ASSAY FOR PREDICTING METHOTREXATE SENSITIVITY IN HUMAN BREAST CANCER

THE LONG-RANGE GOAL OF THIS PROPOSAL IS TO DEVELOP AN IN VITRO TEST TO IMPROVE SELECTION OF THERAPIES FOR INDIVD- UAL BREAST CANCER PATIENTS BY PREDICTING RESPONSE TO METHO- TREXATE. EXISTING ASSAYS FOR BREAST CANCER CELLS CANNOT DE-TERMINE METHOTREXATE SENSITIVITY BECAUSE OF REQUIREMENTS FORFOR FEEDER LAYERS AND MEDIUM COMPONENTS THAT CAN RESCUE SEN-SITIVE CELLS. THIS PROPOSA WILL,THEREFORE, EVALUATE 2 DIF- FERENT APPROACHES: (1) IMPROVING AN EXISTING CLONOGENIC AS- SAY BY MANIPULATING A SERUM-FREE MEDIUM BASED ON MCDB 170, SO THAT METHOTREXAATE TOXICITY IS ACHIEVED; AND (2) DEVELOP-ING A TOTALLY DIFFERENT ASSAY FOR METHOTREXATE RESISTANCE THAT MEASURES UPTAKE OF FLUORESCEIN-CONJUGATED DRUG USING A FLUORESCENCE-ACTIVATED CELL SORTER (FACS). THE LATTER ASSAY DOES NOT REQUIRE CLONAL GROWTH. PHASE I WILL ESTABLISH THE NECESSARY MEDIUM FORMULATION FOR THE CLONAL ASSAY AND SHOW THAT METHOTREXATE DOSE-RESPONSE CURVES ARE FEASIBLE FOR NORMAL AND TUMOR-DERIVED EPITHELIAL CELLS. ADDITIONALLY, PHASE I WILL CONTINUE THE FACS STUDIES AND EXPAND POTENTIAL SOURCES OF CLINICAL MATERIAL AND CORRELATIONS SO THAT DURING PHASE II, BOTH ASSAYS MAY BE CONTINUED AND EVALUATED FOR EFFICACY AND PRACTICALITY. THE ASSAYS DEVELOPED IN PHASE I WILL PROVIDE A MEANS TO ISOLATE METHOTREXATE-RESISTANT CELLS; THEIR MECHANISMS OF RESISTANCE WILL BE EXAMINED DURING PHASE II. THUS, THESE IN VITRO ASSAYS WILL NOT ONLY BE USEFUL FOR PREDICTING PA- TIENT SENSITIVITY TESTING, BUT SHOULD ALSO PROVIDE A BASIC FOR IMPROVED DRUG DESIGN.