Tumor - Host Celi Hybrids in Radiochimeras ( in vivo hybrid / enzyme - deficient solid tumors / ascites tumors / macrophage )

F1 hybrid mice syngeneic or semiallogeneic with respect to the relevant tumor were lethally irradiated and then reconstituted with hemopoietic cells from strain CBAT6T6 mice. After chimerism had been established, the animals were inoculated with solid or ascites tumors. Tumor-host cell hybrids were selected from enzyme-deficient solid tumors by explanting the tumor cell suspension into hypoxanthine-amethopterin-thymidine containing medium. The selection of hybrid cells from ascites tumors was achieved by exploiting the difference between the ascites tumor cells and hybrid cells in their ability to adhere to the surface ofculture vessels. T6T6 chromosomal and H-2 antigenic markers served to distinguish between the hemopoietic cells derived from the donor graft and the cells of the host. All solid tumors tested fused with cells of the irradiated host, whereas ascites tumors fused with repopulating cells ofhemopoietic origin. In two previous publications (1, 2) we have described the successful selective isolation of tumor-host cell hybrids from solid and ascites tumors in Pivo. The tumors studied included polyomaand methylcholanthrene-induced sarcomas, an ascites carcinoma of mammary origin, and sarcoma lines derived from spontaneous transformation of normal fibroblasts. Unequivocal evidence was obtained for fusion between the tumor cells and host cells. Hybrid cells were isolated containing tumor-derived chromosomal and antigenic (H-2) markers together with host-derived H-2 or translocation (T6T6) markers. The chromosomal constitutions of the selected hybrid line were often only slightly reduced in comparison with what might be expected from the fusion of one tumor cell with one host cell. The type of host cell involved in the fusion is unknown. In order to assess the biological significance of the phenomenon, it was obviously important to define this, at least in outline. Radiation chimeras in which the host and the repopulating cells carried different markers appeared to present a possible approach to this question. MATERIALS AND METHODS The experimental design was as follows. F1 hybrid mice syngeneic or semiallogeneic with respect to the test tumor were lethally irradiated and then reconstituted with CBAT6T6 hemopoietic cells. After chimerism had been established, the animals were inoculated with solid or ascites tumors. Tumor-host cell hybrids were selected as previously described (1, 2). T6T6 chromosomal and H-2 antigenic markers served to distinguish between the hemopoietic cells derived from the embryonic liver graft and the cells of the host. Production of Chimeras. C3H X DBA/2, C3H X C57B1, and C3H X C57 leaden F. hybrid mice, 6-8 weeks old, were lethally irradiated with 800 R (3). About 5 X 105 hemopoietic cells obtained from the liver of newborn CBAT6T6 mice were suspended in 0.5 ml of sterile Eagle's minimal essential medium and inoculated into the tail vein of an irradiated recipient. The development of chimerism was confirmed by examining metaphase plates of spleen or lymph node cells. As a rule, lymphopoietic and hemopoietic organs were repopulated with T6T6 marker positive cells within 2-3 weeks. Tumors. (a). Solid tumors. A9HT and B82HT are highly malignant variants selected from the L cell sublines, A9 and B82 (4). Like the original in vitro lines from which they were derived, A9HT and B82HT are enzyme-deficient; A9HT lacks hypoxanthinq guanine phosphoribosyltransferase (HGPRT), while B82HT lacks thymidine kinase (TK). Due to these enzyme deficiencies both lines are unable to grow on hypoxanthine-amethopterin-thymidine (HAT) medium. With inoculla of 4 X 104 to 4 X 104 cells, both lines grew progressively in newborn x-irradiated (4 J kg) syngeneic or semisyngeneic C3H mice and killed 85-95% of the recipients (4). Both lines carry the H_2k histocompatibility complex, dharacteristic of the C3H mouse strain from which the original L-cell line was derived. The karyotypes of A9HT and B82HT are similar to those of A9 and B82, but show slightly reduced chromosome numbers (5). The modal chromosome number of A9HT and B82HT are about 53, compared with 57 for A9 and B82. The 23-28 biarmed chromosomes present in both lines served as chromosomal markers. (b). Ascites tumors. The ascitic form of the SEWA sarcoma was used. SEWA was originally induced by polyoma virus in an A.SW mouse (6) and carries the H-2s histocompatibility complex. It also carries the polyoma-specific transplantation antigen. It has a near-diploid chromosome constitution with a mode of 43 and a narrow range of 42-44 subtelocentric or telocentric chromosomes. The majority of the metaphase plates contain a very long telocentric marker, a marker with a secondary constriction, and several minute chromosomes or fragments (7). SEYF is a polyoma-induced fibrosarcoma of an A.BY mouse, converted to the ascites form. It contains the H-2b complex and the polyoma-specific transplantation antigen (8). It has an aneuploid chromosome constitution ranging between 57 and 69 chromosomes, with a mode of 67. Between 6 and 10 biarmed chromosomes are present (mode: 7); these serve as chromosomal markers. The TA3Ha tumor is the ascitic form of a spontaneous mammary adenocarcinoma of the A/Sn strain (9). It carries the H-2" isoantigen complex. It has a near diploid chromosome number with a mode of 41. An 8-azaguanine-resistant variant designated TA3Bimpwas produced in Oxford. This variant line is deficient in hypoxanthine-guanine-phosphoribosyltransferase and does not grow on HAT medium. 148 Abbreviation: HAT, hypoxanthine-amethopterin-thymidine. Tumor-Host Cell Hybrids in Radiochimeras 149 Chromosome Preparations. Metaphase spreads of cultured cells were prepared by the air-drying technique (10). Colcemid (GIBCO) was added to the culture in a final concentration of 0.04 ,ug/ml 4-5 hr before the cells were harvested. After hypotonic treatment (0.075 M KCl solution for 5 min), the pellet was fixed in acetic acid-methanol (3:1), spread on chilled wet slides, air dried, and stained with alkaline Giemsa solution. The total number of chromosomes and the proportion of biarmed chromosomes were estimated by direct counting under the microscope. Randomly selected metaphase plates were photographed, and karyograms prepared for detailed analysis. Isoantisera and Adsorption Tests. Groups of 10-12 adult mice of the appropriate genetic constitution were immunized with pooled cell suspensions prepared from spleen, kidney, and liver of mice carrying the requisite H-2 complex. The animals were injected subcutaneously once every 2 weeks for 12-14 weeks. The activity and specificity of all isoantisera were checked by cytotoxicity tests against lymph node cells of the appropriate genotype as described (11). To measure the concentration on H-2 antigens on the surface of the cell lines, quantitative adsorption tests were done as described previously (11). The ratio of bound to free antibody, (100-P)/P, was calculated by the method of Reif (12).