Metcalf Science Center, Chemistry Department, Boston University.
Pulmonary and Critical Care Medicine, Brigham and Women's Hospital, Harvard Medical School;
J Vis Exp. 2021 Aug 2(174). doi: 10.3791/62793.
Macropinocytosis is a highly conserved, actin-dependent endocytic process that allows the uptake of extracellular material, including proteins and lipids. In proliferating cells, macropinocytosis can deliver extracellular nutrients to the lysosome, processed into critical macromolecule building blocks. Recent studies have highlighted the dependence of multiple cancers on macropinocytosis, including breast, colorectal and pancreatic cancer. Ras mutations are thought to be the driver events behind macropinocytosis initiation, leading to the activation of cellular anabolic processes via the mTORC1 signaling pathway. Interestingly, mTORC1 can also be activated by macropinocytosis independently of Ras. Therefore, macropinocytosis represents a metabolic vulnerability that can be leveraged to target macropinocytic tumors by limiting their access to nutrients therapeutically. In Tuberous Sclerosis Complex (TSC) and Lymphangioleiomyomatosis (LAM), mTORC1-hyperactivation leads to enhanced macropinocytosis and metabolic reprogramming. Here, we describe a flow cytometry-based protocol to assess macropinocytosis in mammalian cells quantitatively. TSC2-deficient MEFs are employed, which exhibit aberrant activation of mTORC1 and have been shown to have increased macropinocytosis compared to TSC2-expressing cells. Cells treated with pharmacologic inhibitors of macropinocytosis are incubated with fluorescently labeled, lysine-fixable, 70 kDa dextran, or fluorescently labeled bovine serum albumin (BSA) assayed by flow cytometry. To date, robust image-based techniques have been developed to quantitatively assess macropinocytosis in tumor cells in vitro and in vivo. This analysis provides a quantitative assessment of macropinocytosis in multiple experimental conditions and complements existing image-based techniques.
巨胞饮作用是一种高度保守的、肌动蛋白依赖性的内吞作用过程，它允许细胞摄取细胞外物质，包括蛋白质和脂质。在增殖细胞中，巨胞饮作用可以将细胞外营养物质运送到溶酶体，被加工成关键的大分子构建块。最近的研究强调了多种癌症对巨胞饮作用的依赖性，包括乳腺癌、结直肠癌和胰腺癌。Ras 突变被认为是巨胞饮作用起始的驱动事件，通过 mTORC1 信号通路导致细胞合成代谢过程的激活。有趣的是，mTORC1 也可以独立于 Ras 被巨胞饮作用激活。因此，巨胞饮作用代表了一种代谢脆弱性，可以通过限制营养物质的摄取来靶向巨胞饮肿瘤，从而达到治疗目的。在结节性硬化症（TSC）和淋巴管平滑肌瘤病（LAM）中，mTORC1 的过度激活导致巨胞饮作用增强和代谢重编程。在这里，我们描述了一种基于流式细胞术的方法来定量评估哺乳动物细胞中的巨胞饮作用。我们使用 TSC2 缺陷的 MEF 细胞，这些细胞表现出 mTORC1 的异常激活，并且已经被证明与表达 TSC2 的细胞相比，具有增加的巨胞饮作用。用巨胞饮作用的药理学抑制剂处理的细胞与荧光标记的、赖氨酸固定的、70 kDa 葡聚糖或荧光标记的牛血清白蛋白（BSA）孵育，然后通过流式细胞术进行分析。迄今为止，已经开发了强大的基于图像的技术来定量评估体外和体内肿瘤细胞中的巨胞饮作用。这种分析提供了对多种实验条件下巨胞饮作用的定量评估，并补充了现有的基于图像的技术。
