Quantified Using Flow Cytometry Expression of a Mouse Lung Tumor-associated Antigen Heterogeneity and Clonal Variation Related to Cell Surface

Previous reports have established that line 1, a spontaneous BALB/c lung carcinoma, expresses a M, 180,000 tumor-associ ated surface antigen (TSP-180). In this study, using a monoclonal antibody and flow cytometry to quantify cell surface TSP-180 expression, we found that essentially all cells in a tissue cultureadapted line 1 population express TSP-180, but that the amount of TSP-180 expressed by cells is quite heterogeneous. Variation in amount of TSP-180 was found to be in part related to cell size heterogeneity, and to the expression of TSP-180 being cell cycledependent. The amount of surface-expressed TSP-180 corre lated somewhat with cell size, and was greater on the average for cells in the G2 cell cycle compartment. However, cells of a defined size and specific cell cycle stage still showed marked heterogeneity of expression. Even though the average amount of TSP-180 expressed per cell decreased during in vitro propa gation, little change in heterogeneity was observed. To explore whether any TSP-180-related heterogeneity resulted from herit able variation of expression, 263 limiting dilution-derived line 1 clones were analyzed. The majority displayed, shortly after clon ing, heterogeneous TSP-180 profiles and mean TSP-180 levels similar to those observed for the parent tumor. Occasionally, however, clones were isolated that again appeared as hetero geneous as the parent, but differed by as much as 3-fold in mean TSP-180 expression. Extensive passage did not substantially increase the low probability of isolating clones which differed in expression of TSP-180. Differences in TSP-180 expression among clones were found to be relatively stable upon passage, typically maintained after recloning, and large enough to influence clonal susceptibility to TSP-180-directed antibody and comple ment-mediated lysis. Heritable variation in TSP-180 expression among some clones was also shown to be independent of differences related to cell size, cell cycle, or expression of another line 1 surface antigen (P-100). We concluded that although clones demonstrating large heritable differences in TSP-180 expression can occasionally be isolated, line 1 TSP-180 heterogeneity is predominantly nonheritable, being similar to that present in re cently cloned lines, quite stable during in vitro passage, and not totally accounted for by cell cycle or cell size variation.