Rates of development of methotrexate resistance in heterogeneous mouse mammary tumor cell cultures

Our previous studies have indicated that the expression by tumor cells of sensitivity to chemotherapeutic drugs such as methotrexate can be affected by the presence of other tumor cells; thus, otherwise methotrexate-resistant cells may respond to that drug in the presence of methotrexate-sensitive cells. In order to determine whether tumor heterogeneity also affects the emergence of drug resistance, we measured the rate of development of methotrexate resistance in mixed monolayer cultures of three mammary tumor subpopulation lines (66, 168TFAR, 4T07) that differ in degree of sensitivity to methotrexate. Cultures were treated weekly with 80 nM or 200 nM methotrexate. Each individual cell line was re-isolated from the mixture by passage in selective medium and then assayed for methotrexate sensitivity. Cultures of each of the three lines were treated and assayed in parallel. Few differences in the rate of development of methotrexate resistance were seen among cells from mixtures and cells cultured alone; line 4T07 appeared to become resistant somewhat more rapidly in mixtures. In untreated mixed cultures, line 66, the line least sensitive to methotrexate, gradually became dominant; this process was accelerated in treated cultures. One methotrexate-resistant subline from each parent cell line was tested to determine the mechanisms by which methotrexate resistance was increased. Two lines appeared to have increased levels of dihydrofolate reductase, and one exhibited decreased methotrexate transport as well. The third cell line had neither mechanism. Others have shown that tumor heterogeneity can act as a brake on the rate of development of new metastatic or immunogenic variants. Our data indicate that, at least in the model system we have tested, the rate of development of extrinsic drug resistance is substantially independent of pre-existing clonal diversity.