Drug resistance remains a major clinical problem despite advances in targeted therapies. In recent years, methods to culture cancer cells in three-dimensional (3D) environments to better mimic native tumors have gained increasing popularity. Nevertheless, unlike traditional two-dimensional (2D) cell cultures, analysis of 3D cultures is not straightforward. Most biochemical assays developed for 2D cultures have to be optimized for use with 3D cultures. We addressed this important problem by presenting a simple method of quantitative size-based analysis of growth and drug responses of 3D cultures of cancer cells as tumor spheroids. We used an aqueous two-phase system to form consistently sized tumor spheroids of colorectal cancer cells. Using spheroid images, we computed the size of spheroids over time and demonstrated that growth of spheroids from this analysis strongly correlates with that using a PrestoBlue biochemical assay optimized for 3D cultures. Next, we cyclically treated the tumor spheroids with a MEK inhibitor, trametinib, for 6-day periods with a recovery phase in between. This inhibitor was selected because of mutation of colon cancer cells in the MEK/ERK pathway. We used size measurements to evaluate the efficacy of trametinib and predict development of resistance of colon cancer cells during the cyclical treatment and recovery regimen. This size-based analysis closely matched the biochemical analysis of drug responses of spheroids. We performed molecular analysis and showed that resistance to trametinib emerged due to feedback activation of the PI3K/AKT signaling pathway. Therefore, we combined trametinib with a PI3K/AKT inhibitor, dactolisib, and demonstrated that size-based analysis of spheroids reliably allowed quantifying the effect of the combination treatment to prevent drug resistance. This study established that size measurements of spheroids can be used as a straightforward method for quantitative studies of drug responses of tumor spheroids and identifying drug combinations that block resistance.

中文翻译：

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基于尺寸大小的肿瘤球体分析和对治疗的反应。

尽管靶向疗法取得了进展，但耐药性仍然是主要的临床问题。近年来，在三维（3D）环境中培养癌细胞以更好地模拟天然肿瘤的方法越来越受欢迎。然而，与传统的二维（2D）细胞培养不同，对3D培养的分析并不简单。针对2D培养物开发的大多数生化分析必须针对与3D培养物一起使用进行优化。我们通过提出一种简单的基于大小的定量分析方法来分析癌细胞的3D培养物（如肿瘤球体）的生长和药物反应，从而解决了这一重要问题。我们使用了水相两相系统来形成大小一致的结直肠癌细胞肿瘤球体。使用球状图像，我们计算了随时间变化的球体大小，并证明此分析中球体的生长与使用针对3D培养优化的PrestoBlue生化测定法密切相关。接下来，我们用MEK抑制剂曲美替尼对肿瘤球体进行了为期6天的循环治疗，其间处于恢复阶段。选择该抑制剂是因为结肠癌细胞在MEK / ERK途径中发生了突变。我们使用尺寸测量来评估曲美替尼的功效，并预测周期性治疗和恢复方案期间结肠癌细胞的耐药性发展。这种基于大小的分析与球体药物反应的生化分析非常匹配。我们进行了分子分析，结果表明，由于PI3K / AKT信号通路的反馈激活，出现了对曲美替尼的耐药性。因此，我们将曲美替尼与PI3K / AKT抑制剂dactolisib组合使用，并证明了基于大小的球体分析可靠地量化了组合治疗预防耐药性的效果。这项研究确定了球体的尺寸测量可以用作定量研究肿瘤球体药物反应和鉴定阻止耐药性的药物组合的直接方法。