Virus Oncogenesis and Tumor Immunogenicity in the Mouse Mammary Tumor System1
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[CANCER RESEARCH 34, 1319 1324, June 1974] Virus Oncogenesis and Tumor Immunogenicity in the Mouse Mammary Tumor System1 Jan Vaage and Daniel Medina Department of Cancer Therapy Development, Pondville Hospital, Walpole, Massachusetts 02081 [J. K.J, and Department oj Cell Biology, Baylor College of Medicine, Houston. Texas 77025 [D. M.\ SUMMARY INTRODUCTION Mammary tumorigenesis and mammary tumor trans Several factors are known to be involved in the etiology of plantation immunogenicity were examined in breeding fe spontaneous mammary tumors in mice; most important males of 2 syngeneic sublines of the C3H strain: in among them are viral (2, 5, 15, 16), hormonal (16), and mammary tumor virus (MTV) plus nodule-inducing virus- genetic (8, 15) factors. In the C3H strain, 2 mammary infected C3H/Sed mice; and in MTV-free, nodule-inducing tumor viruses have been recognized: the MTV3 of high and virus-infected C3Hf/Sed mice. early oncogenic activity; and the NIV, of high but late Ninety-seven % of the C3H mice (29 of 30) developed oncogenic activity (11). The oncogenic effect of the in mammary tumors at an average age of 280 days. Of 43 M/ero-transmitted NIV is distinguishable in the C3Hf different C3H tumors tested for transplantation immunoge mouse, which was established as a syngeneic subline of C3H nicity in the C3H strain of origin, 13 (30%) had non-MTV- by foster nursing to exclude the milk-transmitted MTV, and associated tumor-specific transplantation antigens (TSTA). the high but late mammary tumor incidence in C3Hf mice Of 5 primary C3H hosts that developed multiple mammary becomes apparent in animals with a very long normal tumors, 4 animals developed both TSTA-positive and life-span in the infection-free conditions of a hygienic and TSTA-negative tumors. All C3H tumors tested (20 of 20) pathogen-free environment (11). for antigenicity in the C3Hf strain had MTV-associated In MTV-induced mouse mammary tumors, immunogenic transplantation antigens. reactivity (1, 10, 13, 20, 21, 23) and immunogenic cross- Sixty-nine % of the C3Hf mice (34 of 49) developed reactivity (20, 21, 23, 24) have been demonstrated in mammary tumors at an average age of 708 days. Of 16 MTV-free syngeneic C3Hf hosts where the MTV and/or different C3Hf tumors tested for antigenicity in the C3Hf cell products controlled by the viral genome probably strain of origin, 1 (6%) had TSTA. constitute common VATA's. In contrast, C3H mice, neona- Mammary tumorigenesis and mammary tumor trans tally infected with MTV, have been shown to be immuno- plantation immunogenicity were examined in expiants of 2 logically tolerant of the MTV (10, 12), and they can separate MTV-free C3Hf/Ki hyperplastic alveolar nodule consequently be expected to respond only to mammary (HAN) outgrowth lines transplanted to the cleared mam tumor immunogens other than the viral antigens and/or cell mary fat pads of syngeneic MTV-free C3Hf/Ki mice and products controlled by the viral genome.4 Such non-MTV- transplanted to the cleared mammary fat pads of syngeneic associated and non-cross-reacting TSTA's in MTV-induced MTV-infected C3H/KÃŒmice.Tumor development in HAN mouse mammary carcinomas have been demonstrated by line 1 was 44% at 297 days in C3Hf recipients and 100% at. both in vivo (14, 20, 21, 23) and in vitro (7) test procedures. 175 days in C3H recipients. Tumor development in HAN Non-virus-associated transplantation immunogenicity may line 2 was 75% at 225 days in C3Hf recipients and 87% at be expected in about 1of 4 MTV-induced mammary tumors 142 days in C3H recipients. One of 9 HAN line 1 tumors (21, 23). C3Hf mice most probably respond like C3H mice acquired TSTA during development in the C3Hf/Ki strain: to TSTA, but in the C3Hf mice this immune response is 0 of 10 acquired TSTA during development in the C3H/KÃŒ strain. Each tumor tested had acquired virus-associated transplantation antigens during development in the C3H/KÃŒ 'The abbreviations used are: MTV, mammary tumor virus; NIV, strain. One of 14 HAN line 2 tumors acquired TSTA during nodule-inducing virus; VATA, virus-associated transplantation antigen: TSTA, tumor-specific transplantation antigen: HAN, hyperplastic alveo development in the C3Hf/Ki strain; 0 of 15 acquired TSTA lar nodule: i.d., intradermally. during development in the C3H/KÃŒstrain. 4Reported demonstrations of anti-MTV immune factors in immuni/ed MTV-infected mice are considered to be due to a complete or partial loss of tolerance to MTV-associated antigens in occasional MTV-infected mice 'This work was supported in part by USPHS Grants ÇA 130I8. [see review by Blair (3)) or may be due to immune factors that do not CAI 1944. and CAI326I. impede tumor growth in vivo. However, the clear difference between C3H 2Recipient of Cancer Research Scholar Award from the American and C3Hf mice in their response to VATA has shown that immune Cancer Society, Massachusetts Division, which supported part of this tolerance to the VATA is a general characteristic of the C3H strain. Both work. C3H and C3Hf mice are assumed to be tolerant of the in ¡//era-transmitted Received November 15, 1973: accepted February 19, 1974. NIV. JUNE 1974 1319 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1974 American Association for Cancer Research. J. Vaage and D. Medina obscured by the much stronger response to the ever-present Tumor tissue was removed from freshly killed or live VATA. anesthetized donor animals, and skin and necrotic tissue The possibilities that the non-virus-associated tumor were removed before the tissue was placed in tissue culture transplantation immunogenicity could be due to medium in a sterile Petri dish kept on a bed of crushed ice. histocompatibility variation in the donor or could be due to Disruption of tumor tissue to obtain suspensions of dis the presence of antigenic variants of the MTV or even persed tumor cells was accomplished by a mechanical unrelated infectious agents were excluded by repeated procedure described in a previous publication (22). demonstrations that a mouse may develop both immuno- In all of the experiments reported here, s.c. sensitization genic and nonimmunogenic tumors at the same time (23). procedures were done on the right side of the animal and s.c. The present investigation has attempted to compare the challenge implantations were done on the left side. Chal frequency of non-virus-associated immunogenicity in mam lenge implantation s.c. of presensitized and control mice mary tumors induced by the action of the MTV with the was by injection of viable (trypan blue-negative) tumor cells frequency of non-virus-associated immunogenicity in mam suspended in tissue culture medium and consisting predomi mary tumors induced by the action of the NIV and also to nantly of single cells. The proportion of trypan blue-nega examine the frequency and variability of immunogenicity in tive cells in the tumor cell suspension was usually about mammary tumors arising in multiple expiants of mammary 25%. tissue from a single preneoplastic source. Statistical Analyses. For comparison of tumor incidence after challenge, the \2 test was used to evaluate the significance of differences in the total number of tumors MATERIALS AND METHODS between groups of mice. Differences between groups were considered significant only when p values of comparison Mice. The animals used in these experiments were as were 0.05 or smaller. follows. Group 1, was composed of 12-week-old female mice of inbred strains C3Hf/Sed and C3H/Sed from the defined-flora, pathogen-free breeding colonies (mice carry RESULTS ing only the following enteric bacteria: Closlridium sp., Peptostreptococcus sp., Bacillus sp., Bacteroides sp.) main Tumor Development in C3H/Sed and C3Hf/Sed Mice. tained by the Section of Experimental Radiotherapy at the Table 1 shows the incidence and age of mammary carci University of Texas M.D. Anderson Hospital and Tumor noma development in defined-flora, pathogen-free breeding Institute at Houston and by the Department of Radiation females of C3H/Sed and C3Hf/Sed mice. Foundation line Medicine, Massachusetts General Hospital. The C3Hf/Sed breeding mice were kept 1 pair to a cage in sterile air mice came originally from the germfree colony of Burdette isolators until retirement after 4 to 5 litters, at an average at the M.D. Anderson Hospital; he in turn had acquired his age of about 26 weeks. Retired female breeders are kept in mice from the germfree colony of Pilgrim at the University filter-top cages in filtered air environment at 74°Finregular of Utah, Salt Lake City, Utah. Pilgrim established his animal rooms and observed regularly for tumor develop germfree mice by cesarian section from C3Hf/Crgl mice. ment and general condition for the rest of their lives. C3H/Sed mice were established by foster nursing All the MTV-positive C3H/Sed female mice, with 1 C3Hf/Sed on C3H/PÕ. Group 2 was composed of 8- to exception, developed a mammary carcinoma at an average 12-week-old male and female mice from inbred strains age of 280 days. No gross evidence of other pathological C3H/KÕand C3Hf/Ki maintained in a closed mouse colony conditions was found in these mice at postmortem examina in the Department of Cell Biology at Baylor College of tion. The exceptional C3H/Sed female mouse is at the time Medicine at Houston. of writing 650 days of age and apparently disease free. Her Tumors. Female C3H and C3Hf mice with spontaneous offspring did not enter foundation line breeding, and it is recently arisen mammary carcinomas were routinely re therefore uncertain whether she has biologically active moved from the breeding colony.