology of the mouse mammary tumor virus (MTT) : Identification in vitro of mouse antibodies against, MTV. Cancer Rer. 26, 647-651. BOREK, F., and SILVERSTEIN, A. M. (1961). Characterization and purification of ferritin-antibody globulin conjugates. J. Zmmunol. 87, 555561. BROWN, E. R., and BITTNER, J. J. (1961a). Fluorescent antibody reactions against the mouse mammary tumor agent. Proc. Sot. Exptl. Biol. Med. 106, 303-306. BROWN, E. R., and BITTNER, J. J. (1961b). Adaptation of fluorescent microscopy to determination of genetic variation in mouse mammary tumor agent,. Proc. Sot. Exptl. Biol. Med. 106, 844-847. CASLEY-SMITH, J. R. (1962). The displacement of ferritin molecules by the microtome knife. J. Microscopic 1, 335-342. CHOPPIN, P. W., and STOECKENIUS, W. (1965). Interactions of ether-disrupted influenza A2 virus with erythrocytes, inhibitors, and antibodies. Virology 22, 482-492. COMPANS, R. W., HOLMES, K. T-., DALES, S., and CHOPPIN, P. W. (1966). An electron microscopic study of moderate and virulent virus-cell interactions of the parainfluenza virus ST’5. Virology 30, 411-426. COONS, A. H., LEDUC, E. H., and CONNOLLY, J. M. (1955). Studies on antibody production. I. A method for the histochemical demonstration of specific autibody and its application to a study AMANO,

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in preparation.