Despite the remarkable clinical efficacy demonstrated by molecularly targeted cancer therapeutics, the benefits are typically temporary due to the emergence of acquired drug resistance. This has spurred a massive effort by the cancer research community to identify mechanisms used by cancer cells to evade treatment. Among the various methodologies developed and employed to identify such mechanisms, the most commonly used approach has been to model acquired resistance by exposing cancer cells in culture to gradually increasing concentrations of drug over an extended period of time. Here, we employed a less commonly used variation on this approach, wherein resistant cells are selected by immediately exposing cancer cells to a continuous, high concentration of drug. Using this approach, we isolated clones representing three distinct mechanisms of resistance to inhibition of MET kinase activity from a single clonally derived cancer cell line. The emergent clones had acquired resistance through engagement of alternative receptor tyrosine kinases either through upregulation of FGF3 or HBEGF or increased MAPK signaling through an activating V600E mutation in BRAF. Importantly, these mechanisms were not identified using the conventional “ramp-up” approach in previous studies that employed the same cell line. These results suggest that the particular nature of the selection scheme employed in cell culture modeling studies can determine which potential resistance mechanisms are identified and which ones may be missed, highlighting the need for careful consideration of the specific approach used to model resistance in cultured cells. Significance: Through modeling resistance to MET kinase inhibition in cultured cancer cells using single-step, high-dose selection, these findings highlight that the specific nature of the selection protocol impacts which resistance mechanisms are identified. See related commentary by Floros et al., [p. 25][1] [1]: /lookup/volpage/80/25?iss=1

中文翻译：

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一步，大剂量选择方案揭示了癌细胞中获得的对致癌激酶抑制的抗性的不同机制。

尽管分子靶向的癌症治疗剂显示出显着的临床疗效，但由于获得性耐药的出现，其获益通常是暂时的。这激起了癌症研究界的巨大努力，以鉴定癌细胞逃避治疗的机制。在开发和用于识别这种机制的各种方法中，最常用的方法是通过使培养物中的癌细胞在延长的时间段内逐渐暴露于逐渐升高的药物浓度来模拟获得性耐药。在此，我们采用了一种不太常用的方法，其中通过立即将癌细胞暴露于连续的高浓度药物中来选择耐药细胞。使用这种方法，我们从单个克隆衍生的癌细胞系中分离出代表三种不同的抗MET激酶活性抑制机制的克隆。出现的克隆通过FGF3或HBEGF的上调或通过激活BRAF中的V600E突变而增加的MAPK信号传导，通过替代受体酪氨酸激酶的参与获得了抗性。重要的是，在采用相同细胞系的先前研究中，并未使用常规的“加速”方法识别这些机制。这些结果表明，在细胞培养建模研究中采用的选择方案的特殊性质可以确定鉴定出哪些潜在的耐药机制，而哪些可能会遗漏，这突出表明需要仔细考虑用于对培养的细胞进行耐药性建模的特定方法。意义：通过使用单步高剂量选择对培养的癌细胞中MET激酶抑制的抗性进行建模，这些发现表明，选择方案的特殊性质会影响所确定的抗性机制。参见Floros等人的相关评论，[p。25] [1] [1]：/ lookup / volpage / 80/25？iss = 1