Laboratory of Biochemistry, Chulabhorn Research Institute, Chulabhorn Graduate Institute, Bangkok 10210, Thailand.
Oncol Rep. 2018 Apr;39(4):1765-1774. doi: 10.3892/or.2018.6258. Epub 2018 Feb 12.
It is now widely accepted that the tumor microenvironment influences the fate of cancer cells and plays crucial roles in regulating tumor dormancy and chemoresistance. The standard cell culture system on plastic surfaces does not account for cell interactions with the extracellular matrix (ECM), and is thus a less reliable approach to analyze cellular activity ex vivo. In the present study, A549 lung cancer cells were cultured in a semi-solid growth substrate (Matrigel) to mimic the tumor microenvironment and to investigate the role played by ECM proteins, as well as to evaluate the mechanism of cell-ECM communication. A549 cells embedded in semi-solid Matrigel exhibited dormant cell characteristics, including decreased cell proliferation, migration and invasion rates, compared with the corresponding cells cultured on plastic plates. Exposure of A549 cells to Matrigel leads to resistance against conventional chemotherapeutic drugs (etoposide, paclitaxel, vinblastine, doxorubicin and 2-deoxy-D-glucose). Cell cycle distribution analysis indicated that a larger percentage of the cells embedded within semi-solid Matrigel was arrested in the G0/G1 phase. RT-qPCR analysis revealed that A549 cells cultured in semi-solid Matrigel exhibited a marked decrease in the expression levels of genes that are related to tumor progression and invasion (uPA, uPAR, MMP2, MMP7, MMP9 and CXCR4). The effects of altering various signaling pathways, such as p-ERK, p-Akt and p-STAT3, were evaluated, in order to assess whether these pathways could account for the observed responses of the cells. The inhibition of ERK1/2 and Akt activation using specific inhibitors induced G0/G1 arrest and drug resistance. These results demonstrated that Matrigel drove A549 cells into a drug-resistant dormancy state, most likely through inhibition of the ERK1/2 and PI3K/Akt pathways. Cell culture within semi-solid Matrigel offered a simple in vitro model for studying the mechanisms responsible for tumor dormancy and drug resistance. These studies may lead to therapeutic approaches that can eliminate dormant tumor cells and prevent disease recurrence.
现在人们普遍认为肿瘤微环境会影响癌细胞的命运，并在调节肿瘤休眠和化疗耐药方面发挥关键作用。在塑料表面的标准细胞培养系统中，没有考虑到细胞与细胞外基质（ECM）的相互作用，因此，这种方法不太可靠，无法在体外分析细胞的活性。在本研究中，A549 肺癌细胞在半固体生长基质（Matrigel）中培养，以模拟肿瘤微环境，并研究 ECM 蛋白所起的作用，以及评估细胞与 ECM 通讯的机制。与在塑料板上培养的相应细胞相比，嵌入半固体 Matrigel 中的 A549 细胞表现出休眠细胞的特征，包括细胞增殖、迁移和侵袭率降低。A549 细胞暴露于 Matrigel 会导致其对常规化疗药物（依托泊苷、紫杉醇、长春新碱、多柔比星和 2-脱氧-D-葡萄糖）产生耐药性。细胞周期分布分析表明，更多比例的细胞嵌入半固体 Matrigel 中停滞在 G0/G1 期。RT-qPCR 分析显示，在半固体 Matrigel 中培养的 A549 细胞中，与肿瘤进展和侵袭相关的基因（uPA、uPAR、MMP2、MMP7、MMP9 和 CXCR4）的表达水平显著降低。评估了改变各种信号通路（如 p-ERK、p-Akt 和 p-STAT3）的效果，以评估这些通路是否可以解释细胞的观察反应。使用特异性抑制剂抑制 ERK1/2 和 Akt 激活可诱导 G0/G1 期阻滞和耐药性。这些结果表明，Matrigel 使 A549 细胞进入耐药休眠状态，很可能是通过抑制 ERK1/2 和 PI3K/Akt 通路。在半固体 Matrigel 中培养细胞为研究肿瘤休眠和耐药机制提供了一个简单的体外模型。这些研究可能会导致能够消除休眠肿瘤细胞并预防疾病复发的治疗方法。
