Automatic enumeration and characterization of heterogeneous clonal progression in cell transformation.

Most human tumors are clonal in origin, although the cells may be diverse in their properties. Since the tumors evolve through progressive stages over decades of time, it is possible that the conditions that induced the tumor transform many cells, but that selective overgrowth of the fastest growing lead to a clonal population of identified tumor cells. We studied the progression of neoplastic transformation in clones from a population in which about 10% of the cells formed well-defined transformed foci. A few of the clones produced many large foci, but most of the clones produced no foci or only one focus. Maintenance of the nonproducing and low-producing clones under the growth constraint of confluence and low serum concentration, which promotes transformation, led to the production of large numbers of small foci by all of them. Visual inspection revealed considerable heterogeneity in size and density among the foci from each clone, and this was quantitated by computer scanning. Subclonal analysis of focus formation was done on one of the clones after it had undergone further growth constraint to promote transformation. As in the original cloning, some of the subclones produced many large foci, but most produced none. Another round of growth constraint was imposed on the nonproducing subclones, which then became producers of many small foci varying in size and density. The results indicate that most if not all cells in the population respond to growth constraint by undergoing transformation. Though there is wide variation in the degree of transformation, the results are consistent with the view that an entire field of cells exposed to carcinogenic conditions in an animal undergoes some progression toward neoplasia but that heterogeneity of the response followed by selective neoplastic growth may lead to a clonal origin of the clinically detected tumor.