Liver cell membrane alloantigens as cellular markers in genotypic mosaic rat livers undergoing chemically induced hepatocarcinogenesis.

We have developed a new marker system utilizing rat alloantigens as genetic cellular markers for hepatocytes in genotypic mosaic livers of rats undergoing hepatocarcinogenesis. This marker system has been designed to facilitate the eventual purification of subpopulations of hepatocytes from such livers which contain precursor cells of carcinomas. Thus, a lineage relationship between cells of early putative premalignant liver lesions and those of carcinomas may ultimately be established, and the process of cancer development may be biochemically probed. A donor-to-host liver cell transplantation procedure was used. Donor liver cells were prepared by collagenase dissocia tion from rats treated with a carcinogenic regimen consisting of diethylnitrosamine (200 mg/kg, i.p.) followed by an experi mental regimen involving dietary 2-acetylaminofluorene (0.02%) and partial hepatectomy. Host rats subjected to the 2-acetylaminofluorene/partial hepatectomy regimen were grafted orthotopically i.v. with donor liver cells via a mesenteric vein of the hepatic portal system. Cryostat sections of host rat livers were prepared 10 to 13 days after donor liver cell transplantation for histochemical localization of y-glutamyltranspeptidase-positive (GT+) hepatocyte colonies. Genotypic mosaic host rat livers were further analyzed by indirect immunofluorescence in serial cryostat sections to identify hepato cyte colonies on the basis of their strain-specific alloantigens. Syngeneic liver cell transplantation between WF donor and host rats was successful, as judged by GT+ liver colony pro duction in host rats, as has been reported previously for F344 rats. The physiological response of WF rats to the 2-acetyl aminofluorene/partial hepatectomy regimen paralleled that of F344 rats. Allogeneic liver cell transplantation between WF and F344 rats did not result in GT+ liver colony production. How ever, WF x F344F! hybrid host rats were capable of supporting GT+ liver colony production when grafted with donor liver cells from either parental strain (WF or F344). Polyvalent F344 anti-WF alloantiserum was prepared by immunizing F344 rats with WF skin and spleen cells. The resulting serum reacted positively by indirect immunofluorescence with cryostat liver sections as well as collagenase-dissociated hepatocytes from WF and WF x F344Fi hybrid rats. No reaction of the alloantiserum with F344 liver tissue or isolated hepatocytes was observed. Serial cryostat sections were prepared from livers of WF x ' This investigation was supported by Grants CA-24818, CA-07175, and CA09020, awarded by the National Cancer Institute, United States Department of Health, Education, and Welfare; and by American Cancer Society Institutional Research Grant IN-35U-2. These studies were presented in part at the 72nd annual meeting of the American Society for Cancer Research, Washington, D. C. (8). 2 Research Scholar of the Foundation for Cancer Research, Chicago. Received April 28, 1981; accepted October 13, 1981. F344F, hybrid host rats grafted with F344 donor liver cells. One section was used to localize GT+ liver colonies, and a contiguous section was processed with F344 anti-WF alloan tiserum by indirect immunofluorescence to localize WF-specific alloantigens possessed by the WF x F344Fi hybrid host liver tissue. Presumptive F344 donor origin liver colonies appeared as dark nonfluorescent (FA") areas of hepatocytes surrounded by brightly fluorescent F, hybrid host liver. These colonies could be stained FA+ along with the surrou: ding liver tissue if the indirect immunofluorescence was done using WF anti-F344 alloantiserum, indicating that they were of ,T344 donor origin. Some F344 donor origin colonies did not express the GT+ phenotype and were classified as y-glutamyltranspeptidasenegative. With the aid of tissue architectural landmarks, 276 individual colonies were analyzed from host rats killed on Days 10 to 13 following liver cell transplantation. Ninety-seven% of the colonies appeared to be of F344 donor origin (92% FA" GT+; 5% FA~ y-glutamyltranspeptidase-negative); 3% were FA+ GT+, and apparently of F, hybrid host origin. Rat alloan tigens expressed on the surface of hepatocytes may offer the potential for purification of donor-origin putative premalignant hepatocytes from genotypic mosaic host rat livers at sequential stages of hepatocarcinogenesis.