Immunological approaches to the purification of putative premalignant hepatocytes from genotypic mosaic rat livers

Abstract

Surface-localized rat RT1 alloantigens on isolated hepatocytes have been used to achieve partial purification of putative premalignant liver cells from rats undergoing chemically induced hepatocarcinogenesis. Genotypic mosaic rat livers were constructed by transplantation of carcinogen-altered F-344 (RT1Iv1) or WF (RT1u) donor liver cells into livers of WF X F-344 F1 host rats, whose liver cells bear alloantigens of both parental strains: WF and F-344, RT1u and RT1Iv1, respectively (J. M. Hunt et al., Cancer Res., 42: 227-236, 1982). Donor and host origin hepatocytes were thus distinguishable immunologically with anti-RT1u or anti-RT1Iv1 alloantisera. Donor rats were treated with diethylnitrosamine (200 mg/kg i.p.) followed by an experimental regimen of dietary 2-acetylaminofluorene and partial hepatectomy. Standard host rats received only the 2-acetylaminofluorene-partial hepatectomy regimen. At 10 to 21 days after transplantation, mosaic host rat livers typically contained 7% donor origin hepatocytes, 96% of which were positive histochemically for gamma-glutamyl transpeptidase. Host origin hepatocytes could be effectively purified by affinity chromatography ("panning") of isolated hepatocytes. To obtain donor origin hepatocytes, the putative progenitor cells of liver carcinomas in these mosaic livers, two approaches were used. Alloantibody-mediated rosette formation followed by sedimentation through Ficoll/metrizamide resulted in a 4- to 10-fold enrichment for donor origin hepatocytes isolated from mosaic livers. Similarly, a 5- to 11-fold enrichment for donor origin hepatocytes was achieved by specific alloantibody-mediated cytolysis of host hepatocytes with rabbit complement followed by purification of viable donor origin cells by sedimentation on metrizamide cushions. Hepatocellular carcinomas which developed in the genotypic mosaic host rat livers were excised 17 to 21 months after donor liver cell transplantation and passaged s.c. or i.m. in histocompatible rats. The transplantable tumors were typed for strain of origin by indirect immunofluorescence using rat alloantisera, and five of six tumors displayed antigenicity reflecting donor strain origin. We conclude, therefore, that the transplanted donor liver cell populations contain cellular precursors of hepatocellular carcinomas which may be isolable using combinations of the purification strategies described.