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Disaggregation studies of xenograft solid tumors grown from pure or admixed clonal subpopulations from a heterogeneous human colon adenocarcinoma.

Two clonal tumor subpopulations (designated as A and D) obtained originally from a heterogeneous human colon adenocarcinoma (DLD-1) were used to produce xenograft solid tumors in nude mice. First, disaggregation studies were performed to determine the optimal choice of enzyme and time of dissociation for the pure A and D neoplasms, using cell yield (cells/mg/min) and colony forming efficiency (CFE) assays. The enzymes investigated were: 0.5 or 0.2% trypsin, and two cocktails containing pronase (0.5 or 0.05%), collagenase (0.02%), and DNAse I (0.02%). For the 0.5% trypsin treatments, the cell yield from A and D tumor fragments increased until about 30 min, at which time a plateau in cell yield was reached. A plateau in CFE was also reached at this time. In contrast, the cell yields for the 0.2% trypsin treatment did not reach a plateau within the time of the dissociation (120 min), and the CFEs were lower than with the 0.5% trypsin. Whereas no differences in cell yield or CFE were found between the enzyme cocktail studies (0.5% trypsin vs. 0.05% pronase), the cell yield and the CFE from the clone D carcinomas were significantly less than that found with the 0.5% trypsin (the cell yield and CFE from clone A tumors were identical for 0.5% trypsin or enzyme cocktail). These data indicate that, while these clonal neoplasms have somewhat different responses to enzyme disaggregation, it is possible to select an enzyme treatment and treatment time that is appropriate for use on both A and D tumors (i.e., 0.5% trypsin). After determination of an acceptable enzyme procedure, 'reconstructed' heterogeneous tumors produced from an initial injection bolus of 50% clone A and 50% clone D cells were disaggregated as a function of time (days 12-83 postinjection). Over this period, we found that the cell yield decreased exponentially, with a half-time (T1/2) of 20.5 +/- 7.3 days (95% confidence limits), with a maximum extrapolated cell yield at time zero of about 1.2 X 10(5) cells/mg. The CFE was essentially constant over the duration of the assay period. Moreover, it was found that the percentage of clone A cells appeared to decrease exponentially (T1/2 = 20.5 +/- 11.5 days, 95% confidence limits) until about 40 days postinjection. After this time an equilibrium mixture consisting of about 10% clone A cells and 90% clone D cells was reached.(ABSTRACT TRUNCATED AT 400 WORDS)