Analysis of antigenic heterogeneity within individual 3-methylcholanthrene-induced mouse sarcomas

A method of establishing sublines by trocar transplantation in vivo was used to analyze possible cellular heterogeneity with regard to tumor-specific antigens in individual methylcholanthrene-induced sarcomas. Differences in antigenic specificity were found in at least 1 of the 9 pairs of sublines obtained, respectively, from opposite poles of 9 different primary tumors. No evidence suggested such differences among 7 pairs of sublines similarly derived from later transplant generations. The lack of variation in antigenic specificity between members of subline pairs obtained from later transplant generation tumors suggested that antigenic specificity was a stable characteristic. Even when derived from later transplant generations, the sublines of a particular tumor sometimes differed in growth potential, and/or immunizing capacity, and/or responsiveness to immunity. There was little or no correlation between variations in the two parameters: immunizing capacity and responsiveness to immunity. These findings led to the interpretation that immunizing capacity and responsiveness to immunity behaved like partially independent variables, and that variations in these parameters probably depended on factors other than, or in addition to, cellular antigen content per se. The marked, random, spacial heterogeneity revealed within the tumors in cellular growth rate and antigenic properties suggests that changes seen during serial tumor transplantation are probably due to random clonal variation and subsequent selection.     

Immunogenic neoplastic clones derived in vitro from an originally non-immunogenic BALB/c fibrosarcoma

A non-immunogenic fibrosarcoma (SDC2), obtained by spontaneous neoplastic transformation of BALB/c fibroblasts cultured within a diffusion chamber kept in the peritoneal cavity of (BALB/c x C3Hf)F1 mice, was maintained in tissue culture for 10 passages. Three different clones were then derived from the in vitro neoplastic population. Each of the clones, the original in vivo tumor, and its in vitro line taken at the 10th passage (before cloning) were tested for the presence of individual tumor-associated tramsplantation antigens (TATA) by in vivo growth and excision assay. Contrary to the original SDC2 neoplasm, its in vitro line and 2 out of 3 clones (CL1-SDC2 and CL3-SDC2) were immunogenic, whereas the other clone (CL6-SDC2) displayed no immunogenicity. In addition, CL1-SDC2 and CL3-SDC2 were able to induce a reciprocal cross-protection in in vivo transplantation tests, thus showing common TATA; no cross-reactions were found between these clones and 2 other chemically-induced immunogenic sarcomas. The results suggest an antigenic heterogeneity in the original population of the nonimmunogenic SDC2 sarcoma, with the presence of antigenic but sub-immunogenic neoplastic cells.     

Drug-mediated immunogenic changes of virus-induced leukemia in vivo.

LSTRA and RBL-5 lymphomas induced by Moloney and Rauscher leukemia viruses, respectively, were used to determine whether antigenically altered tumors induced by 5-(3,3-dimethyl-1-triazeno)-imidazole-4-carboxamide in vivo would retain their original antigenic properties and/or have new antigenic properties. The tumors became highly immunogenic in the syngeneic hosts after 4 to 8 transplant generations with drug treatment. Syngeneic mice could be protected against challenge with the parental tumor by presensitization with the drug-altered sublines while unrelated tumor lines were incapable of protecting them. The drug-altered subline of LSTRA was used for treatment of the LSTRA in conjunction with chemotherapy, and this immunochemotherapy produced significant increases in number of survivors and increases in median survival time compared to either treatment alone. Tolerance studies indicated that there are novel antigens and parental tumor antigens associated with the drug-treated sublines.     

Increased immunogenicity of two lymphoma lines after drug treatment of athymic (nude) mice.

Highly immunogenic sublines of L1210 and LSTRA lymphomas were obtained from athymic (nude) mice treated with 4(5)-(3,3-dimethyl-1-triazeno)imidazole-5(4)carboxamide (DIC) in vivo. Conventional mice, compatible with the parent tumor, rejected the DIC-treated sublines and were relatively resistant to a subsequent challenge with the parent lines. The DIC-treated sublines were not rejected by athymic mice, which indicated that the transplantation resistance to these tumors in conventional mice was thymus-cell dependent. In addition, there was marginal or no increase of tumor-cell immunogenicity when the parent lines were passaged in nude mice without DIC treatment. This indicated that the DIC-dependent immunogenic changes in DIC-treated leukemic conventional mice could not be ascribed merely to protection by naturally occurring antigenic clones that resulted from DIC-induced immunodepression.     

Enhanced efficacy of tumor cell vaccines transfected with secretable hsp70

Tumor immunotherapy has exploited the ability of heat shock proteins to chaperone precursors of antigenic peptides to antigen-presenting cells and to activate efficiently an immune response against tumor-associated antigens. The most common strategy is based on the purification of heat shock protein-peptide complexes from tumor cell lines or from tumor surgical samples for in vivo administration. In this article, we have modified the murine-inducible hsp70 into a secreted protein and engineered tumor cells to secrete constitutively their antigenic repertoire associated with the hsp70 protein. In vitro studies showed that the relocalization of hsp70 from the cytoplasm to the secretory pathway did not modify the ability of hsp70 to interact with peptides derived either from natural tumor-associated antigens or model antigens, and that antigen-presenting cells specifically took up the secreted hsp70 and presented the chaperoned epitopes to T cells. In vivo studies showed that tumors secreting hsp70 displayed increased immunogenicity, with induction of a strong and specific CTL response. Mice injected with hsp70-secreting tumors showed increased survival and impaired tumor take compared with mice bearing parental tumors. More than 70% of mice rejected tumor cells secreting hsp70 through mechanisms that involve T lymphocytes and natural killer cells, with the induction of a memory response in the case of T lymphocytes. Moreover, hsp70 secretion increased the immunogenic potential of tumor cell vaccines.     

Heterogeneity in surface antigen of cell lines and clones from a murine carcinoma

Cell lines of the murine Lewis lung tumor (3LL) were established from primary tumor site (PT) and from lung metastatic foci (Met). Expression of histocompatibility antigens was assessed by immunofluorescence with an anti-H-2b antibody and by tumorigenicity of the cells in different strains of mice. Analysis for tumor-associated transplantation antigens (TATA) were performed by challenging mice with living tumor cells after pretreatment with irradiated cells or by in vivo tumor neutralization assays with different tumor cells as target cells and T lymphocytes from immunized animals as effector cells. The 3LL tumor was found to be heterogeneous in respect to tumor growth rate in vivo, metastatic capacity in vivo, expression of normal histocompatibility antigens and expression of "TATA". Moreover, monoclonal antibody B1 (H11-115) binding to a Met cell surface antigen, but not to normal cells, was obtained. The antibody bound to 3LL tumor cell lines from primary tumor (PTH, PTV), from a mixture of metastatic foci (MetH, MetV), from different metastatic foci originating from 3 different mice, and to 21 subclones derived from one of the lines established from a metastatic focus. Analysis of the 3LL sublines demonstrated a significant antigenic heterogeneity within each tumor population as well as among the different sublines.     

Some immunogenic acid biochemical properties of tumor-associated transplantation antigens (TATA) obtained in soluble form or solubilized from two methylcholanthrene-induced sarcomas, Meth A and CI-4

The subcellular distribution of the tumor-associated transplantation antigens (TATAs) of two highly immunogenic methylcholanthrene-induced sarcomas, Meth A and CI-4, were compared. Most of the TATA of CI-4 was found in the soluble fraction (cytosol) of the cell while the TATA from Meth A was variably distributed between the membrane and cytosol. The soluble fraction TATAs from both tumors were very immunogenic and their strong immunity could not be influenced by administering antigen in a variety of protocols that altered the immune response in other systems. The soluble fraction and membrane-associated TATAs from both tumors could be specifically bound to liposomes in reconstitution experiments and the antigenicity of all of the TATAs was significantly enhanced when they were incorporated into these artificial membranes.     

Immunosensitivity and histocompatibility antigens in drug-altered leukemic cells.

New antigenic properties of experimental lymphomas have been reported previously following in vivo treatment with antitumor agents. 5-(3,3-Dimethyl-1-triazeno)imidazole-4-carboxamide (DIC) induced new antigenic characteristics on L1210 and L5178Y lymphomas, that were previously investigated in studies in animals compatible with the original untreated parental tumors. Here the L1210/DIC and L5178Y/DIC susceptibility to the cytotoxic effects of allogeneic and xenogeneic lymphocytes and sera obtained from animals sensitized to DBA/2 histocompatibility antigens were studied. The original and the DIC tumors showed the same sensitivity to anti-DBA/2 cellular and humoral cytotoxicity. The immune response electied in allogeneic mice by the original and DIC sublines was evaluated by in vitro cell-mediated and humoral cytotoxic assay. Beyond the immune response to histocompatibility antigens, a specific, anti-DIC-antigen immunoreaction was not found. Inhibition assay of the cell-mediated cytotoxicity and absorption of the humoral cytotoxicity demonstrated that DIC-induced antigens are not reciprocally related in cell-surface concentration to the natural DBA/2 histocompatibility antigens associated with tumor cells of DIC lines. An experiment was conducted in which specific activity against the DIC-treated L5178Y/DIC cells was observed with anti-L5178Y/DIC rabbit immune serum absorbed with the parental L5178Y lymphoma. This finding provides additional support to previous studies indicating that treatment with DIC induced new antigens on the lymphoma cells.     

L1210/DTIC antigenic subline: Studies at the clone level

Treatment of murine tumors with the anti-tumor agent 5-(3,3 dimethyl-1-tri-azeno)imidazole-4-carboxamide (DTIC) has resulted in the induction of antigenic specificities not found in parental cells. After the withdrawal of DTIC treatment, the antigenic sublines maintained the new properties indefinitely, as a heritable character although the mechanism of induction and the molecular nature of the new antigens are essentially unknown. Seven clones from the murine immunogenic leukemia L1210/DTIC were selected and studied in some detail. Three of the seven clones showed an increased immunogenicity in vivo since two clones (D5 and D7) were rejected by syngeneic mice and one (D6) prolonged the life span for 3 weeks (untreated tumours killed the mice in 7 days). The seven clones and the L1210/DTIC were recognized and lysed by in vivo primed, in vitro stimulated (with L1210/DTIC) lymphocytes. Therefore, the seven clones shared antigens with the L1210-DTIC immunogenic subline. Secondary stimulated lymphocytes to clone D5, and clone D7 were able to lyse D5, D6 and D7 cells, respectively, but were unable to recognize the remaining clones. From in vivo and in vitro studies, all the L1210/DTIC sublines are composed of cells carrying two or more distinct antigens. Each cell clone expressed one set of antigens. It was concluded that only a few antigens expressed in different cells were induced by a DTIC treatment of L1210 leukemia.     

Occurrence of shared nonviral tumor-associated transplantation-type antigens among C3H/He mammary carcinomas

The occurrence of shared nonviral tumor-associated antigens among 18 immunogenic C3H/He mammary carcinomas was investigated in vivo. By cross-immunization and s.c. challenge between pairs of tumors in 37 different combinations, six of the tumors were found to cross-immunize with one to three other tumors. A minimum of three different antigenic specificities were found to be shared among cross-reacting tumors.     

Detection of private and common tumor-associated antigens in murine sarcomas induced by different chemical carcinogens.

Two fibrosarcomas of similar histological type, induced in C3Hf mice by either methylcholanthrene or 3,4-benz(a)pyrene, were shown to have individually unique tumor-rejection antigens in classical transplantation-type experiments. By contrast, sera of autochthonous mice, which resisted only transplants of the immunizing sarcoma, were found to contain complement-dependent cytotoxic antibodies, specific for both sarcomas, in vitro. The existence of individually unique as well as common tumor-associated antigens in chemically-induced murine sarcomas is suggested. The private "tumor transplantation-type" antigens elicited tumor rejection responses in vivo. The common tumor-associated antigens, although immunogenic in autochthonous hosts, inducing the production of tumor-specific antibodies, failed to induce transplantation cross-resistance in vivo. This study supports the contention that, in carcinogen-induced murine tumors, and perhaps in human neoplasms as well the evaluation of humoral (and cell-mediated) immune responses in vitro may not reflect tumor rejection-type immune responses in vivo.     

Increase in immunogenicity of a pulmonary squamous-cell carcinoma, propagated in vitro

The chemically induced, non-immunogenic lung squamous-cell carcinoma (MSC-10) propagated in vitro gradually loses tumorigenicity in immunocompetent hosts with increasing in vitro passage. This was found to be related to an increase in antigenicity, since immunosuppressed hosts (thymectomy plus 600 rads whole body X-irradiation) supported the growth of tumor cells, whereas immunocompetent recipients did not. The antigens involved in rejection are not heterologous serum proteins present in culture media since the cell line grown in isologous serum is also rejected. Immunization with the in vitro tumor line partially protected against the parental in vivo line, therefore the antigens involved must be present on both tumor lines. Inoculation of the cultured cell line into normal or immunosuppressed hosts produced tumors with the same histological characteristics as those of the in vivo tumor line. We concluded that by in vitro culture the weakly antigenic carcinoma becomes more immunogenic and thereby capable of inducing transplantation resistance. The cultured tumor cells retain their antigenic specificity and histologic characteristics while increasing their antigenic potency.     

Role of anchorage in the expression of tumorigenicity of untransformed mouse cell lines

Cultured mouse cells were tested for tumorigenicity in nude mice with both a conventional assay (injection of cell suspensions) and a new test involving implantation of cells grown on gelatin sponges. Sublines of Balb/3T3 cells, obtained from different sources, varied in their tumorigenic potential with either assay. One subline (A) formed distinctive precancerous nodules only in the sponge assay; these nodules often became progressive after a latent period of 3-4 months. However, suspensions of cells of this subline also caused tumors after a similar latent period, but no nodular phase preceded tumor formation. Another subline of Balb/3T3 (M) has failed to form tumors in either assay. The Balb/3T3 sublines did not differ in vitro properties, such as low saturation density, failure to grow in methylcellulose, and monolayer morphology. A second experimental approach involved tests on nude BALB/c mouse-embryo fibroblasts at various passage levels. The cells were passaged from primary culture, through crisis, to heteroploid, established cell lines. Tumorigenicity was demonstrable earlier in the sponge assay, at which time in vitro parameters putatively associated with malignant behavior were unchanged. Possible relationships with the in vivo phenomenon of solid-surface sarcomagenesis are discussed.     

Demonstration of cross-reacting tumor rejection antigens in chemically induced respiratory tract carcinomas in rats.

Experiments were performed to determine whether chemically induced rat respiratory tract carcinomas, which possess considerable immunogenicity, contain cross-reacting antigens. In vivo studies showed that effective cross-protection can be induced with four of the five respiratory tract cancers studied, suggesting that these tumors have common tumor rejection antigens. In vitro cytotoxicity studies with sera from tumor-immune hosts showed cross-reactivity among three of the carcinomas tested.     

Cell-mediated and humoral immunity studies of murine and hamster sarcoma virus-induced producer and non-producer tumors in syngeneic animals

Murine sarcoma virus (MSV)-induced transformed tumor cells, which are releasers and non-releasers of virus, were investigated by transplantation immunity and a variety of serological assays. Transplantation immunity demonstrated that tumor-associated transplantation antigens could be detected on producer cells and were also apparent, but to a lesser degree, on non-releaser cells of BALB/c mouse origin. Some lymphocyte microcytotoxicity tests employing hamster MSV tumors failed to detect any in vitro immunity. Studies employing immunofluorescence, complement fixation and virus focus neutralization tests demonstrated that Harvey-MSV producer cells of hamster origin were strongly immunogenic in inducing antibody formation in syngeneic hamsters. By contrast, no antibody could usually be detected by these techniques following immunization with non-producer Harvey-MSV or immunization with either Moloney-MSV non-producer cells or those non-producer cells that had been made releasers by treatment with IUDR in syngeneic hosts. The results suggest that humoral antibody, as detected by the techniques employed, is probably produced largely in response to infectious MSV contained by producer tumor cells and is not directed against new cell neoantigens, whereas transplantation immunity is probably, in part, a response to non-virion neoantigens.     

Tumor-bound immunoglobulins: an in vivo phenomenon of masked specificity.

We examined the specificity of the lg "coating" on murine tumor cells grown in vivo. Cells were treated in vitro for release of cell-bound lg from ascites tumors. Such uncoated cells showed an increased expression of tumor-associated antigens and a parallel decrease in intensity of the lg coat, but displayed no changes in the expressions of other normal membrane antigens. This was shown by a complement-dependent cytotoxicity assay and radioimmunoassay. These changes were attenuated if the tumor originated from animals that had been irradiated before tumor inoculation. No alterations were found with corresponding cells propaged in vitro and submitted to the same treatments. Our findings and others suggest tumor-specific antibodies among the lg coats detected on tumor cells grown in vivo.     

The Rauscher-MuLV-induced leukemia, RBL-5, bears two tumor-associated transplantation antigens expressed on distinct molecules

Tumor cells frequently express on their surface a new antigenic determinant which renders them immunogenic in the host animal. When immunity to this antigen results in rejection of a syngeneic tumor transplant, it is referred to as a tumor-associated transplantation antigen (TATA). RBL-5 is a Rauscher murine leukemia virus (MuLV)-induced leukemia of C57B1/6 origin that is potently immunogenic and shares a TATA with other tumors induced by the closely related Friend and Moloney-MuLVs (FMR-TATA). We have recently isolated a 175 kilodalton (kd) glycoprotein (gp175) which has all the properties expected of the FMR-TATA (Rogers et al., 1984). When this molecule was separated from a purified total glycoprotein fraction by DEAE chromatography, the remaining glycoproteins still contained a highly immunogenic TATA. Control experiments involving radioimmunoassay and immunoprecipitation with rabbit anti-gp175 indicated that this immunogenicity was not due to residual gp175 or breakdown products of gp175. We therefore conclude that RBL-5 expresses at least two distinct TATAs: gp175 and another glycoprotein distinguished from gp175 by its elution from a diethylaminoethyl-cellulose (DE52) column. These results, from a completely in vivo system, support data with other tumors obtained by in vitro methods and indicate that tumor cells may express several immunogenic molecules.     

Chemoimmunotherapy of L1210 leukemia with adriamycin, cyclophosphamide, and OK-432, and their effects on the generation of antitumor immunity

The synergistic effects of combined chemotherapy with adriamycin (ADR) and cyclophosphamide (CY) on L1210 tumors in mice were potentiated by use of a streptococcal preparation, OK-432, in a time- and dose-dependent way. Some mice were cured by treatment with the three agents, and resisted a later challenge by L1210 but not P388 leukemia cells. This immunity was blocked by administration of antimacrophage agents or CY. The effects of OK-432 were also studied with mice sensitized by L1210 cells attenuated with mitomycin C. OK-432 potentiated syngeneic and semi-syngeneic transplantation resistance in vivo and augmented primary and secondary cytotoxicity mediated by spleen cells in vitro. In vivo administration of ADR and CY together enhanced in vivo tumor transplantation resistance and in vitro cytotoxicity was blocked, but this inhibition was reversed by injection of OK-432. The results suggested that OK-432 acted by increasing the activity of cytotoxic spleen cells against L1210 cells, which are fast-growing and poorly immunogenic, and that this cytotoxicity killed tumor cells that survived the chemotherapy.     

Mammary tumor virus oncogenesis and tumor immunogenicity in three sublines of the C3H mouse.

Mammary tumorigenesis and mammary tumor transplantation immunogenicity have been studied and compared in three sublines of the C3H strain: in standard mammary tumor virus (MTV-S)-infected C3H/He mice; in MTV-S-infected C3H/Ki mice; and in MTV-S-free C3Hf/He mice. The age at the appearance of the first tumor, the growth rate of the tumors in their first transplant generation, and the immunogenicity of each tumor in syngeneic female recipients have been determined for the first tumor to appear in each of 25 breeding females from each of the three sublines. Two statistically significant trends were evident among the tumor characteristics compared in the three sublines: (a) an early appearance of tumors was related to the presence of the MTV-S. The genetically dissimilar sublines, C3H/He and C3H/Ki, both infected with the MTV-S, developed mammary tumors at an average age of about 10 months, 11 months before MTV-S-free C3Hf/He mice; (b) the tumor characteristics of immunogenicity and growth stimulation were related to host genetic factors. The genetically similar sublines, C3H/He and C3Hf/He, developed similar proportions of immunogenic and growth-stimulating mammary tumors; the genetically divergent C3H/Ki subline developed tumors that were not immunogenic and tended to be strongly growth stimulating.     

Differential cell surface antigen expression on metastatic variant lymphoma cell lines.

In the present investigation we have studied the cell surface antigenicity of a syngeneic murine metastatic lymphoma model system. This system is comprised of a highly malignant and metastatic RAW117-H10 subline and the less malignant/metastatic parental RAW117-P cell line. Using rabbit antisera raised against whole tumor cells we have been able to identify two major glycoprotein antigens which are differentially expressed on the cells. Although these antigens have a similar molecular weight (70 kD), they are antigenically distinct as determined by in vitro cytotoxicity assays. The levels of glycosylation on these glycoproteins were also found to be different. Increased expression of one of the antigenic components on the metastatic RAW117-H10 cells appeared to be associated with metastasis in this tumor model system.