Leith J T, Faulkner L E, Bliven S F, Lee E S, Glicksman A S, Dexter D L
Invasion Metastasis. 1985;5(6):317-35.
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)
最初从异质性人类结肠腺癌（DLD-1）获得的两个克隆肿瘤亚群（命名为A和D）被用于在裸鼠中产生异种移植实体瘤。首先，进行了细胞解离研究，以确定针对纯A和D肿瘤的酶的最佳选择以及解离时间，采用细胞产量（细胞/毫克/分钟）和集落形成效率（CFE）测定法。所研究的酶包括：0.5%或0.2%的胰蛋白酶，以及两种含有链霉蛋白酶（0.5%或0.05%）、胶原酶（0.02%）和脱氧核糖核酸酶I（0.02%）的混合酶。对于0.5%胰蛋白酶处理，A和D肿瘤片段的细胞产量在约30分钟前增加，此时细胞产量达到平台期。此时CFE也达到平台期。相比之下，0.2%胰蛋白酶处理的细胞产量在解离时间（120分钟）内未达到平台期，且CFE低于0.5%胰蛋白酶处理的情况。虽然在混合酶研究（0.5%胰蛋白酶与0.05%链霉蛋白酶）之间未发现细胞产量或CFE的差异，但克隆D癌的细胞产量和CFE显著低于0.5%胰蛋白酶处理的情况（0.5%胰蛋白酶或混合酶处理时，克隆A肿瘤的细胞产量和CFE相同）。这些数据表明，虽然这些克隆肿瘤对酶解离的反应略有不同，但有可能选择一种适用于A和D肿瘤的酶处理方法和处理时间（即0.5%胰蛋白酶）。在确定了可接受的酶处理程序后，将由初始注射剂量为50%克隆A细胞和50%克隆D细胞产生的“重建”异质性肿瘤作为时间（注射后12 - 83天）的函数进行解离。在此期间，我们发现细胞产量呈指数下降，半衰期（T1/2）为20.5±7.3天（95%置信限），在时间零点的最大外推细胞产量约为1.2×10⁵细胞/毫克。在测定期间，CFE基本保持恒定。此外，发现克隆A细胞的百分比似乎呈指数下降（T1/2 = 20.5±11.5天，95%置信限），直到注射后约40天。在此之后，达到了由约10%克隆A细胞和90%克隆D细胞组成的平衡混合物。（摘要截短至400字）
