In vitro development of oncogenicity in cell lines established from tracheal epithelium preexposed in vivo to 7,12-dimethylbenz(a)anthracene.

Abstract

The purpose of these studies was to determine whether carcinogenesis initiated in vivo in tracheal epithelium of rats progresses in vitro upon culturing of the epithelium. Transplanted rat tracheas were exposed to either 150 or 640 µg of 7,12-dimethylbenz( a )anthracene over a period of 2 weeks. Epithelial cell lines were derived from tracheas exposed in this manner and were tested periodically in vivo for development of oncogenicity and for phenotypic changes associated with neoplastic transformation in vitro . Three cell lines were obtained from tracheas preexposed to 150 µg 7,12-dimethylbenz( a )anthracene. Tumorigenicity was demonstrated only in the late passages of 2 cell lines maintained to 400 days or more in vitro . With these cells in vivo tumor latency was 115 to 255 days. Eight of the 9 cell lines derived from tracheas preexposed to 640 µg 7,12-dimethylbenz( a )anthracene have become tumorigenic. Several of these were found to be oncogenic after approximately 200 days in vitro and yielded palpable tumors within 9 to 80 days after inoculation. In several instances inoculation of cells from early passage into immunosuppressed isogenic recipients resulted in the development of keratinic cysts or tumors that regressed. Inoculation of later passages of the same cell lines resulted in the development of invasive tumors. Loss of anchorage dependence of growth was, in most cases, a reliable phenotypic marker for development of oncogenicity, as was colony-forming efficiency on plastic. Growth rate, saturation density, and cell and colony morphology were not. All of the cell lines were epithelial as indicated by ultrastructural characteristics, the production of keratin in vitro , and/or the formation of squamous cell carcinomas upon inoculation in vivo . The cell lines had stable morphological characteristics distinguishing each cell line from any other by growth pattern, cell shape, and degree of keratinization. Our data indicate that the process of carcinogenesis initiated in epithelial cells in vivo continues in in vitro culture. Thus it should be possible to study in detail both the evolution of the neoplastic process and factors affecting its progression.