Brief Communication: Moloney Sarcoma Virus-Induced Tumors in Athymic
(Nude) Mice: Growth Pattern and Antibody Responses 1 Stephen Davis
2.3
SUMMARY-Tumor growth and antibody production were evaluated in nude (nu/nu) mice and their heterozygous normal (+/nu) littermates after inoculation of Moloney sarcoma virus (MSY). Sarcoma-bearing nude mice developed progressively growing tumors, whereas 53% of their normal counterparts showed tumor regressions. By indirect membrane fluorescence, significant amounts of IgG antibody to MSY could be detected in thymus-bearing but not in nude mice.-J Natl Cancer Inst 54: 793-
794,1975.
MOLONEY SARCOMA VIRUS (MSV) injected into adult mice causes tumors to develop at the site of inoculation. Most of these tumors eventually regress, presumably due to host immunologic mechanisms directed against virus antigens (1). With the use of in vitro systems, it was shown that both thymus-derived (T) and bone-marrow-derived (B) lymphocytes are involved in the suppression of tumor growth. T lymphocytes have been shown to be active as effector cells, whereas it has been proposed that B cells interact with antibody in mediating tumor-cell lysis (2). The in vivo significance of these findings is not yet clear. However, Gorczynski (3) protected sublethally irradiated mice from development of progressively growing tumors with T cells obtained from MSV-inoculated mice whose tumors had regressed. Similarly, antibodies from regressor animals, passively administered to naive hosts, have protected against subsequent challenge with syngeneic tumor cells (4). To further evaluate the role of thymus-dependent immunologic reactions to MSV antigens in vivo, nude mice were inoculated with MSV. These mice are homozygous for the nu allele (nulnu) at the nude locus, with resultant thymic aplasia (5). They are thus unable to mediate thymus-dependent immunologic functions. I compared tumor growth and antibody production in nude mice with those in normal mice heterozygous at the nude locus (+/nu). MATERIALS AND METHODS
Virus.-The Moloney strain of MSV (MSV-M) was obtained from the Program Resources and Logistics Segment, Special Virus-Cancer Program, National Cancer Institute, as pool SVRP 215. The virus titer by focus assay on secondary mouse embryo culture was 2 X 107 focus-forming units (FFU) 1001 (6). Animals.-Nude (nulnu) mice and their heterozygous normal (+Inu) littermates of both sexes were inoculated. All mice were 4-6 weeks old and were bred at the Frederick Cancer Research Center, Frederick, Maryland. Inoculations.-Mice were inoculated intramuscularly in the thigh of the hind leg with 10 6 FFU of MSVMImI. The growth of tumors induced by this virus
was monitored by palpation and measurement twice weekly. Mice were individually bled at day 35, or when tumors were 20 moo in diameter. Serum was stored at -20 0 C. MSB cell line.-This cell line was established from tumors induced in C57BL mice with MSV-M (6). The cells were maintained in RPMI-1640 plus 10% fetal calf serum and were passaged once a week by exposure to 0.25% trypsin. Antibody titers.-The indirect membrane fluorescence test (MF) was used to determine antibody production. It was performed as previously described (4), with the use of fluorescein-conjugated goat antimouse IgM (Meloy Laboratories, Springfield, Va.) and goat antimouse IgG (Hyland Laboratories, Los Angeles, Calif.). A minimum of 100 MSB cells was scored in each preparation to determine the percentage of MF-positive cells that showed sector or full-ring fluorescence. The last serum dilution (expressed as a reciprocal) giving 10% MF-positive cells was chosen as the serum titer. RESULTS Growth of MSY-Induced Tumors
Previous experience with MSV-induced tumors has shown that a high percentage regress (4). Since the precise in vivo role of T lymphocytes in these regression phenomena is yet to be determined, it was of interest to compare MSV-induced tumor growth in normal and athymic (nude) mice. Text-figure 1 shows that tumor growth was similar in both groups of mice until day 14. At this time thymus-bearing mice had suppressed tumor growth, as shown by the plateau of mean tumor size. In these experiments 14 of 30 (47%) ~ormal and all (41/41) nude mice developed progressIvely growing tumors. Of 50 nude mice inoculated with MSV, 41 developed tumors. The remaining animals failed to develop tumors but died within 21 days of injection. Histologic examination of the spleens in the latter group failed to disclose any gross abnormalities. Antibody Production to MSY-Associated Antigens
IgM and IgG titers to MSV-induced tumor antigens were individually quantitated in 33 nude (nulnu) and 29 heterozygous normal (+/nu) mice on day 35 after virus inoculation or when tumor size reached 20 moo. All normal mice had detectable IgG antibody as measured by indirect MF. The Received October 16, 1974; accepted December 10 1974. Viral Biology Branch, National Cancer Institute, N~tional Institutes of Health, Public Health Service, U.S. Department of Health, Education, and Welfare, Bethesda, Md. 20014. 3 I thank Dr. Gary R. Pearson and Dr. Charles W. Boone for their invaluable contributions. 1
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 54, NO.3, MARCH 1975
793
DAVIS
794 14
12
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TEXT-FIGURE I.-Growth of MSV-induced tumors in nude mice (A----A) and their heterozygous normallittermates (e-e). Numbers in parentheses represent number of tumorbearing dead mice at indicated intervals.
IgG antibody titer in these animals ranged from 4 through .16 (mean titer, 7.2). Of the 33 sera tested from nude mice, only 2 had detectable IgG levels at an antibody titer of 2. The latter 2 sera failed to give positive MF with antimouse IgM. None of the remaining sera from nude mice clearly demonstrated any IgM antibody, though 5 sera were questionably positive with undiluted serum (titer 1). With indirect MF, no IgM antibody could be detected in normal mice; similar results have been reported by others (7). DISCUSSION
Both T and B lymphocytes are involved in the immunologic response against MSV-induced tumors in vitro (2). However, the immune response mediated by T cells is believed primarily responsible for the tumor regression observed. B-cell activity increases after regression and persists for long periods thereafter in the absence of demonstrable T-cell activity. Possibly, therefore, the B-cell response is significant in the long-term remission of MSV-induced tumor growth. This study was initiated to help define the function of the T cell in the primary immunologic response to MSV antigens by evaluation of tumor growth and antibody production in nude (athymic) mice. At the dose of virus inoculum used in these experiments, all nude mice developed progressively growing tumors. These data are contrary to the findings of Stanton et al. (8), who found regressors in various strains of
neonatally thymectomized mice. However, these discrepancies may not be mutually exclusive since, at their inoculum dose, 100% of normal (thymusbearing) mice were regressors, whereas in myexperiments, 53% of the thymus-bearing mice demonstrated tumor regression by day 35. The inabiliW of nude mice to suppress tumor growth appears to result from a deficiency in T cells; however, the pattern of tumor growth observed in nude mice may be influenced in some manner by the "wasting-like" syndrome they tend to develop at an early age. The absence of IgG antibodies with MSV specificities in nude mice indicates that the antibody response to MSV antigens is T-cell dependent. The thymus dependency of antibody production to MSV antigens is further confirmed by the development of significant IgG antibody titers in the heterozygous normal littermates. Since, in mice, the IgG response to certain antigens is more sensitive to the presence of a thymus than is the IgM response (9), the failure to clearly demonstrate MSV-specific antibody of the IgG class suggests the complete dependency of the thymus in the primary antibody response to MSV. REFERENCES (1) HELLSTROM KE, HELLSTROM I: Cellular immunity against tumor antigen. Adv Cancer Res 12 :167-223, 1969 (2) LAMON EW, ANDERSSON B, WIGZELL H, et al: The immune response to primary Moloney sarcoma virus tumors in Balb/c mice: Cellular and humoral activity of long term regressors. Int J Cancer 13 :91-104, 1974 (3) GORCZYNSKI RM: Evidence for in vivo protection against murine-sarcoma virus-induced tumors by T lymphocytes from immune animals. J Immunol 112:533-539, 1974 (4) PEARSON GR, REDMON LW, BASS LR: Protective effect of immune sera against transplantable Moloney-virus induced sarcoma and lymphoma. Cancer Res 33: 171178, 1973 (5) FLANAGAN SP: "Nude," a new hairless gene with pleiotropic effects in the mouse. Genet Res 8 :295-309, 1966 (6) MASSICOT JG, WOODS WA, CHIRIGOS MA: Cell line derived from a murine sarcoma virus (Moloney pseudotype)-induced tumor: Cultural, antigenic, and virological properties. App1 Microbio1 22: 119-121, 1971 (7) LAMON EW, KLEIN E, ANDERSSON B, et al: The humoral antibody response to a primary viral neoplasm (MSV) through its entire course in Balb/c mice. Int J Cancer 12 :637-645, 1973 (8) STANTON MF, LAW LW, TING RC: Some biologic, immunogenic, and morphologic effects in mice after infection with a murine sarcoma virus. II. Morphologic studies. J Natl Cancer Inst 40:1113-1129, 1968 (9) TAYLOR RB, WORTIS HH: Thymus dependence of antibody response: Variation with dose of antigen and class of antibody. Nature (Lond) 220:927-929, 1968
(Nude) Mice: Growth Pattern and Antibody Responses 1 Stephen Davis
2.3
SUMMARY-Tumor growth and antibody production were evaluated in nude (nu/nu) mice and their heterozygous normal (+/nu) littermates after inoculation of Moloney sarcoma virus (MSY). Sarcoma-bearing nude mice developed progressively growing tumors, whereas 53% of their normal counterparts showed tumor regressions. By indirect membrane fluorescence, significant amounts of IgG antibody to MSY could be detected in thymus-bearing but not in nude mice.-J Natl Cancer Inst 54: 793-
794,1975.
MOLONEY SARCOMA VIRUS (MSV) injected into adult mice causes tumors to develop at the site of inoculation. Most of these tumors eventually regress, presumably due to host immunologic mechanisms directed against virus antigens (1). With the use of in vitro systems, it was shown that both thymus-derived (T) and bone-marrow-derived (B) lymphocytes are involved in the suppression of tumor growth. T lymphocytes have been shown to be active as effector cells, whereas it has been proposed that B cells interact with antibody in mediating tumor-cell lysis (2). The in vivo significance of these findings is not yet clear. However, Gorczynski (3) protected sublethally irradiated mice from development of progressively growing tumors with T cells obtained from MSV-inoculated mice whose tumors had regressed. Similarly, antibodies from regressor animals, passively administered to naive hosts, have protected against subsequent challenge with syngeneic tumor cells (4). To further evaluate the role of thymus-dependent immunologic reactions to MSV antigens in vivo, nude mice were inoculated with MSV. These mice are homozygous for the nu allele (nulnu) at the nude locus, with resultant thymic aplasia (5). They are thus unable to mediate thymus-dependent immunologic functions. I compared tumor growth and antibody production in nude mice with those in normal mice heterozygous at the nude locus (+/nu). MATERIALS AND METHODS
Virus.-The Moloney strain of MSV (MSV-M) was obtained from the Program Resources and Logistics Segment, Special Virus-Cancer Program, National Cancer Institute, as pool SVRP 215. The virus titer by focus assay on secondary mouse embryo culture was 2 X 107 focus-forming units (FFU) 1001 (6). Animals.-Nude (nulnu) mice and their heterozygous normal (+Inu) littermates of both sexes were inoculated. All mice were 4-6 weeks old and were bred at the Frederick Cancer Research Center, Frederick, Maryland. Inoculations.-Mice were inoculated intramuscularly in the thigh of the hind leg with 10 6 FFU of MSVMImI. The growth of tumors induced by this virus
was monitored by palpation and measurement twice weekly. Mice were individually bled at day 35, or when tumors were 20 moo in diameter. Serum was stored at -20 0 C. MSB cell line.-This cell line was established from tumors induced in C57BL mice with MSV-M (6). The cells were maintained in RPMI-1640 plus 10% fetal calf serum and were passaged once a week by exposure to 0.25% trypsin. Antibody titers.-The indirect membrane fluorescence test (MF) was used to determine antibody production. It was performed as previously described (4), with the use of fluorescein-conjugated goat antimouse IgM (Meloy Laboratories, Springfield, Va.) and goat antimouse IgG (Hyland Laboratories, Los Angeles, Calif.). A minimum of 100 MSB cells was scored in each preparation to determine the percentage of MF-positive cells that showed sector or full-ring fluorescence. The last serum dilution (expressed as a reciprocal) giving 10% MF-positive cells was chosen as the serum titer. RESULTS Growth of MSY-Induced Tumors
Previous experience with MSV-induced tumors has shown that a high percentage regress (4). Since the precise in vivo role of T lymphocytes in these regression phenomena is yet to be determined, it was of interest to compare MSV-induced tumor growth in normal and athymic (nude) mice. Text-figure 1 shows that tumor growth was similar in both groups of mice until day 14. At this time thymus-bearing mice had suppressed tumor growth, as shown by the plateau of mean tumor size. In these experiments 14 of 30 (47%) ~ormal and all (41/41) nude mice developed progressIvely growing tumors. Of 50 nude mice inoculated with MSV, 41 developed tumors. The remaining animals failed to develop tumors but died within 21 days of injection. Histologic examination of the spleens in the latter group failed to disclose any gross abnormalities. Antibody Production to MSY-Associated Antigens
IgM and IgG titers to MSV-induced tumor antigens were individually quantitated in 33 nude (nulnu) and 29 heterozygous normal (+/nu) mice on day 35 after virus inoculation or when tumor size reached 20 moo. All normal mice had detectable IgG antibody as measured by indirect MF. The Received October 16, 1974; accepted December 10 1974. Viral Biology Branch, National Cancer Institute, N~tional Institutes of Health, Public Health Service, U.S. Department of Health, Education, and Welfare, Bethesda, Md. 20014. 3 I thank Dr. Gary R. Pearson and Dr. Charles W. Boone for their invaluable contributions. 1
2
JOURNAL OF THE NATIONAL CANCER INSTITUTE, VOL. 54, NO.3, MARCH 1975
793
DAVIS
794 14
12
~
:2 ;;:; 10 N
iii cr::
o
:2 8 ::> f-
z
«
~ 6
°L'f--(
..L.--_--------'-_ _
14
-----'_~_.L_I
_ _----'-1_
21
28
35
TIME IDAYS)
TEXT-FIGURE I.-Growth of MSV-induced tumors in nude mice (A----A) and their heterozygous normallittermates (e-e). Numbers in parentheses represent number of tumorbearing dead mice at indicated intervals.
IgG antibody titer in these animals ranged from 4 through .16 (mean titer, 7.2). Of the 33 sera tested from nude mice, only 2 had detectable IgG levels at an antibody titer of 2. The latter 2 sera failed to give positive MF with antimouse IgM. None of the remaining sera from nude mice clearly demonstrated any IgM antibody, though 5 sera were questionably positive with undiluted serum (titer 1). With indirect MF, no IgM antibody could be detected in normal mice; similar results have been reported by others (7). DISCUSSION
Both T and B lymphocytes are involved in the immunologic response against MSV-induced tumors in vitro (2). However, the immune response mediated by T cells is believed primarily responsible for the tumor regression observed. B-cell activity increases after regression and persists for long periods thereafter in the absence of demonstrable T-cell activity. Possibly, therefore, the B-cell response is significant in the long-term remission of MSV-induced tumor growth. This study was initiated to help define the function of the T cell in the primary immunologic response to MSV antigens by evaluation of tumor growth and antibody production in nude (athymic) mice. At the dose of virus inoculum used in these experiments, all nude mice developed progressively growing tumors. These data are contrary to the findings of Stanton et al. (8), who found regressors in various strains of
neonatally thymectomized mice. However, these discrepancies may not be mutually exclusive since, at their inoculum dose, 100% of normal (thymusbearing) mice were regressors, whereas in myexperiments, 53% of the thymus-bearing mice demonstrated tumor regression by day 35. The inabiliW of nude mice to suppress tumor growth appears to result from a deficiency in T cells; however, the pattern of tumor growth observed in nude mice may be influenced in some manner by the "wasting-like" syndrome they tend to develop at an early age. The absence of IgG antibodies with MSV specificities in nude mice indicates that the antibody response to MSV antigens is T-cell dependent. The thymus dependency of antibody production to MSV antigens is further confirmed by the development of significant IgG antibody titers in the heterozygous normal littermates. Since, in mice, the IgG response to certain antigens is more sensitive to the presence of a thymus than is the IgM response (9), the failure to clearly demonstrate MSV-specific antibody of the IgG class suggests the complete dependency of the thymus in the primary antibody response to MSV. REFERENCES (1) HELLSTROM KE, HELLSTROM I: Cellular immunity against tumor antigen. Adv Cancer Res 12 :167-223, 1969 (2) LAMON EW, ANDERSSON B, WIGZELL H, et al: The immune response to primary Moloney sarcoma virus tumors in Balb/c mice: Cellular and humoral activity of long term regressors. Int J Cancer 13 :91-104, 1974 (3) GORCZYNSKI RM: Evidence for in vivo protection against murine-sarcoma virus-induced tumors by T lymphocytes from immune animals. J Immunol 112:533-539, 1974 (4) PEARSON GR, REDMON LW, BASS LR: Protective effect of immune sera against transplantable Moloney-virus induced sarcoma and lymphoma. Cancer Res 33: 171178, 1973 (5) FLANAGAN SP: "Nude," a new hairless gene with pleiotropic effects in the mouse. Genet Res 8 :295-309, 1966 (6) MASSICOT JG, WOODS WA, CHIRIGOS MA: Cell line derived from a murine sarcoma virus (Moloney pseudotype)-induced tumor: Cultural, antigenic, and virological properties. App1 Microbio1 22: 119-121, 1971 (7) LAMON EW, KLEIN E, ANDERSSON B, et al: The humoral antibody response to a primary viral neoplasm (MSV) through its entire course in Balb/c mice. Int J Cancer 12 :637-645, 1973 (8) STANTON MF, LAW LW, TING RC: Some biologic, immunogenic, and morphologic effects in mice after infection with a murine sarcoma virus. II. Morphologic studies. J Natl Cancer Inst 40:1113-1129, 1968 (9) TAYLOR RB, WORTIS HH: Thymus dependence of antibody response: Variation with dose of antigen and class of antibody. Nature (Lond) 220:927-929, 1968
