Dynamics of neoplastic development in carcinogen-exposed tracheal mucosa.

Abstract

The qualitative and quantitative cellular changes occurring in tracheal epithelium in vivo during the process of carcinogenesis were investigated by use of an in vitro assay which measures the formation of epithelial foci (EF); this method is called EF assay. The cell or cells giving rise in vitro to an EF which survives and proliferates for at least 1 month after tracheal cells have been seeded into culture dishes was defined as the epithelial focus-forming unit (EFFU). Viable epithelial cells were harvested from heterotopic tracheal grafts immediately and at 2, 4, and 8 months after they had been exposed in vivo for 4 weeks to 165 µg of dimethylbenz( a )anthracene. This carcinogen exposure was known to induce a 9% incidence of invasive tracheal carcinomas. The cell suspensions obtained from individual tracheas (20 tracheas per time point) were seeded into culture dishes, and the number of EF was scored 1 month later. Normal tracheal cells and cells from tracheas exposed to 100 µg of 12- O -tetradecanoylphorbol-13-acetate produced no EF. In contrast, 80 to 90% of the cultures of all tracheas, exposed to dimethylbenz( a )anthracene 8 months prior to the collection of cells, developed one or more EF. During the first 4 months after termination of dimethylbenz( a )anthracene exposure, the number of EF per trachea remained constant at 3 to 4. Between 4 and 8 months, the number of EF per trachea increased 3- to 5-fold. Three different types of EF could be distinguished experimentally: EF that could not be subcultured (EF); EF that could be subcultured but did not grow in soft agarose (EFs,ag−); and EF that could be subcultured and that also grew in soft agarose (EFs,ag+). During the 8 months after exposure, EF that can be subcultured (EFs) increased from 53 to 84%, and EFs,ag+ increased from 23 to 57%. Simultaneously, the frequency of EF dropped from 47 to 16%. The relative frequency of EFs,ag− remained the same with time. Studies with “mixed-population” cell cultures derived from the same tracheas with which the EF assays were carried out showed similar trends, namely, an increase in subculturability and tumorigenicity in cultures established at increasingly longer time intervals after carcinogen exposure. Our findings suggest the existence, in the epithelium of carcinogen-exposed tracheas, of EFFU's endowed with different in vitro growth capacities. The data are consistent with a gradual conversion of EFFU's with limited growth capacity to EFFU's with neoplastic growth capacity as a function of time after carcinogen exposure (EFFUo → EFFUs,ag− → EFFUs,ag+). A tumor induction study, in which tracheal transplants were exposed during a 4-week period to 165 µg dimethylbenz( a )anthracene, showed an incidence of 9% invasive tracheal carcinomas. In comparison, the EF assay demonstrated that at 8 months 80% of such tracheas contain cells with neoplastic potential (EFFUs,ag+). This suggests that, left in the host animals, only a fraction of these cells actually succeed in establishing a malignant tumor.