Efforts to use fresh human
sarcoma cells for evaluating
antifolate resistance with an in situ thymidylate synthesis assay using 5-[3H]
deoxyuridine were unsuccessful because of low thymidylate synthesis activity in enzymatically disaggregated
tumors. By incubating
tumor cell
suspensions in supplemented RPMI-1640 medium with 10%
fetal bovine serum for 3 days, activity of the in situ thymidylate synthesis assay markedly increased (1.42 versus 0.03 pmol/h/10(7) cells), thus allowing 75% of samples to be evaluated for
antifolate sensitivity. By criteria developed with a
methotrexate-resistant and -sensitive cell line, this assay indicated that most
sarcomas are naturally resistant to
methotrexate (12 of 15). Natural resistance to
10-ethyl-10-deazaaminopterin and
trimetrexate was also observed in 60% of the samples (nine of 15, respectively). The results from the 3-day in situ assay were confirmed by specific tests for resistance mechanisms in most
sarcoma samples. The resistance mechanisms detected were impaired polyglutamylation, an increased level of
dihydrofolate reductase, and amplification of this gene. These results encourage further exploration of this assay to predict response to
antifolates in individual patients and to evaluate efficacy of new
antifolates as candidates for clinical trial.