炎症癌相关成纤维细胞介导结直肠癌细胞耐药的机制研究

Mechanism of inflammatory cancer-associated fibroblast-mediated drug resistance in colorectal cancer cells

背景与目的:结直肠癌(colorectal cancer,CRC)是常见的消化系统恶性肿瘤之一,但是其产生肿瘤耐药的机制仍不清楚。肿瘤微环境(tumor microenvironment,TME),尤其是其中的癌相关成纤维细胞(cancer-associated fibroblast,CAF),在肿瘤的发生、发展及耐药过程中都发挥重要作用。本研究旨在探讨炎症CAF(inflammatory CAF,iCAF)对CRC细胞耐药的影响及可能的分子机制。方法:提取来自上海中医药大学附属普陀医院(2022年8月——2022年9月)诊治的CRC患者并经手术切除的CRC组织以获得原代CAF[获得上海中医药大学附属普陀医院伦理委员会批准,编号:PTEC-A-2023-5(S)-1],按照CAF的表面标志物血小板源性生长因子受体α(platelet derived growth factor receptor alpha,PDGFRA)对原代细胞进行分选,筛选出iCAF。使用不添加血清的培养基分别对人结肠成纤维细胞(human intestinal fibroblast,HIF)和iCAF进行培养以获取HIF条件培养基(HIF-conditioned medium,HIF-CM)和iCAF条件培养基(iCAFconditioned medium,iCAF-CM)。根据处理方式将结肠癌细胞分为对照组(不处理)、实验组1(加入HIF-CM)和实验组2(加入iCAF-CM)。观察经HIF或iCAF刺激后CRC细胞的存活率变化、凋亡率、蛋白水平、mRNA水平变化及对Wnt/β-catenin信号转导通路的影响。结果:经iCAF刺激后,CRC细胞的半数抑制浓度(half inhibition concentration,IC50)较对照组和HIF-CM组升高(P<0.05)。与对照组和HIF-CM组相比,iCAF-CM组肿瘤细胞的凋亡率明显下降,凋亡蛋白caspase-3的表达量下降,抗凋亡蛋白Bcl-2、Bcl-xL和survivin表达量均上升(P<0.05)。iCAF-CM组Wnt/β-catenin信号转导通路被激活。结论:iCAF可以介导CRC细胞产生耐药,其机制与激活Wnt/β-catenin信号转导通路有关。

Background and purpose:Colorectal cancer(CRC)is one of the common malignancies,but the mechanism by which it develops resistance to drug remains unclear.The tumor microenvironment(TME),especially cancer-associated fibroblast(CAF),plays an important role in the occurrence,development and drug resistance of tumors.This study aimed to investigate the effect of inflammatory cancer-associated fibroblasts(iCAF)on drug resistance in CRC cells and its possible mechanism.Methods:The primary CAFs were collected from CRC patients underwent surgery in Putuo Hospital,Shanghai University of Traditional Chinese Medicine from Aug.2022 to Sep.2022,and the primary cells were sorted according to the surface marker of CAF[approved by the Ethics Committee of Putuo Hospital Affiliated to Shanghai University of Traditional Chinese Medicine:PTEC-A-2023-5(S)-1],platelet derived growth factor receptor alpha(PDGFRA),to screen iCAF.Human intestinal fibroblast(HIF)and iCAF cells were cultured using serum-free medium to obtain conditioned medium.According to the treatment method,colon cancer cells were divided into control group(no treatment),experimental group 1(treated with HIF-CM)and experimental group 2(treated with iCAF-CM).We observed the changes in the survival rate and apoptotic rate of CRC cells,the changes in protein and mRNA levels and the effect on the Wnt/β-catenin signaling pathway after stimulation with HIF-CM or iCAF-CM.Results:After iCAFs stimulation,the half inhibition concentration(IC50)of CRC cells was higher compared with the control group and HIF-CM group(P<0.05).Compared with the control group and HIF-CM group,the apoptotic rate of tumor cells in iCAF-CM group decreased significantly,the expression of caspase-3 was decreased,and the expressions of Bcl-2,Bcl-xL and survivin were increased(P<0.05).The Wnt/β-catenin signaling pathway was activated in the iCAF-CM group.Conclusion:iCAFs can mediate drug resistance in CRC cells,and the mechanism is related to the activation of Wnt/β-catenin signaling pathway.