Changing Transplantation Characteristics with Serial in Vivo Passage of C3H/He Mammary Carcinomas1

The transplantation immunogenicity of spontaneous C3H/He mammary carcinomas was studied by means of the surgery-challenge procedure during serial in vivo passages in syngeneic mice. Reciprocal cross-sensitization and challenge tests between late transplant genera tions and early transplant generations (from liquid N, storage) of the same tumors showed that factors respon sible for transplantation resistance and factors causing stimulated tumor growth were present in the tumors at the same time as independent variables. The immunogenicity and the immunosensitivity of tumors were seen as de pendent variables. The relative prominence of the char acteristics of immunogenicity and growth stimulation changed with continuous in vivo passsages. Transplanta tion immunogenicity was tumor specific in four of five tumors tested. Growth stimulation was, in each of four combinations tested, not tumor specific. INTRODUCTION Previous studies (7) have shown that individual mammary carcinomas appearing in MTV-S2-infected C3H/He mice may arise with characteristics that induce a detectable tumor-specific graft rejection response in the host (immunogenic) or with characteristics that will cause nonspecific stimulation of the growth of additional or subsequent tumor implants (growth stimulating). These and other character istics such as growth rate and transplantability may change during serial in vivo passages. Whereas the nature of growth-stimulating factors associated with some tumors is not well understood,3 it has been demonstrated that the immunogenicity of C3H/He mammary tumors in C3H/He hosts is not related to immunogens controlled by the exogenous MTV-S but to immunogens determined by host genes and/or by the endogenous germ-line virus with low oncogenic activity (MTV-L) (4, 9, 10). Because the diverse growth characteristics of mammary tumors may undergo significant changes during in vivo growth (7), it seemed important to investigate further the changes during serial passage, which may affect the rejec tion response of the hosts. Several tumors tested for transplantation immunogenicity and described previously (7) were selected for this study. 1 Supported by Grant CA-15960 awarded by the National Cancer Institute, Department of Health, Education and Welfare. The work was started at the Massachusetts General Hospital and completed at the Pondville Hospital. 2 The abbreviations used are: MTV-S, mammary tumor virus-standard (exogenous, milk-transmitted virus with high oncogenic activity). 3 J. Vaage, S. Agarwal. and C. A. Apffel. Effect of Progressive Neoplastic Growth on the Decarboxylation of DL-Ornithine-1-14C by Neoplastic Cells, submitted for publication. Received March 10, 1978; accepted June 27. 1978. MATERIALS AND METHODS Mice. The tumor donors were pedigree breeding female C3H/He mice, force-bred from the age of 6 weeks and removed from breeding at the appearance of the first mammary tumor or after the fifth litter. The experimental mice were 10to 12-week-old females produced in full sibmated line breeding of C3H/He and C3Hf/He mice. All the mice were from the defined-flora, pathogen-free breeding colony (mice carry only the following nonpathogenic en teric bacteria: Clostridium sp., Peptostreptococcus sp., Bacillus sp., and Bacteroides sp.) maintained by the De partment of Cancer Therapy Development at Pondville Hos pital, Walpole, Mass. Tumors. The primary mammary carcinomas were excised soon after they became palpable (usually removed at 3 to 5 mm) with wide margins to prevent recurrences. Most of the tumor donors were kept after surgery and observed for the development of further (not recurrent) tumors, which were also excised and tested. Tumor Implantation. Tumors (5 to 10 mm) were removed from live anesthetized donor animals, and skin and necrotic tissue were removed before the tissue was placed in culture medium and cut into small pieces in a Petri dish kept on a bed of crushed ice. Implantation of two 1-cu mm pieces of living tumor tissue s.c. was used to sensitize. Tumors from treated animals were never used. Removal of sensitizing tumors implanted s.c. was done under pentobarbital anes thesia. Unsensitized control animals were also anesthetized before challenge. The sensitizing tumors were removed when their average size per group was 8 to 10 mm. This required from 3 to 6 weeks, depending on the growth rate of individual tumors. Because host immunity is already detect able 5 days after the implantation of an immunogenic tumor (5) and because concomitant immunity is strong until a tumor gets quite large (8), this procedure gives consistent results with immunogenic tumors. In all of the experiments reported here, s.c. sensitization procedures were done on the right side of the animals, and s.c. challenge implantations were done on the left side. The challenge of presensitized (10/group) and control (10/ group) mice, which was done on the day of the removal of the sensitizing implants with the use of cells prepared from the excised tumors, was carried out by means of injection of 5 x 105 cells (usually about 20% viable by trypan blue tests) in single-cell suspension, at both the left shoulder and the hip of each animal. The purpose of the double challenge implants was to observe the consistency of indi vidual host responses. Disruption of tumor tissue to obtain suspensions of dispersed cells was accomplished with the use of 105 mesh polyester cloth (HC-1-105 screen cloth; TETKO, Inc., Elmsford, N. Y.) by means of a mechanical 3264 CANCER RESEARCH VOL. 38 on April 14, 2017. © 1978 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from Changes in Serially Transplanted Tumors procedure described in previous publications (5, 6). In each separate experiment the challenge of all the mice was completed within the briefest possible time with the use of cells from the same suspension, kept on crushed ice. The incidence of tumors at the s.c. injection sites was checked at the same time in all groups at 7-day intervals, and the tumor size was measured with calipers. By mea surement of 2 bisecting diameters of each tumor and with the use of only the lesser to express tumor size, the possible error of measuring the added diameters of more than 1 focus of growth along the needle path of implantation was avoided. Each experiment was terminated before the mice became cachectic because of progressive s.c. tumor growth. The incidence and mean tumor size at the last measurement are presented in the tables. Statistical Analyses. The effect of pretreatment on s.c. tumor growth is described as the ratio of average size times incidence in treated mice divided by the average size times incidence in controls. For comparison of tumor growth between treated groups and between treated and control groups, Student's f test was used. Differences between groups were considered significant when p was 0.05 or less. RESULTS The Relationship between Induction of Transplantation Immunity and Stimulation of Growth. For examination of the nature of a shift from immunogenicity to growth stimu lation observed in serial passages of some tumors (7), 3 such tumors were tested in crisscross sensitization-challenges between immunogenic and growth-stimulating gen erations of the same tumor. Tumors in early transplant generations were reimplanted into mice from tissue stored in liquid N.,and were tested against later transplant gener ations as shown in Table 1. The results demonstrate the following, (a) Each of the 3 tumors used in an early genera tion (generation 2 or 3) to sensitize would induce protection against the challenge with the tumor in its next generation (p < 0.05). (b) Each tumor, when becoming less immunogenic in later transplant generations, would also become less susceptible to rejection in a sensitized mouse (less immunosensitive). For example, Tumor 9-13, used in gen eration 2 to sensitize, produced a protection against chal lenge that was more clearly expressed against generation 3 (more immunosensitive) than against challenge with gen eration 9 (less immunosensitive) (0.1 > p > 0.05). GeneraTable 1 Cross-sensitizing and challenge between mammary carcinomas in different transplant generations In each test 2 groups of mice were each sensitized with s.c. implants of 1 of 2 tumors. On the day of surgical removal of the implants, cell suspensions of the 2 tumors were used to challenge equal halves of each sensitized group. The vertical columns for each challenge tumor represent the following: (a) incidence of tumors per total number of implants; (to)average tumor size in mm; and (c) growth ratio derived from average size times incidence for treated mice divided by the average size times incidence for control mice. Challenge tumors C3H/He hosts C3Hf/He hosts Sensitizing tumors No. of tu mors Av. size (mm) Ratio (treated/ control) No. of tu mors Av. size (mm) Ratio (treated/ control) No. of tuAv. size No. of tuAv. size mors (mm) mors (mm) 9-13,g2 9-13,g8 Unsensitized 9-13,g2 9-13,g12 Unsensitized 9-13,g3 9-13,g13 Unsensitized 9-16,g2 9-16,g6 Unsensitized 14/20 15/20 20/20