Immunologieal Approaches to the Purification of Putative Premalignant Hepatocytes from Genotypic Mosaic Rat Livers1

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 con structed by transplantation of carcinogen-altered F-344 (n77'"1) or WF (RT1U)donor liver cells into livers of WF x F-344 F, host rats, whose liver cells bear alloantigens of both parental strains: WF and F-344, RT1U and RT1M, respectively (J. M. Hunt ef al., Cancer Res., 42: 227-236, 1982). Donor and host origin hepa tocytes were thus distinguishable immunologically with anti-RT1u or anti-RT1'v1 alloantisera. Donor rats were treated with diethylnitrosamine (200 mg/kg i.p.) followed by an experimental regimen of dietary 2-acetylaminofluorene and partial hepatectomy. Stand ard 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 hepa tocytes, 96% of which were positive histochemically for yglutamyl 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 sedimen tation through Ficoll/metrizamide resulted in a 4to 10-fold enrichment for donor origin hepatocytes isolated from mosaic livers. Similarly, a 5to 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 car cinomas which may be isolable using combinations of the purifi cation strategies described. 1Supported by NIH Grants CA-24818, CA-07175, CA-09020, and CA-37150 and by American Cancer Society Institutional Research Grant IN-35U-2. Presented in part at the 74th Annual Meeting of the American Association for Cancer Research, San Diego, CA (7). 2 Present address: Department of Pathology and Laboratory Medicine, University of Texas Medical School, P. O. Box 20708, Houston, TX 77225. 3To whom requests for reprints should be addressed. 4 Present address: Genesis Laboratories, Inc., Edina, MN 55435. Received 3/2/84; revised 1/2/85; accepted 1/18/85. INTRODUCTION The genotypic mosaic liver model for chemically induced rat hepatocarcinogenesis is being developed to facilitate the purifi cation and characterization of viable premalignant liver cells which are members of a cellular lineage leading to hepatocellular carcinomas (10). The experimental system for this model involves the i.v. transplantation of carcinogen-altered premalignant paren tal-strain donor liver cells into livers of Ft hybrid host rats. The host rats can be subjected to experimental regimens which promote the selective proliferation of transplanted donor liver cells. Orthotopic transplantation (12) of putative premalignant liver cells was developed using a highly selective regimen for host rats, namely, dietary AAF5 in combination with PH AAF-PH regimen) (See Chart 1). The AAF-PH regimen appears to act as a very potent promoter of hepatocarcinogenesis (20-22) in host rats. The striking failure of several alternative promoter regimens (18, 20), e.g., dietary phénobarbital, 1,1-bis(p-chlorophenyl)2,2,2-trichlorobenzene, benzene hexachloride, 2,6-di-fert-butyl4-methyl-phenol, and Aroclor 1254,6 to yield GT+ donor liver colony production in host rats underscores the utility of the strongly selective AAF-PH regimen in yielding detectable num bers of donor origin liver cells in the host rats at early stages of hepatocarcinogenesis and, as reported here, donor origin carci nomas as the end point. Rat alloantigens encoded by genes of the rat RT1 major histocompatibility complex are strain specific (3, 11) and can be serologically detected on isolated liver cells (6, 8,10). The RT1 genotypes of the inbred strains used, F-344 and WF, and fî77/v1 and RT1", respectively. Rats bred as Fi hybrids of these 2 strains would thus be hétérozygotes, possess the RTr/RTI"1 genotype, and express the Class I major histocompatibility complex gene products encoded by the RT1.A loci (6, 11) of both parental strains. Carcinogen-altered donor liver cells were generated in rats of the F-344 (RTI^), WF (RT1"), or WF x F-344 F, (RT1U/ RTI^) strains. The F, hybrid rats serve as recipient host rats which can accept "grafts" of transplanted donor liver cells from either parental rat strain or from the F, hybrid strain. Further, the use of parental-strain donor rats makes possible the identification of donor origin liver cells in the F, hybrid host rat genotypic mosaic livers, as has been demonstrated previously in cryostat histological sections by indirect immunofluorescence using polyvalent alloantisera (10). 5The abbreviations used are: AAF, 2-acetylaminofluorene; DEN, diethylnitrosamine; FA, fluorescent antibody-staining reaction by indirect immunofluorescence (+ or -); GT, histochemical enzyme phenotype (+ or -) for i-glutamyl transpepti dase; PAA, buffer containing bovine serum albumin (0.2%) and sodium azide (0.1 %) in phosphate-buffered saline; PBS, phosphate-buffered saline containing (per liter) 8.0 g NaCI, 0.2 g KCI, 2.16 g Na2HPO4-7H20, and 0.2 g KH2PO4; PH, two-thirds partial hepatectomy. 6J. M. Hunt, M. T. Buckley, and B. A. Laishes, manuscript in preparation. CANCER RESEARCH VOL. 45 MAY 1985