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Failure of tumor cell nuclei to induce tumor isoimmunity
Failure of Tumor Cell Nuclei to Induce Tumor lsoimmunity YOSEF H. PILCH, M.D., Bethesda, Maryland ALFRED S. KETCHAM, M.D., Bethesda, Maryland
The purpose of the present study was to isolate tumor cell nuclei so free of contamination with intact cells that no tumor transplantation would occur even when very large numbers of nuclei were administered to highly susceptible hosts, and then to use such nuclear preparations as antigens in studies of tumor isoimmunity. Two tumors were studied: the Cloudman S91 melanoma and the Krebs K2 ascites tumor. A number of acceptable methods were available for the isolation of cell nuclei from normal mammalian tissues. The substantial literature on these methods and their applications in biologic research has been comprehensively reviewed by Schneider and Hogeboom [I], Dounce [Z], and Allfrey [8]. These methods all involve the controlled mechanical disruption of cells in an appropriate suspension medium and the subsequent segregation of the nuclear fraction by differential centrifugation, filtration, countercurrent distribution, or floatation. However, tumor cells generally are much more resistant to mechanical disruption than are normal cells [4,5], and methods for the isolation of nuclei devised for use with normal tissues are often unsatisfactory when applied to tumor cells. A variety of methods for the isolation of tumor cell nuclei has been employed with varying degrees of success. This problem has recently been the subject of a review by Dounce [6]. This resistance of tumor cells to mechanical disruption makes the contamination of preparations of cell nuclei with unbroken cells a more difficult problem in the case of tumor tissues than in the case of normal tissues. If such preparations of tu-
From the Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014. Reprint requests should be addressed to Dr. Pilch. Surgery Branch, National Cancer Institute. National Institutes of Health, Bethesda, Maryland 20014.
Vol.
118,July1969
mor cell nuclei were to be injected in large doses into susceptible hosts, tumor growth at the site of inoculation might be expected to occur due to the transplantation of viable intact tumor cells. Such preparations, obviously, could not be used to study the ability of tumor cell nuclei to induce tumor isoimmunity. It was therefore necessary to devise a method for preparing intact tumor cell nuclei essentially free of viable whole cells. As early as 1939, Stoneburg [7] isolated nuclei from the rat carcinosarcoma 256 (Walker). These nuclei were prepared by treatment of the tissue with 5 per cent citric acid followed by pepsin digestion. Two years later, Marshak [8] isolated nuclei from the Lawrence-Gardner lymphoma, mouse sarcoma 180, and the Walker carcinoma 256 by treating minced tumor tissue with 5 per cent citric acid and grinding the tissue with a mortar and pestle. Nuclei could be isolated in a similar fashion with 5 per cent acetic acid but not with 5 per cent boric acid. Although a substantial percentage of the nuclei had attached cytoplasmic fragments, these could be eliminated by repeated differential centrifugation. The degree of contamination with intact cells is not stated. In 1942 Haven and Levy [9] isolated nuclei from the Walker carcinosarcoma by treatment with 2 per cent citric acid without pepsin digestion or grinding. The following year Dounce [IO] prepared nuclei from the Walker carcinosarcoma as well as from the rat hepatoma 31. The Walker tumor cell nuclei were isolated in 1.5 per cent citric acid, cellular disruption being accomplished in a Waring blender. The final pH of the mixture was about 3.0. In 1945 von Euler et al. [III prepared nuclei from the Jensen rat sarcoma by treatment with citric acid at pH 4 without mechani49
Pilch and Ketcham
The following year cal homogenization. Schneider [Ia] reported the isolation of nuclei from p-dimethylaminoazobenzene-induced rat hepatomas in alkaline water (pH 9.5) using a Potter-Elvehjem [I$] homogenizer. Price et al. [14] in 1949 prepared nuclei from azo-dye-induced rat hepatomas in a similar manner using hypertonic suerose (0.88 M) as the isolation medium. In 1950 Schneider and Hogeboom [IS] reported on the isolation of nuclei from mouse hepatoma 98/15 using the PotterElvehjem homogenizer and an isotonic (0.25 M) sucrose medium. Most recently Dounce et al. [I61 has described the isolation of Walker carcinoma nuclei in 0.44 M sucrose containing 0.005 M CaCl, using a new type of glass homogenizer. All of these methods, regardless of their relative merits in other regards, share one common shortcoming. They all provide preparations of nuclei containing significant numalthough the exbers of whole cells [S,li,l5,26], act ratios of intact cells to nuclei are rarely mentioned. Material
and Methods
The Cloudman S91 melanoma (Fig. 1) was carried in female CDBA/R hybrid mice. The K2 ascites tumor (Fig. 2) was carried in its ascitic form in both female CDBA/F, hybrid mice and female GH/HeN mice. Both of these K2 tumor lines were studied separately and each was treated as a separate and distinct tumor. 50
The tissue homogenizer* used was that described in 1955 by Dounce et al. [z,~s]. This homogenizer consists of a glass homogenizing vessel and two ball-type homogenizing pestles, identical in appearance, but providing different clearances. The loosely fitting pestle provides a clearance of 0.001 to 0.0015 inch at the circumference of the ball, whereas the tightly fitting pestle provides a clearance of 0.0005 inch or slightly less. This relatively tight fit makes it possible to disrupt a very high percentage of the cells without significant damage to the nuclei. Preparation of Nuclei from S91 Melanoma. Female CDBA/FI hybrid mice bearing the S91 melanoma in their hind limbs were killed by neck dislocation. Tumors were aseptically excised and necrotic portions removed. Tumor tissue was weighed, minced, and immediately chilled. From this point all operations were performed at 0 to 2”~. using chilled glassware and solutions. The isolation procedure employed is a modification of that described by Dounce [.%‘I. The tumor tissue was mixed with a volume of 0.25 M sucrose containing 0.005 M CaCh equal to five times the weight of the tumor tissue. An initial homogenization was performed using the loose fitting pestle. The homogenizer rested in a large rubber stopper in an ice bath [S]. About ten to fifteen strokes sufficed to effect initial homogenation. The pH was then lowered to 6.0 to 6.2 with citric acid and homogenization was resumed using
*Available from Kontes Glass Co., Vineland, New Jersey. The AmericanJournal
of Surgery
Tumor Cell Nuclei and Tumor lsoimmunity
the tight fitting pestle until microscopic
examination of the homogenate revealed only a very rare intact cell. Twenty-five to thirty strokes were required. The homogenate was filtered through four thicknesses of 60 grade cheese cloth, then through 4 thicknesses of 120 grade cheese cloth, and finally through one thickness of double-napped flannelette. The homogenate was then transferred into 40 ml. glass centrifuge tubes, underlayed with an equal volume of 0.34 M sucrose without CaCh and centrifuged in the cold at 1,800 x g for ten minutes. The supernate was discarded and the sediment resuspended in 0.25 M sucrose without CaCL using a few strokes of the loose fitting pestle. This suspension was transferred to centrifuge tubes, underlayed with an equal volume of 0.34 M sucrose, and centrifuged for ten minutes at 900 x g. Again the supernate was discarded, the sediment resuspended in 0.26 M sucrose with the aid of the loose fitting pestle, underlayed with 0.34 M sucrose, and centrifuged, this time at 700 x g for ten minutes. The supernate was discarded, and the nuclei resuspended in a convenient volume of 0.25 M sucrose. Preparation of Nuclei from KZ Aedtes Tumor. The procedure here was the same whether the tumor was obtained from CDBA/F1 mice or from GH/HeN mice. A&tic fluid was aseptically removed from tumor-bearing mice by abdominal paracentesis and centrifuged in the cold at 200 to 250 x g for five minutes. More vigorous centrifugation caused severe clumping of cells. The cells were then washed three times with Earles balanced salt solution, centrifuging at 200 x g for five minutes on each occasion. The packed tumor cells were weighed and suspended in 5 volumes of 1.5 per cent citric acid. The cells were then homogenized with the tight fitting pestle. No initial homogenization with the loose fitting pestle was required. The progress of the homogenization was followed microscopically and terminated when only a very rare intact cell could be found. From forty-five to iifty strokes were required to effect complete disruption of cells. The pH of the homogenate varied between 2.9 and 3.2. No filtration was necessary since the ascites tumor contained no fibrous elements. The homogenate was centrifuged for ten minutes at 700 x g, the supernate discarded, and the sediment suspended in 0.01 M citric acid with a few passes of the loose fitting pestle. The suspension was then centrifuged for ten minutes at 560 x g. The nuclei were washed two additional times with 0.01 M citric acid, centrifuging at 400 x g for ten minutes initially and then at 250 x g for ten minutes. The supernate was discarded on each occasion. The nuclei were then suspended in a convenient volume ef 0.25 M sucrose. Vol.118,July1969
Results When S91 melanoma was homogenized in 0.44 M or 0.88 M sucrose at pH 6 to 6.2 both with and without calcium, poor nuclear preparations were obtained. There was poor separation of cytoplasm from the nuclei and numerous intact cells (15 per cent or more) persisted. When as many as fifty passes of the tight fitting pestle were used, the percentage of intact cells decreased but considerable destruction of nuclei occurred. S91 melanoma nuclei isolated in 0.25 M sucrose without calcium gave evidence of considerable damage. This is consistent with the observation that calcium increases the resistance of nuclei to mechanical disruption [ 1-4,161. When S91 melanoma nuclei were prepared in 0.25 sucrose with 0.005 M CaCl, by the method described, excellent results were obtained. The nuclei were remarkably uniform in size and shape with excellent preservation of morphology and were generally free of adherent cytoplasm. Such a preparation is illustrated in Figures 3 and 4. Whole cell contamination of these preparations was less than 1 per cent. It was then necessary to determine whether these preparations of nuclei were sufficiently free of viable whole cells that tumor transplantation would not occur when large doses were injected by various routes into highly susceptible hosts. One million S91 melanoma nuclei in 0.5 or 0.25 cc. of 0.25 M sucrose were injected into each of four groups of six week old female CDBA/F, mice. Each group consisted of twelve mice. One group was inoculated intraperitoneally, one intravenously (tail vein), one intra-arterially (tail artery), and one intramuscularly (thigh). Two nuclear preparations were used : nuclei isolated according to the method described containing less than 1 per cent whole cells, and nuclei prepared in 0.44 M sucrose containing approximately 15 per cent intact cells. The results are tabulated in Table I. It is apparent that when significant contamination with whole cells occurred, tumors were transplanted. In the mice inoculated intra-arterially and intravenously, tumors arose in the lungs, whereas with intraperitoneal and intramuscular inoculation, tumors arose locally. However, when substantially whole cell free preparations of nuclei were used, no tumors arose regardless of the 51
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4
3
Nuclei of S91 melanoma prepared by the technic described. Three nuclei have undergone auFig. 3. cytoplasm, and nuclear shape and morphology are well tolysis. The nuclei are quite free of adherent preserved. There is essentially no contamination with whole cells. (Methyl green-pyronin [PappenheimSaathoff] stain; original magnification X1010.) A high power of the same preparation of S91 melanoma nuclei shown in Figure 3. Note the chroFig. 4. matin detail, the intact nuclear membranes, and the well preserved nucleoli. (Methyl green-pyronin [Pappenheim-Saathoff] stain; original magnification X 1650.)
TABLE
I
Frequency Melanoma
of Tumor Transplantation Observed in Female CDi3A/FI Nuclei Prepared by Different Methods Per cent of Whole Cells
Method of Isolation of Nuclei 0.25 M Sucrose + 0.005M CaClz (method scribed in text)
0.44 M sucrose
de-
106 S91
No. of Mice in Group
No. of Mice Developing Tumors
Intraperitoneal Intra-arterial Intravenous Intramuscular
12 12 12 12
0 0 0 0
15
lntraperitoneal Intra-arterial Intravenous Intramuscular
12 12 12 12
11 10 11 9
route of administration. It is evident that, although some intact cells ( were transplanted, the number of viable intact cells remaining was insufficient to cause tumor growth in the recipient hosts. In fact very few (2 to 5 per cent) of the occasional whole cells present in these preparations were able to exclude trypan blue. Therefore, most of the intact cells transplanted were probably not viable. To determine whether this exposure to large numbers of tumor cell nuclei had conferred upon the recipient animals any degree of antitumor immunity, all the animals receiving the whole cell free preparations of nuclei were inoculated intramuscularly six weeks later 52
Route of Injection
Mice Receiving
with 30,000 viable S91 melanoma cells.* A control group of thirty stock mice was also inoculated with 30,000 melanoma cells at this time. The results appear in Table II. Obviously, prior inoculation with tumor cell nuclei had not altered the resistance of these animals to transplants of this particular tumor. The K2 ascites tumor cells proved to be much more resistant to mechanical disruption *Prepared by passing S91 melanoma tissue suspended in normal saline through a Snell wtosieve [IT] and counting the cell suspension thus prepared in a hemocytometer. Viability was determined bg the exclusion of 0.5 per cent tmpan blue dge. The AmericanJournal
of Surgery
Tumor Cell Nuclei and Tumor lsoimmunity
TABLE
II
Frequency of Tumor Takes in Female CD13A/F1 Mice Inoculated with 30,000 S91 Melanoma Cells Six Weeks after Receiving lo6 S91 Melanoma Nuclei
Routeof Administration of Nuclei ___ lntraperitoneal Intra-arterial Intravenous Intramuscular Control (no nuclei)
No. of Mice in Group ~~._ 12 12 12 12 30
No. of Mice Developing Tumors 11 10 12 12 29
ihan the S91 melanoma cells. All attempts at the isolation of nuclei from this tumor in sucrose media failed regardless of the concentration of sucrose employed or the presence or absence of calcium. Even with over fifty strokes of the tight fitting pestle, over 35 per cent of the cells remained intact and the remaining nuclei retained cytoplasmic attachments. When these cells were homogenized in distilled water acidified to pH 3.8 with 1.0 M citric acid, only an 83 per cent whole cell free preparation of nuclei could be obtained. It was necessary to resort to homogenization in diluted citric acid to effect complete disruption of cells. Citric acid concentrations between 1.5 and 4 per cent were equally effective. With 4 per cent citric acid the pH of the homogenate
with lower approximately 2.5. However, concentrations of citric acid, pH’s of between 2.9 and 3.2 could be utilized. There was no difference between the behavior of the K2 ascites tumor maintained in female CDBA/F1 hybrid mice from that maintained in female C,H/HeN mice. Nuclei of the K2 tumor prepared from the C,H/HeN strain by this method described are illustrated in Figures 5 and 6. Again the nuclei are round, uniform, morphologically intact, and quite free of adherent cytoplasm. The percentage of whole cell contamination in these preparations was very low, varying between 1 and 3 per cent. As in the case of the S91 melanoma nuclei, the ability of the various K2 nuclear preparations to transplant tumor was examined. The results of these experiments appear in Table III. It is obvious that, when whole cell contamination was substantial, tumor was transplanted whereas when preparations of nuclei substantially free of viable intact cells were injected, no tumors arose. Of the whole cells present in these preparations (10,000 to 30,000), less than 2 per cent were viable (able to exclude trypan blue). The numbers of viable intact cells present in these inocula were obviously insufficient to cause tumor transplantation. Again, the mice that had received the prepa-
was
Nuclei of K2 ascites tumor carried in a female CsH/HeN mouse prepared by the method described. Fig. 5. The nuclei are quite free of whole cell contamination. Nuclear shape and morphology are well preserved. (Methyl green-pyronin [Pappenheim-Saathofl stain; original magnification X380.) Fig. 6. A higher power of the same preparation are quite free of adherent cytoplasm. Chromatin Saathoffj stain; original magnification x1010.)
Vol.118,July1969
of K2 ascites tumor nuclei shown in Figure 5. The nuclei detail is excellent. [Methyl green-pyronin [Pappenheim-
53
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TABLE III
Frequency of Tumor Transplantation Observed in Female CDBA/FI and Female C3H/HeN Mice Receiving 106KnAscites Tumor Nuclei Prepared by Different Methods
Melhodof Isolation of Nuclei 1.5% Citric acid (method described in text)
Per cent of Strain of Tumor WholeCells Cellsof Origin 3
3
0.25 M sucrose + 0.005 M CaClz
CDBA/Fl
CsH/HeN
QH/HeN
CDBA/Fl
CDBA/F,
17
&H/HeN
&H/HeN
15
CDBA/Fl
CDBA/Fl
33
&H/HeN
CaH/HeN
35
CDBA/F,
CDBA/Fl
rations of whole cell free K2 nuclei were inoculated six weeks later with 30,000 viable tumor cells from the K2 tumor of the isologous strain.* On this occasion, since the tumor involved was an ascites tumor, one half the animals in each group were inoculated intramuscalarly and the other half intraperitoneally. Again control groups of untreated mice were similarly inoculated with 30,000 tumor cells. The results are outlined in Table IV. No increase in host resistance to a subsequent *Tumor cells were sedimented from ~citic fluid, washed three time8 in Earles balanced salt solution, resuspended in BSS, counted in a hemocytometer, and viability determined by the exclusion of 0.5 per cent trgpan blue. 54
CsH/HeN
CDBA/F,
4% Citric acid
Distilled water brought to pH 3.8 with 1.0 M citric acid
CaH/HeN
Strain of Recipient Mice
Routeof Injection
NO.of Mice in Groups
No.of Mice Developing Tumors
lntraperitoneal Intra-arterial Intravenous Intramuscular lntraperitoneal Intra-arterial Intravenous Intramuscular
20
0
20 20 20 20 20 20 20
0 0 0 0 0 0 0
lntraperitoneal Intra-arterial Intravenous Intramuscular lntraperitoneal Intra-arterial Intravenous Intramuscular
20 20 20 20 20 20 20 20
0 0 0 0 0 0 0 0
lntraperitoneal Intra-arterial Intravenous Intramuscular lntraperitoneal Intra-arterial Intravenous Intramuscular
20 20 20 20 20 20 20 20
20 18 19 19 20 19 18 17
Intraperitoneal Intra-arterial Intravenous Intramuscular lntraperitoneal Intra-arterial Intravenous Intramuscular
20 20 20 20 20 20 20 20
20 20 20 20 20 20 20 20
tumor transplant could be shown to have been induced by prior treatment with tumor cell nuclei.
Comments The technical problem which presented itself in this study was to obtain nuclei so free of viable intact. cells that they would be incapable of causing tumor transplantation even when injected into susceptible hosts in large numbers. Since most tumor cells are more resistant to mechanical rupture than cells of normal mammalian tissues, and since preparations substantially free of intact cells were desired, it was obvious that more vigorous shearing forces would have to be applied to the The American
Journal of Surgery
Tumor
TABLE
IV
Frequency
of Tumor Takes in Female CDBA/h
K2 Tumor Cells Six Weeks after Receiving
Experimental
Donor Strain of
Group
WholeCells
Route of Administration
Cell Nuclei and Tumor
and Female C3H/HeN
Mice Inoculated
lsoimmunity
with 30,000
UP K2 Nuclei
No. of Mice in Group
No. of Mice Developing Tumors
Cells TumorCells lntraperitoneally Intramuscularly lntraperitoneally Intramuscularly Tumor
Tumor
lntraperitoneal Intra-arterial Intravenous Intramuscular
10 10 10 10
10 10 10 10
10 10 10 10
10 9 10
of Nuclei
Tumor
9
1.5% Citric acid nuclei prepared from C3H/HeN strain
C3H/HeN
1.5% Citric acid nuclei prepared from CDBA/Fl strain
CDBA/Fl
lntraperitoneal Intra-arterial Intravenous Intramuscular
10 10 10 10
10 10 10 10
10 10 10 10
10 10 10 10
4% Citric acid nuclei prepared from C3H/HeN strain
C3H/HeN
lntraperitoneal Intra-arterial Intravenous Intramuscular
10 10 10 10
10 10 10 10
10 10 10 10
10 9 10 10
4% Citric acid nuclei prepared from CDBA/F1 strain
CDBA/F1
lntraperitoneal I ntra-arterial Intravenous Intramuscular
10 10 10 10
10 10 10 10
10 10 10 10
10 10 10 9
Control
C3H/HeN
15
15
15
14
Control
CDBA/F,
15
15
15
14
cells, and applied in such a way as to avoid rupturing significant numbers of nuclei. This was accomplished by using the Dounce homogenizer and by lowering the pH of the homogenizing media with citric acid. The very small clearance of the tight pestle (0.0005 inch or less) made it possible to disrupt a very high percentage of cells and yet cause little damage to the nuclei. If such a tight clearance were applied with a cylindrical rather than a ball type pestle (such as the cylindrical pestles of the Potter-Elvehjem types of homogenizers), the friction resulting would make it almost impossible to move the plunger and create intense local heating. In the Dounce homogenizer this clearance is along the circumference of a circle (the widest diameter of the ball), and, since the shearing force is applied only along the circumference of this circle, friction is reduced to a minimum. The pestle therefore moves freely and no appreciable local heating occurs [Z]. There was a considerable difference in the resistance to mechanical disruption between the two tumors studied, and, therefore different homogenizing media were necessary for each tumor. Vol.118.July1969
It is well established that cells resistant to breakage in sucrose or other media at pH values near neutrality can be broken in citric acid media at low pH’s. It is also known that by lowering the pH of the medium with citric acid or by adding calcium chloride to the medium the fragility of nuclei can be reduced [8-10,28-SO], thereby permitting the use of more vigorous mechanical shearing forces. By the addition of 0.005 M CaC12 to the sucrose medium and by lowering the pH to between 6.02 and 6.2 with citric acid, it was possible to obtain essentially complete disruption of the S91 melanoma cells without rupturing significant numbers of nuclei. By using diluted citric acid at pH 2.8 to 3.2 as a medium, it was possible to obtain substantially whole cell free preparations of K2 nuclei without significant nuclear damage. Nuclei prepared in this manner could be inoculated in large numbers into susceptible hosts without causing tumor transplantation. The possibility that nuclei might contain tumor antigens was first suggested in 1949 by Arnesen, Goldsmith, and Dulaney 121 I. They isolated nuclei from the spleens of nor55
Pilch and Ketcham
ma1 and leukemic AKm mice by a modification of the Dounce [16] procedure using M/125 citric acid in 8.5 per cent sucrose. These nuclei were found to be poor antigens since only low antibody titers to the nuclei were produced upon immunizing rabbits with these preparations. In addition, the antibodies formed crossreacted heavily with cytoplasm obtained from both normal and leukemic spleen cells. However, it should be pointed out that the nuclei used in this study were probably significantly contaminated with whole cells and cytoplasmic fragments. The marked cross-reactivity observed in this study is, therefore, not at all surprising. Nuclei prepared by the methods herein described might provide much more nearly pure nuclear antigen preparations. More recently, Rapaport et al. [22] have studied the subcellular fractions of human leukocytes as sources of transplantation antigens, and Monaco, Wood, and Russell [23] have examined the subcellular fractions of mouse spleen cells for the presence of homograft antigens. In both these studies the nuclear fractions were found to be essentially devoid of transplantation antigens. However, studies investigating the ability of subcellular fractions of tumor cells to induce tumor isoimmunity in syngeneic systems are lacking. In the studies reported herein, no tumor isoimmunity could be demonstrated in animals who had received a single inoculation of l,OOO,000 tumor cell nuclei six weeks prior to challenge with 30,000 viable tumor cells. However, these animals received only a single injection without the use of adjuvant materials. This was true in’ both the tumor-host systems studied. It is also possible that with a prolonged course of multiple injections, possibly employing an adjuvant material, some degree of tumor isoimmunity might have been produced. It is also possible that the two tumors studied were not particularly antigenic and that if additional tumor-host systems were examined, some antigenic activity within their nuclei might be observed. However, it seems likely that tumor-specific antigens are not present in the nuclei of the two tumors studied. Since tumor antigens may be similar to or a special class of transplantation antigens, the results of the present study are in agreement with the observations of Rapaport et al. [22] and Monaco, Wood, and Russell [23] who were 56
unable to demonstrate transplantation antigens within mammalian cell nuclei. These antigens must, therefore, reside elsewhere in the cell, either on the cell membrane or within the cytoplasm. Studies are currently underway in this laboratory to examine tumor cell membranes and cytoplasmic constituents for the presence of tumor-specific antigens. Summary Methods were devised for the isolation of tumor cell nuclei substantially free of contamination by viable whole cells. Such preparations were found to be incapable of producing tumor transplantation when injected in large numbers and by different routes into susceptible hosts. The ability of such nuclei to induce tumor isoimmunity was studied in two different tumor-host systems. Nuclei prepared in this manner failed to induce tumor isoimmunity when injected into syngeneic hosts. In both cases, mice receiving lo6 tumor cell nuclei evidenced no increase in resistance to subsequent tumor transplants of 30,000 viable whole cells from the same tumor inoculated six weeks later. References 1.
2.
3.
4. 5.
6.
SCHNEIDER, W. C. and HOGEBOOM, G. H. Cytochemical studies of mammalian tissues. The isolation of cell components by differential centrifugation: a review. Cancer Res., 11: 1, 1951. DOUNCE,A. L. The isolation and composition of cell nuclei and nucleoli. In: The Nucleic Acids. Edited by Chargaff, E. and Davidson, J. N. New York, 1955. New York Academic Press. ALLFREY,V. The isolation of subcellular components. In: The Cell. Edited by Brachet, J. and Mirsky, A. E. New York, 1959. New York Academic Press. DOUNCE,A. L. Enzyme systems of isolated cell nuclei. Ann. New York Acad. Sci., 50: 982, 1950. VON EULER, H., FISHER, I., HASSELQUIST, H., and JAARMA, M. Stability of isolated cell nuclei in different media: enzyme systems in nuclei. Ark. Kemi, Mineralocri. I I and Geologi, 12: 1, 1946. ’ DOUNCE,A. L. The isolation of nuclei from tumor cells. Exper. Cell Res., Suppl., 9: 126,1963.
7. 8.
9.
STONEBURG, C. A. Lipids of cell nuclei. J. Biol. Chem., 129: 189, 1939. MARSHAK, A. Psz uptake by nuclei. J. Gen. Physiol., 25, 275, 1941. HAVEN, F. L. and Lm, S. R. Phospholipids The American Journal of Surgery
Tumor Cell Nuclei and Tumor lsoimmunity
10. 11.
12.
13. 14.
15.
16.
of tumor cell and nuclei. Cancer Res., 2: 797,1942. DOUNCE, A. L. The desoxyribonucleic acid content of isolated nuclei of tumor cells. J. Biol. Chem., 147: 235, 1943. VON EULW, H., HANN, H., HASSELQUIST,H., JAARMA, M., and LUNDIN, M. Studies on the isolation and characterization of cell nuclei from calf thymus, hog liver and rat Jensen sarcoma. Svensk Kemisk Tidshrift, 57: 217,1945. SCHNEIDER,W. C. Intracellular distribution of enzymes. II. The distribution of succinic dehydrogenase, cytochrome oxidase adenosine triphosphatase and phosphorus compounds in normal rat liver and-in rat hepatomas. Cancer Res.. 6: 685. 1946. POTTER,V. R. and EJ&EHJE~, C. A. A modified method for the study of tissue oxidations. J. Biol. Chem., 144; 495, 1936. PRICE, J. M.. MILLER. J. A.. MILLER. E. C.. and WEBE~, G. M. Studies’ on the intracel: lular composition of liver and liver tumor from rats fed 4-dimethylaminoazobenzene. Cancer Res., 9: 96, 1949. SCHNEIDER,W. C. and HOGEBOOM, G. H. Intracellular distribution of enzymes. VI. The distribution of succinoxidose and cytochrome oxidose activities in normal mouse liver and in mouse hepatoma. J. Nat. Cancer inst., 10: 969, 1950. DOIJNCE,A. L., WITTER, R. F., MONTY, K. J., PATE, S., and COTTONE,M. A. A method
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17.
18. 19.
20.
21.
22.
23.
for isolating intact mitochondria and nuclei from the same homogenate, and the influence of mitochondrial destruction on the properties of cell nuclei. J. Biophys. & Biochem. Cwtol.. 1: 139. 1955. SNELL, C. D: C&sieve permitting sterile preparation of suspensions of tumor cells for transplantation. J. Nat. Cancer Inst., 13: 1511,1953. DOUNCE, A. L. Further studies on isolated cell nuclei of normal rat liver. J. Biol. Chem., 151: 221,1943. MIRSKY, A. E. and POLLISTER,A. W. Chromosin, a desoxyribose nucleoprotein complex of the cell nucleus. J. Gen. Physiol., 30: 117,1946. SCHNEIDER,R. M. and PETERMAN,M. L. Nuclei from normal and leukemic mouse spleen. I. The isolation of nuclei in neutral medium. Cancer Res., 10: 751, 1950. ARNESEN,K., GOLDSMITH,Y., and DULANEY, A. D. Antigenic properties of nuclei segregated from spleens of normal and leukemic mice. Cancer Res., 9: 669, 1949. RAPAPORT,F. T., LAWRENCE,H. S., CONVERSE, J. M., and MULHOLLAND,J. H. Leukocyte fractions as skin homograft antigens in man. S. Forum, 14: 146,1963. MONACO,A. P., WOOD,M. L., and RUSSELL,P. S. A simple method for the preparation of cell free transplantation antigens. Physical and biological properties. S. Forum, 15: 133, 1964.
57
The purpose of the present study was to isolate tumor cell nuclei so free of contamination with intact cells that no tumor transplantation would occur even when very large numbers of nuclei were administered to highly susceptible hosts, and then to use such nuclear preparations as antigens in studies of tumor isoimmunity. Two tumors were studied: the Cloudman S91 melanoma and the Krebs K2 ascites tumor. A number of acceptable methods were available for the isolation of cell nuclei from normal mammalian tissues. The substantial literature on these methods and their applications in biologic research has been comprehensively reviewed by Schneider and Hogeboom [I], Dounce [Z], and Allfrey [8]. These methods all involve the controlled mechanical disruption of cells in an appropriate suspension medium and the subsequent segregation of the nuclear fraction by differential centrifugation, filtration, countercurrent distribution, or floatation. However, tumor cells generally are much more resistant to mechanical disruption than are normal cells [4,5], and methods for the isolation of nuclei devised for use with normal tissues are often unsatisfactory when applied to tumor cells. A variety of methods for the isolation of tumor cell nuclei has been employed with varying degrees of success. This problem has recently been the subject of a review by Dounce [6]. This resistance of tumor cells to mechanical disruption makes the contamination of preparations of cell nuclei with unbroken cells a more difficult problem in the case of tumor tissues than in the case of normal tissues. If such preparations of tu-
From the Surgery Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014. Reprint requests should be addressed to Dr. Pilch. Surgery Branch, National Cancer Institute. National Institutes of Health, Bethesda, Maryland 20014.
Vol.
118,July1969
mor cell nuclei were to be injected in large doses into susceptible hosts, tumor growth at the site of inoculation might be expected to occur due to the transplantation of viable intact tumor cells. Such preparations, obviously, could not be used to study the ability of tumor cell nuclei to induce tumor isoimmunity. It was therefore necessary to devise a method for preparing intact tumor cell nuclei essentially free of viable whole cells. As early as 1939, Stoneburg [7] isolated nuclei from the rat carcinosarcoma 256 (Walker). These nuclei were prepared by treatment of the tissue with 5 per cent citric acid followed by pepsin digestion. Two years later, Marshak [8] isolated nuclei from the Lawrence-Gardner lymphoma, mouse sarcoma 180, and the Walker carcinoma 256 by treating minced tumor tissue with 5 per cent citric acid and grinding the tissue with a mortar and pestle. Nuclei could be isolated in a similar fashion with 5 per cent acetic acid but not with 5 per cent boric acid. Although a substantial percentage of the nuclei had attached cytoplasmic fragments, these could be eliminated by repeated differential centrifugation. The degree of contamination with intact cells is not stated. In 1942 Haven and Levy [9] isolated nuclei from the Walker carcinosarcoma by treatment with 2 per cent citric acid without pepsin digestion or grinding. The following year Dounce [IO] prepared nuclei from the Walker carcinosarcoma as well as from the rat hepatoma 31. The Walker tumor cell nuclei were isolated in 1.5 per cent citric acid, cellular disruption being accomplished in a Waring blender. The final pH of the mixture was about 3.0. In 1945 von Euler et al. [III prepared nuclei from the Jensen rat sarcoma by treatment with citric acid at pH 4 without mechani49
Pilch and Ketcham
The following year cal homogenization. Schneider [Ia] reported the isolation of nuclei from p-dimethylaminoazobenzene-induced rat hepatomas in alkaline water (pH 9.5) using a Potter-Elvehjem [I$] homogenizer. Price et al. [14] in 1949 prepared nuclei from azo-dye-induced rat hepatomas in a similar manner using hypertonic suerose (0.88 M) as the isolation medium. In 1950 Schneider and Hogeboom [IS] reported on the isolation of nuclei from mouse hepatoma 98/15 using the PotterElvehjem homogenizer and an isotonic (0.25 M) sucrose medium. Most recently Dounce et al. [I61 has described the isolation of Walker carcinoma nuclei in 0.44 M sucrose containing 0.005 M CaCl, using a new type of glass homogenizer. All of these methods, regardless of their relative merits in other regards, share one common shortcoming. They all provide preparations of nuclei containing significant numalthough the exbers of whole cells [S,li,l5,26], act ratios of intact cells to nuclei are rarely mentioned. Material
and Methods
The Cloudman S91 melanoma (Fig. 1) was carried in female CDBA/R hybrid mice. The K2 ascites tumor (Fig. 2) was carried in its ascitic form in both female CDBA/F, hybrid mice and female GH/HeN mice. Both of these K2 tumor lines were studied separately and each was treated as a separate and distinct tumor. 50
The tissue homogenizer* used was that described in 1955 by Dounce et al. [z,~s]. This homogenizer consists of a glass homogenizing vessel and two ball-type homogenizing pestles, identical in appearance, but providing different clearances. The loosely fitting pestle provides a clearance of 0.001 to 0.0015 inch at the circumference of the ball, whereas the tightly fitting pestle provides a clearance of 0.0005 inch or slightly less. This relatively tight fit makes it possible to disrupt a very high percentage of the cells without significant damage to the nuclei. Preparation of Nuclei from S91 Melanoma. Female CDBA/FI hybrid mice bearing the S91 melanoma in their hind limbs were killed by neck dislocation. Tumors were aseptically excised and necrotic portions removed. Tumor tissue was weighed, minced, and immediately chilled. From this point all operations were performed at 0 to 2”~. using chilled glassware and solutions. The isolation procedure employed is a modification of that described by Dounce [.%‘I. The tumor tissue was mixed with a volume of 0.25 M sucrose containing 0.005 M CaCh equal to five times the weight of the tumor tissue. An initial homogenization was performed using the loose fitting pestle. The homogenizer rested in a large rubber stopper in an ice bath [S]. About ten to fifteen strokes sufficed to effect initial homogenation. The pH was then lowered to 6.0 to 6.2 with citric acid and homogenization was resumed using
*Available from Kontes Glass Co., Vineland, New Jersey. The AmericanJournal
of Surgery
Tumor Cell Nuclei and Tumor lsoimmunity
the tight fitting pestle until microscopic
examination of the homogenate revealed only a very rare intact cell. Twenty-five to thirty strokes were required. The homogenate was filtered through four thicknesses of 60 grade cheese cloth, then through 4 thicknesses of 120 grade cheese cloth, and finally through one thickness of double-napped flannelette. The homogenate was then transferred into 40 ml. glass centrifuge tubes, underlayed with an equal volume of 0.34 M sucrose without CaCh and centrifuged in the cold at 1,800 x g for ten minutes. The supernate was discarded and the sediment resuspended in 0.25 M sucrose without CaCL using a few strokes of the loose fitting pestle. This suspension was transferred to centrifuge tubes, underlayed with an equal volume of 0.34 M sucrose, and centrifuged for ten minutes at 900 x g. Again the supernate was discarded, the sediment resuspended in 0.26 M sucrose with the aid of the loose fitting pestle, underlayed with 0.34 M sucrose, and centrifuged, this time at 700 x g for ten minutes. The supernate was discarded, and the nuclei resuspended in a convenient volume of 0.25 M sucrose. Preparation of Nuclei from KZ Aedtes Tumor. The procedure here was the same whether the tumor was obtained from CDBA/F1 mice or from GH/HeN mice. A&tic fluid was aseptically removed from tumor-bearing mice by abdominal paracentesis and centrifuged in the cold at 200 to 250 x g for five minutes. More vigorous centrifugation caused severe clumping of cells. The cells were then washed three times with Earles balanced salt solution, centrifuging at 200 x g for five minutes on each occasion. The packed tumor cells were weighed and suspended in 5 volumes of 1.5 per cent citric acid. The cells were then homogenized with the tight fitting pestle. No initial homogenization with the loose fitting pestle was required. The progress of the homogenization was followed microscopically and terminated when only a very rare intact cell could be found. From forty-five to iifty strokes were required to effect complete disruption of cells. The pH of the homogenate varied between 2.9 and 3.2. No filtration was necessary since the ascites tumor contained no fibrous elements. The homogenate was centrifuged for ten minutes at 700 x g, the supernate discarded, and the sediment suspended in 0.01 M citric acid with a few passes of the loose fitting pestle. The suspension was then centrifuged for ten minutes at 560 x g. The nuclei were washed two additional times with 0.01 M citric acid, centrifuging at 400 x g for ten minutes initially and then at 250 x g for ten minutes. The supernate was discarded on each occasion. The nuclei were then suspended in a convenient volume ef 0.25 M sucrose. Vol.118,July1969
Results When S91 melanoma was homogenized in 0.44 M or 0.88 M sucrose at pH 6 to 6.2 both with and without calcium, poor nuclear preparations were obtained. There was poor separation of cytoplasm from the nuclei and numerous intact cells (15 per cent or more) persisted. When as many as fifty passes of the tight fitting pestle were used, the percentage of intact cells decreased but considerable destruction of nuclei occurred. S91 melanoma nuclei isolated in 0.25 M sucrose without calcium gave evidence of considerable damage. This is consistent with the observation that calcium increases the resistance of nuclei to mechanical disruption [ 1-4,161. When S91 melanoma nuclei were prepared in 0.25 sucrose with 0.005 M CaCl, by the method described, excellent results were obtained. The nuclei were remarkably uniform in size and shape with excellent preservation of morphology and were generally free of adherent cytoplasm. Such a preparation is illustrated in Figures 3 and 4. Whole cell contamination of these preparations was less than 1 per cent. It was then necessary to determine whether these preparations of nuclei were sufficiently free of viable whole cells that tumor transplantation would not occur when large doses were injected by various routes into highly susceptible hosts. One million S91 melanoma nuclei in 0.5 or 0.25 cc. of 0.25 M sucrose were injected into each of four groups of six week old female CDBA/F, mice. Each group consisted of twelve mice. One group was inoculated intraperitoneally, one intravenously (tail vein), one intra-arterially (tail artery), and one intramuscularly (thigh). Two nuclear preparations were used : nuclei isolated according to the method described containing less than 1 per cent whole cells, and nuclei prepared in 0.44 M sucrose containing approximately 15 per cent intact cells. The results are tabulated in Table I. It is apparent that when significant contamination with whole cells occurred, tumors were transplanted. In the mice inoculated intra-arterially and intravenously, tumors arose in the lungs, whereas with intraperitoneal and intramuscular inoculation, tumors arose locally. However, when substantially whole cell free preparations of nuclei were used, no tumors arose regardless of the 51
Pilch and Ketcham
4
3
Nuclei of S91 melanoma prepared by the technic described. Three nuclei have undergone auFig. 3. cytoplasm, and nuclear shape and morphology are well tolysis. The nuclei are quite free of adherent preserved. There is essentially no contamination with whole cells. (Methyl green-pyronin [PappenheimSaathoff] stain; original magnification X1010.) A high power of the same preparation of S91 melanoma nuclei shown in Figure 3. Note the chroFig. 4. matin detail, the intact nuclear membranes, and the well preserved nucleoli. (Methyl green-pyronin [Pappenheim-Saathoff] stain; original magnification X 1650.)
TABLE
I
Frequency Melanoma
of Tumor Transplantation Observed in Female CDi3A/FI Nuclei Prepared by Different Methods Per cent of Whole Cells
Method of Isolation of Nuclei 0.25 M Sucrose + 0.005M CaClz (method scribed in text)
0.44 M sucrose
de-
106 S91
No. of Mice in Group
No. of Mice Developing Tumors
Intraperitoneal Intra-arterial Intravenous Intramuscular
12 12 12 12
0 0 0 0
15
lntraperitoneal Intra-arterial Intravenous Intramuscular
12 12 12 12
11 10 11 9
route of administration. It is evident that, although some intact cells (
Route of Injection
Mice Receiving
with 30,000 viable S91 melanoma cells.* A control group of thirty stock mice was also inoculated with 30,000 melanoma cells at this time. The results appear in Table II. Obviously, prior inoculation with tumor cell nuclei had not altered the resistance of these animals to transplants of this particular tumor. The K2 ascites tumor cells proved to be much more resistant to mechanical disruption *Prepared by passing S91 melanoma tissue suspended in normal saline through a Snell wtosieve [IT] and counting the cell suspension thus prepared in a hemocytometer. Viability was determined bg the exclusion of 0.5 per cent tmpan blue dge. The AmericanJournal
of Surgery
Tumor Cell Nuclei and Tumor lsoimmunity
TABLE
II
Frequency of Tumor Takes in Female CD13A/F1 Mice Inoculated with 30,000 S91 Melanoma Cells Six Weeks after Receiving lo6 S91 Melanoma Nuclei
Routeof Administration of Nuclei ___ lntraperitoneal Intra-arterial Intravenous Intramuscular Control (no nuclei)
No. of Mice in Group ~~._ 12 12 12 12 30
No. of Mice Developing Tumors 11 10 12 12 29
ihan the S91 melanoma cells. All attempts at the isolation of nuclei from this tumor in sucrose media failed regardless of the concentration of sucrose employed or the presence or absence of calcium. Even with over fifty strokes of the tight fitting pestle, over 35 per cent of the cells remained intact and the remaining nuclei retained cytoplasmic attachments. When these cells were homogenized in distilled water acidified to pH 3.8 with 1.0 M citric acid, only an 83 per cent whole cell free preparation of nuclei could be obtained. It was necessary to resort to homogenization in diluted citric acid to effect complete disruption of cells. Citric acid concentrations between 1.5 and 4 per cent were equally effective. With 4 per cent citric acid the pH of the homogenate
with lower approximately 2.5. However, concentrations of citric acid, pH’s of between 2.9 and 3.2 could be utilized. There was no difference between the behavior of the K2 ascites tumor maintained in female CDBA/F1 hybrid mice from that maintained in female C,H/HeN mice. Nuclei of the K2 tumor prepared from the C,H/HeN strain by this method described are illustrated in Figures 5 and 6. Again the nuclei are round, uniform, morphologically intact, and quite free of adherent cytoplasm. The percentage of whole cell contamination in these preparations was very low, varying between 1 and 3 per cent. As in the case of the S91 melanoma nuclei, the ability of the various K2 nuclear preparations to transplant tumor was examined. The results of these experiments appear in Table III. It is obvious that, when whole cell contamination was substantial, tumor was transplanted whereas when preparations of nuclei substantially free of viable intact cells were injected, no tumors arose. Of the whole cells present in these preparations (10,000 to 30,000), less than 2 per cent were viable (able to exclude trypan blue). The numbers of viable intact cells present in these inocula were obviously insufficient to cause tumor transplantation. Again, the mice that had received the prepa-
was
Nuclei of K2 ascites tumor carried in a female CsH/HeN mouse prepared by the method described. Fig. 5. The nuclei are quite free of whole cell contamination. Nuclear shape and morphology are well preserved. (Methyl green-pyronin [Pappenheim-Saathofl stain; original magnification X380.) Fig. 6. A higher power of the same preparation are quite free of adherent cytoplasm. Chromatin Saathoffj stain; original magnification x1010.)
Vol.118,July1969
of K2 ascites tumor nuclei shown in Figure 5. The nuclei detail is excellent. [Methyl green-pyronin [Pappenheim-
53
Pilch and Ketcham
TABLE III
Frequency of Tumor Transplantation Observed in Female CDBA/FI and Female C3H/HeN Mice Receiving 106KnAscites Tumor Nuclei Prepared by Different Methods
Melhodof Isolation of Nuclei 1.5% Citric acid (method described in text)
Per cent of Strain of Tumor WholeCells Cellsof Origin 3
3
0.25 M sucrose + 0.005 M CaClz
CDBA/Fl
CsH/HeN
QH/HeN
CDBA/Fl
CDBA/F,
17
&H/HeN
&H/HeN
15
CDBA/Fl
CDBA/Fl
33
&H/HeN
CaH/HeN
35
CDBA/F,
CDBA/Fl
rations of whole cell free K2 nuclei were inoculated six weeks later with 30,000 viable tumor cells from the K2 tumor of the isologous strain.* On this occasion, since the tumor involved was an ascites tumor, one half the animals in each group were inoculated intramuscalarly and the other half intraperitoneally. Again control groups of untreated mice were similarly inoculated with 30,000 tumor cells. The results are outlined in Table IV. No increase in host resistance to a subsequent *Tumor cells were sedimented from ~citic fluid, washed three time8 in Earles balanced salt solution, resuspended in BSS, counted in a hemocytometer, and viability determined by the exclusion of 0.5 per cent trgpan blue. 54
CsH/HeN
CDBA/F,
4% Citric acid
Distilled water brought to pH 3.8 with 1.0 M citric acid
CaH/HeN
Strain of Recipient Mice
Routeof Injection
NO.of Mice in Groups
No.of Mice Developing Tumors
lntraperitoneal Intra-arterial Intravenous Intramuscular lntraperitoneal Intra-arterial Intravenous Intramuscular
20
0
20 20 20 20 20 20 20
0 0 0 0 0 0 0
lntraperitoneal Intra-arterial Intravenous Intramuscular lntraperitoneal Intra-arterial Intravenous Intramuscular
20 20 20 20 20 20 20 20
0 0 0 0 0 0 0 0
lntraperitoneal Intra-arterial Intravenous Intramuscular lntraperitoneal Intra-arterial Intravenous Intramuscular
20 20 20 20 20 20 20 20
20 18 19 19 20 19 18 17
Intraperitoneal Intra-arterial Intravenous Intramuscular lntraperitoneal Intra-arterial Intravenous Intramuscular
20 20 20 20 20 20 20 20
20 20 20 20 20 20 20 20
tumor transplant could be shown to have been induced by prior treatment with tumor cell nuclei.
Comments The technical problem which presented itself in this study was to obtain nuclei so free of viable intact. cells that they would be incapable of causing tumor transplantation even when injected into susceptible hosts in large numbers. Since most tumor cells are more resistant to mechanical rupture than cells of normal mammalian tissues, and since preparations substantially free of intact cells were desired, it was obvious that more vigorous shearing forces would have to be applied to the The American
Journal of Surgery
Tumor
TABLE
IV
Frequency
of Tumor Takes in Female CDBA/h
K2 Tumor Cells Six Weeks after Receiving
Experimental
Donor Strain of
Group
WholeCells
Route of Administration
Cell Nuclei and Tumor
and Female C3H/HeN
Mice Inoculated
lsoimmunity
with 30,000
UP K2 Nuclei
No. of Mice in Group
No. of Mice Developing Tumors
Cells TumorCells lntraperitoneally Intramuscularly lntraperitoneally Intramuscularly Tumor
Tumor
lntraperitoneal Intra-arterial Intravenous Intramuscular
10 10 10 10
10 10 10 10
10 10 10 10
10 9 10
of Nuclei
Tumor
9
1.5% Citric acid nuclei prepared from C3H/HeN strain
C3H/HeN
1.5% Citric acid nuclei prepared from CDBA/Fl strain
CDBA/Fl
lntraperitoneal Intra-arterial Intravenous Intramuscular
10 10 10 10
10 10 10 10
10 10 10 10
10 10 10 10
4% Citric acid nuclei prepared from C3H/HeN strain
C3H/HeN
lntraperitoneal Intra-arterial Intravenous Intramuscular
10 10 10 10
10 10 10 10
10 10 10 10
10 9 10 10
4% Citric acid nuclei prepared from CDBA/F1 strain
CDBA/F1
lntraperitoneal I ntra-arterial Intravenous Intramuscular
10 10 10 10
10 10 10 10
10 10 10 10
10 10 10 9
Control
C3H/HeN
15
15
15
14
Control
CDBA/F,
15
15
15
14
cells, and applied in such a way as to avoid rupturing significant numbers of nuclei. This was accomplished by using the Dounce homogenizer and by lowering the pH of the homogenizing media with citric acid. The very small clearance of the tight pestle (0.0005 inch or less) made it possible to disrupt a very high percentage of cells and yet cause little damage to the nuclei. If such a tight clearance were applied with a cylindrical rather than a ball type pestle (such as the cylindrical pestles of the Potter-Elvehjem types of homogenizers), the friction resulting would make it almost impossible to move the plunger and create intense local heating. In the Dounce homogenizer this clearance is along the circumference of a circle (the widest diameter of the ball), and, since the shearing force is applied only along the circumference of this circle, friction is reduced to a minimum. The pestle therefore moves freely and no appreciable local heating occurs [Z]. There was a considerable difference in the resistance to mechanical disruption between the two tumors studied, and, therefore different homogenizing media were necessary for each tumor. Vol.118.July1969
It is well established that cells resistant to breakage in sucrose or other media at pH values near neutrality can be broken in citric acid media at low pH’s. It is also known that by lowering the pH of the medium with citric acid or by adding calcium chloride to the medium the fragility of nuclei can be reduced [8-10,28-SO], thereby permitting the use of more vigorous mechanical shearing forces. By the addition of 0.005 M CaC12 to the sucrose medium and by lowering the pH to between 6.02 and 6.2 with citric acid, it was possible to obtain essentially complete disruption of the S91 melanoma cells without rupturing significant numbers of nuclei. By using diluted citric acid at pH 2.8 to 3.2 as a medium, it was possible to obtain substantially whole cell free preparations of K2 nuclei without significant nuclear damage. Nuclei prepared in this manner could be inoculated in large numbers into susceptible hosts without causing tumor transplantation. The possibility that nuclei might contain tumor antigens was first suggested in 1949 by Arnesen, Goldsmith, and Dulaney 121 I. They isolated nuclei from the spleens of nor55
Pilch and Ketcham
ma1 and leukemic AKm mice by a modification of the Dounce [16] procedure using M/125 citric acid in 8.5 per cent sucrose. These nuclei were found to be poor antigens since only low antibody titers to the nuclei were produced upon immunizing rabbits with these preparations. In addition, the antibodies formed crossreacted heavily with cytoplasm obtained from both normal and leukemic spleen cells. However, it should be pointed out that the nuclei used in this study were probably significantly contaminated with whole cells and cytoplasmic fragments. The marked cross-reactivity observed in this study is, therefore, not at all surprising. Nuclei prepared by the methods herein described might provide much more nearly pure nuclear antigen preparations. More recently, Rapaport et al. [22] have studied the subcellular fractions of human leukocytes as sources of transplantation antigens, and Monaco, Wood, and Russell [23] have examined the subcellular fractions of mouse spleen cells for the presence of homograft antigens. In both these studies the nuclear fractions were found to be essentially devoid of transplantation antigens. However, studies investigating the ability of subcellular fractions of tumor cells to induce tumor isoimmunity in syngeneic systems are lacking. In the studies reported herein, no tumor isoimmunity could be demonstrated in animals who had received a single inoculation of l,OOO,000 tumor cell nuclei six weeks prior to challenge with 30,000 viable tumor cells. However, these animals received only a single injection without the use of adjuvant materials. This was true in’ both the tumor-host systems studied. It is also possible that with a prolonged course of multiple injections, possibly employing an adjuvant material, some degree of tumor isoimmunity might have been produced. It is also possible that the two tumors studied were not particularly antigenic and that if additional tumor-host systems were examined, some antigenic activity within their nuclei might be observed. However, it seems likely that tumor-specific antigens are not present in the nuclei of the two tumors studied. Since tumor antigens may be similar to or a special class of transplantation antigens, the results of the present study are in agreement with the observations of Rapaport et al. [22] and Monaco, Wood, and Russell [23] who were 56
unable to demonstrate transplantation antigens within mammalian cell nuclei. These antigens must, therefore, reside elsewhere in the cell, either on the cell membrane or within the cytoplasm. Studies are currently underway in this laboratory to examine tumor cell membranes and cytoplasmic constituents for the presence of tumor-specific antigens. Summary Methods were devised for the isolation of tumor cell nuclei substantially free of contamination by viable whole cells. Such preparations were found to be incapable of producing tumor transplantation when injected in large numbers and by different routes into susceptible hosts. The ability of such nuclei to induce tumor isoimmunity was studied in two different tumor-host systems. Nuclei prepared in this manner failed to induce tumor isoimmunity when injected into syngeneic hosts. In both cases, mice receiving lo6 tumor cell nuclei evidenced no increase in resistance to subsequent tumor transplants of 30,000 viable whole cells from the same tumor inoculated six weeks later. References 1.
2.
3.
4. 5.
6.
SCHNEIDER, W. C. and HOGEBOOM, G. H. Cytochemical studies of mammalian tissues. The isolation of cell components by differential centrifugation: a review. Cancer Res., 11: 1, 1951. DOUNCE,A. L. The isolation and composition of cell nuclei and nucleoli. In: The Nucleic Acids. Edited by Chargaff, E. and Davidson, J. N. New York, 1955. New York Academic Press. ALLFREY,V. The isolation of subcellular components. In: The Cell. Edited by Brachet, J. and Mirsky, A. E. New York, 1959. New York Academic Press. DOUNCE,A. L. Enzyme systems of isolated cell nuclei. Ann. New York Acad. Sci., 50: 982, 1950. VON EULER, H., FISHER, I., HASSELQUIST, H., and JAARMA, M. Stability of isolated cell nuclei in different media: enzyme systems in nuclei. Ark. Kemi, Mineralocri. I I and Geologi, 12: 1, 1946. ’ DOUNCE,A. L. The isolation of nuclei from tumor cells. Exper. Cell Res., Suppl., 9: 126,1963.
7. 8.
9.
STONEBURG, C. A. Lipids of cell nuclei. J. Biol. Chem., 129: 189, 1939. MARSHAK, A. Psz uptake by nuclei. J. Gen. Physiol., 25, 275, 1941. HAVEN, F. L. and Lm, S. R. Phospholipids The American Journal of Surgery
Tumor Cell Nuclei and Tumor lsoimmunity
10. 11.
12.
13. 14.
15.
16.
of tumor cell and nuclei. Cancer Res., 2: 797,1942. DOUNCE, A. L. The desoxyribonucleic acid content of isolated nuclei of tumor cells. J. Biol. Chem., 147: 235, 1943. VON EULW, H., HANN, H., HASSELQUIST,H., JAARMA, M., and LUNDIN, M. Studies on the isolation and characterization of cell nuclei from calf thymus, hog liver and rat Jensen sarcoma. Svensk Kemisk Tidshrift, 57: 217,1945. SCHNEIDER,W. C. Intracellular distribution of enzymes. II. The distribution of succinic dehydrogenase, cytochrome oxidase adenosine triphosphatase and phosphorus compounds in normal rat liver and-in rat hepatomas. Cancer Res.. 6: 685. 1946. POTTER,V. R. and EJ&EHJE~, C. A. A modified method for the study of tissue oxidations. J. Biol. Chem., 144; 495, 1936. PRICE, J. M.. MILLER. J. A.. MILLER. E. C.. and WEBE~, G. M. Studies’ on the intracel: lular composition of liver and liver tumor from rats fed 4-dimethylaminoazobenzene. Cancer Res., 9: 96, 1949. SCHNEIDER,W. C. and HOGEBOOM, G. H. Intracellular distribution of enzymes. VI. The distribution of succinoxidose and cytochrome oxidose activities in normal mouse liver and in mouse hepatoma. J. Nat. Cancer inst., 10: 969, 1950. DOIJNCE,A. L., WITTER, R. F., MONTY, K. J., PATE, S., and COTTONE,M. A. A method
Vol. 118,July lb69
17.
18. 19.
20.
21.
22.
23.
for isolating intact mitochondria and nuclei from the same homogenate, and the influence of mitochondrial destruction on the properties of cell nuclei. J. Biophys. & Biochem. Cwtol.. 1: 139. 1955. SNELL, C. D: C&sieve permitting sterile preparation of suspensions of tumor cells for transplantation. J. Nat. Cancer Inst., 13: 1511,1953. DOUNCE, A. L. Further studies on isolated cell nuclei of normal rat liver. J. Biol. Chem., 151: 221,1943. MIRSKY, A. E. and POLLISTER,A. W. Chromosin, a desoxyribose nucleoprotein complex of the cell nucleus. J. Gen. Physiol., 30: 117,1946. SCHNEIDER,R. M. and PETERMAN,M. L. Nuclei from normal and leukemic mouse spleen. I. The isolation of nuclei in neutral medium. Cancer Res., 10: 751, 1950. ARNESEN,K., GOLDSMITH,Y., and DULANEY, A. D. Antigenic properties of nuclei segregated from spleens of normal and leukemic mice. Cancer Res., 9: 669, 1949. RAPAPORT,F. T., LAWRENCE,H. S., CONVERSE, J. M., and MULHOLLAND,J. H. Leukocyte fractions as skin homograft antigens in man. S. Forum, 14: 146,1963. MONACO,A. P., WOOD,M. L., and RUSSELL,P. S. A simple method for the preparation of cell free transplantation antigens. Physical and biological properties. S. Forum, 15: 133, 1964.
57