[CANCER RESEARCH 47, 1282-1286, March 1, 1987) Phenotypic Modulation during Tumorigenesis by Clones of Transformed Rat Liver Epithelial Cells1 Ming-Sound Tsao2 and Joe W. Grisham Department of Pathology, University of North Carolina, Chapel Hill, North Carolina 27514 ABSTRACT From nine clonal subpopulations (strains) of chemically transformed cultured rat hepatic epithelial cells which were tumorigenic when im planted into 1-day-old isogeneic rats, a cell line was reestablished from each tumor and the cellular properties of the tumor-derived cell lines were compared to those of the corresponding progenitor cells that were implanted to produce the tumors. In seven of eight instances, the cellular DNA content of the tumor-derived cells was virtually identical to the DNA content of the respective progenitor cells, but in one case the tumor cells had twice as much DNA as did their progenitor cells. During the development of tumors in viva, other cellular phenotypic properties often underwent considerable, but variable changes. These changes included the activity of •v-glutamyl transpeptidase, the growth properties on plastic surfaces, and the expression of LDH isozymes. Although there was a relative enhancement in the ability of most of the tumor-derived cells to proliferate or to form colonies in calcium-poor medium, several tumor- derived cell lines had very low colony-forming efficiencies in media containing either normal or low levels of calcium. The most consistent association between phenotypes expressed in vitro and tumorigenicity was the ability of cells to form colonies in soft agar; all tumor-derived lines expressed this phenotype, and with some of them this phenotype was acquired only during the process of tumor formation in vivo. These results demonstrate that further phenotypic and genotypic alterations may occur in vivo during tumor formation by chemically transformed cultured cells following their implantation into isogeneic animals; and some of the alterations that occur in vivo may be necessary for the complete expression of tumorigenicity. Although anchorage-independent growth capacity cannot be used to predict the tumorigenicity of clones of rat liver epithelial cells chemically transformed in vitro, this growth property appears to be invariably induced prior to or during the formation of tumors in vivo by these cells. INTRODUCTION //; vitro studies of chemical carcinogenesis using cultured mammalian cells are useful for elucidating the molecular and biochemical basis of neoplastic transformation and tumor pro gression. To identify steps or lesions which are relevant to neoplasia, /// vitro studies depend largely on the identification of a few specific "marker" phenotypes which can be strongly correlated with tumorigenicity, i.e.. with the ability of cells to form tumors when they are implanted into suitable host ani mals. These "marker" or paratumorigenic phenotypes, which are few in number, have been identified by two methods: (a) cell lines which have been derived from either tumors or normal tissues are tested for the expression of various phenotypes, or (/>)cell lines which have been treated with a carcinogen in vitro are analyzed for their tumorigenicity and the expression of the various phenotypes. Phenotypic characteristics which are tightly correlated with tumorigenicity have been regarded as "markers" of the neoplastic state. Using rat liver epithelial cells, investigations along these two experimental designs have iden- Received 7/11/86; revised 11/19/86; accepted 11/25/86. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work is supported by N1H Grant CA-29323. •' Centennial Fellow of the MRC of Canada when this work was performed. Present address: Department of Pathology. Montreal General Hospital, Mon treal. Quebec, Canada H3G1A4. titled a few "marker" phenotypes of malignant transformation that appear to correlate with tumorigenicity (1-6). However, a careful consideration of these two experimental systems reveals some significant differences between them, and these differ ences must be recognized and accounted for in the interpreta tion of the results. In the first method the cells are derived from liver tumors induced in vivo by a carcinogen, hence these cells are truly neoplastic since they are derived from hepatocellular carcinomas. In the second method the tumorigenicity of the cells at the time of implantation into the host animals is unknown and constitutes the purpose of the experiment. Tumor development from cell lines transformed by chemical treatment in vitro typically has a long latency period in vivo, sometimes more than a year elapsing after implantation before tumors occur. Many changes, both phenotypic and genotypic, conceivably may occur during this long latent period of tu morigenesis, and some of these changes may be necessary for the expression of the malignant phenotype (formation of tu mors). Alternately, cells expressing a particular phenotype could have been selected from a phenotypically heterogeneous population. These possibilities make it imperative that "marker" phenotypes be examined in clonally derived sublines, as well as in the tumor cells which have arisen from implanted cells of the clonal sublines. This strategy allows the phenotypes of the cultured cells that were implanted to be compared with phenotypes of cells from the tumors that the implanted cells produced. Among reported studies of carcinogenesis of cultured rat liver epithelial cells, we have been unable to find examples of this type of experiment. In our continuing study of pheno typic expression during in vitro chemical induction of neoplastic transformation of cultured rat liver epithelial cells, we have previously demonstrated that none of the cellular properties which have previously been identified as "markers" of neoplas tic transformation of rat liver epithelial cells and that were expressed by cultured cells in vitro could be used to accurately predict the tumorigenicity of clonal sublines of rat liver epithe lial cells isolated from a phenotypically heterogeneous parental population (7). These markers included (a) hyperdiploid or hypotetraploid aneuploidy; (A) cell-associated fibronectin; (e) the activity of-y-glutamyl transpeptidase; (d) the ability of cells to grow in calcium-poor medium; and (c) the ability of cells to form colonies in soft agar, as well as several other properties not discussed here. In this paper, we report the phenotypic properties of cell lines derived from tumors which have arisen from the implanted clonal sublines of /// vitro transformed rat liver epithelial cells, and we compare the phenotypic properties of the tumor-derived cells with the properties of the progenitor cells from which the tumors were derived. The results of this study indicate that further modulation in the phenotypic expression of the im planted cells occurs during the prolonged period of tumor latency in vivo. The characteristics of the phenotypic modula tion in vivo suggest that the diverse phenotypic properties of the clonal sublines converge to a more-or-less common pheno type as the implanted cells proliferate to form tumors ;'// vivo. However, only the ability of cells to grow in soft agar was precisely correlated with tumorigenicity. 1282