Chandrasekaran Siddarth, Deng Huanyun, Fang Ye
Biochemical Technologies, Science and Technology Division, Corning Incorporated, Corning, NY 14831, USA.
Integr Biol (Camb). 2015 Mar;7(3):324-34. doi: 10.1039/c4ib00298a. Epub 2015 Jan 27.
PTEN (phosphatase and tensin homolog), a tumour suppressor negatively regulating the PI3K signalling pathway, is the second most frequently mutated gene in human cancers. Decreased PTEN expression is correlated with colorectal cancer metastases and poor patient survival. Three dimensional (3D) multicellular spheroid models have been postulated to bridge the gap between 2D cell models and animal models for cancer research and drug discovery. However, little is known about the impact of PTEN deletion on the invasion of colon cancer spheroidal cells through a 3D extracellular matrix, and current techniques are limited in their ability to study in vitro 3D cell models in real-time. Here, we investigated the migration and invasion behaviours of the colon cancer cell line HCT116 and its PTEN-/- isogenic cell line using three different in vitro assays, wound healing, transwell invasion, and label-free single cell 3D(2) invasion assays enabled by a resonant waveguide grating (RWG) biosensor. Light microscopic and RWG imaging showed that PTEN deletion influences the spheroid formation of HCT116 cells at high seeding density, and accelerates the spontaneous transfer from the spheroid to substrate surfaces. In vitro migration and invasion assays showed that PTEN knockout increases the 2D migration speed of HCT116 cells, and the invasion rate of individual cells through Matrigel or cells in the spheroid through 3D Matrigel; moreover, the PI3K inhibitor treatment drastically reduces the invasiveness of both cell lines. This study suggests that PTEN knockout potentiates the invasiveness of colorectal cancer spheroidal cells through a 3D extracellular matrix, and the label-free single cell assay is a powerful tool for investigating cancer cell invasion, in particular using 3D cell models.
PTEN（磷酸酶和张力蛋白同源物）是一种对PI3K信号通路起负向调节作用的肿瘤抑制因子，是人类癌症中第二常见的突变基因。PTEN表达降低与结直肠癌转移及患者生存率低相关。三维（3D）多细胞球体模型被认为可填补二维细胞模型与动物模型之间的空白，用于癌症研究和药物发现。然而，关于PTEN缺失对结肠癌细胞球体通过三维细胞外基质侵袭的影响知之甚少，且目前的技术在实时研究体外三维细胞模型方面能力有限。在此，我们使用三种不同的体外实验，即伤口愈合实验、Transwell侵袭实验以及由共振波导光栅（RWG）生物传感器实现的无标记单细胞3D（2）侵袭实验，研究了结肠癌细胞系HCT116及其PTEN基因敲除同基因细胞系的迁移和侵袭行为。光学显微镜和RWG成像显示，PTEN缺失在高接种密度下影响HCT116细胞的球体形成，并加速球体向基质表面的自发转移。体外迁移和侵袭实验表明，PTEN基因敲除增加了HCT116细胞的二维迁移速度，以及单个细胞通过基质胶的侵袭率或球体中的细胞通过三维基质胶的侵袭率；此外，PI3K抑制剂处理显著降低了两种细胞系的侵袭性。本研究表明，PTEN基因敲除增强了结直肠癌球体细胞通过三维细胞外基质的侵袭性，且无标记单细胞实验是研究癌细胞侵袭的有力工具，特别是用于三维细胞模型时。
