DEVELOPMENT OF IMPROVED TECHNIQUES FOR THE CLONING OF HUMAN

THE DEVELOPMENT OF HUMAN TUMOR COLONY FORMING ASSAYS (HTCFA) HAS OPENED NEW POSSIBILITIES FOR BASIC AND CLINICAL CANCER RESEARCH. HOWEVER, THE PERFORMANCE OF THE DOUBLE-LAYER AGAR TECHNIQUE CURRENTLY USED BY THE MAJORITY OF INVESTIGATORS HAS BEEN GENERALLY LOW. IN THIS CONTRACT, WE PROPOSE THE FURTHER DEVELOPMENT OF AN ALTERNATIVE TECHNIQUE OF TUMOR CELL CLONING USING GLASS CAPILLARY TUBES INSTEAD OF PETRI DISHES. THE CAPILLARY HTCFA PROMISES IMPORTANT ADVANTAGES OVER THE 2-LAYER AGAR METHOD, MOST SIGNIFICANTLY BY INCREASING THE PLATING EFFICIENCY AND THE OVERALL GROWTH RATE OF TUMOR SPECIMENS. FURTHERMORE, BECAUSE LESS CULTURE MATERIAL AND FEWER CELLS ARE REQUIRED, THE ASSAY CAN BE PERFORMED MORE ECONOMICALLY AND ON SMALL TUMOR BIOPSIES. THE SYSTEM WILL BE OPTIMIZED FOR VOLUME AND CONCENTRATION OF AGAROSE, NUMBER OF CELLS SEEDED, AND THE DURATION OF INCUBATION. TUMOR COLONY GROWTH SUPPORTING ABILITY OF DIFFERENT TYPES OF MEDIA AND MEDIA SUPPLEMENTS WILL BE EVALUATED. QUALITY CONTROL CRITERIA FOR THE CAPILLARY HTCFA WILL BE DEVELOPED AND EVALUATED. IN PHASE II, IN VITRO DRUG SENSITIVITY OF PATIENTS' TUMORS WILL BE CORRELATED RETROSPECTIVELY WITH PATIENTS' RESPONSE TO THERAPY. ALSO IN PHASE II A PROTOTYPE MICROSCOPE FOR AUTOMATED COLONY COUNTING AND DATA ANALYSIS WILL BE PRODUCED AND TESTED.