摘要
目的探讨高转移性结直肠癌细胞HCT116诱导M2型巨噬细胞极化对结直肠癌免疫逃逸的影响及其机制。方法将结直肠癌细胞条件培养基(CM)与佛波酯(PMA)诱导的M0型巨噬细胞共培养后,通过流式细胞术、实时荧光定量聚合酶链反应(qPCR)和酶联免疫吸附试验(ELISA)观察M2型巨噬细胞极化状态;将CMM0、CMSW480-Mφ和CM_(HCT116-Mφ)与HCT116细胞共培养后,通过Western blot和qPCR检测程序性死亡受体配体1(PD-L1)的表达情况,同时将刺激后的HCT116细胞与人源T淋巴细胞共培养,检测HCT116细胞存活情况及上清液中的肿瘤坏死因子-α(TNF-α)和干扰素-γ(IFN-γ)水平;用Western blot、qPCR和ELISA检测SW480和HCT116细胞中Kruüppel样因子4(KLF4)表达差异;用KFL4抑制剂肯帕罗酮(Ken)预处理HCT116细胞后,将CM_(HCT116-Mφ)和CM_((HCT116+Ken)-Mφ)与M0型巨噬细胞共培养,通过流式细胞术、qPCR、ELISA实验观察M2型巨噬细胞极化状态。将CM_(HCT116-Mφ)和CM_((HCT116+Ken)-Mφ)与HCT116细胞共培养后,通过Western blot和qPCR检测PD-L1的表达情况。结果M0型巨噬细胞在CM刺激后,HCT116-Mφ细胞中CD11b+CD206+细胞比例更高,M2型巨噬细胞标志物白细胞介素(IL)-10及转化生长因子-β(TGF-β)表达更高;相比于CMSW480-Mφ组,CM_(HCT116-Mφ)组的PD-L1蛋白表达水平更高;与T淋巴细胞共培养后,CM_(HCT116-Mφ)组细胞存活最多,同时TNF-α和IFN-γ水平最低;加入Ken后,M2型巨噬细胞极化比例及标志物均降低。与CM_(HCT116-Mφ)组相比,CM_((HCT116+Ken)-Mφ)组HCT116细胞PD-L1表达下降。结论高转移性结直肠癌细胞通过分泌KLF4诱导M2型巨噬细胞极化促进结直肠癌细胞PD-L1表达,促进肿瘤免疫逃逸。该研究有望为提高临床免疫治疗效果提供潜在靶点。
Objective To investigate the effect and mechanism of M2 macrophage polarization induced by HCT116 with highly metastatic colorectal cancer cells on immune escape in colorectal cancer.Methods After the co-culture of colorectal cancer cells conditioned medium(CM)and PMA-induced M0 macrophages,the polarization of M2 macrophages was observed by flow cytometry,real-time polymerase chain reaction(qPCR)and enzyme-linked immunosorbent assay(ELISA)experiments.The CM M0,CM SW480-Mφand CM HCT116-Mφwere co-cultured with HCT116 cells,and the expression of programmed death-ligand 1(PD-L1)was detected by Western blot and qPCR.At the same time,the stimulated HCT116 cells were co-cultured with human T lymphocytes to detect the survival of HCT116 cells and the levels of tumor necrosis factor-α(TNF-α)and interferon-γ(IFN-γ).The difference of kruüppel-like factor 4(KLF4)expression between SW480 and HCT116 cells was detected by Western blots,qPCR and ELISA.After pretreatment of HCT116 cells with KFL4 inhibitor Kenpaullone(Ken),the CM HCT116 and CM HCT116+Ken were co-cultured with M0 macrophages and the polarization of M2 macrophages was observed by flow cytometry,qPCR and ELISA.The CM_(HCT116-Mφ)and CM_((HCT116+Ken)-Mφ)was co-cultured with HCT116 cells,and the PD-L1 expression of HCT116 cells was detected by Western blot and qPCR.Results After the stimulation of M0 macrophages with CM,the proportion of CD11b+CD206+cells in HCT116-Mφcells was higher,and the expression of M2 macrophage markers interleukin(IL-10)and transforming growth factor-β(TGF-β)were higher.Compared with the CM SW480-Mφgroup,the PD-L1 protein expression level was higher in the CM_(HCT116-Mφ)group.After co-culture with T lymphocytes,the cell survival rate are the most in CM_(HCT116-Mφ)group,while the levels of TNF-αand IFN-γwere the lowest.After the addition of Ken,the polarization ratio and markers of M2 macrophages decreased.Compared with CM_(HCT116-Mφ)group,the expression of PD-L1 in HCT116 cells of the CM_((HCT116+Ken)-Mφ)group decreased.Conclusion Highly metastatic colorectal cancer cells induce polarization of M2 macrophages by secreting KLF4,promote PD-L1 expression in colorectal cancer cells,facilitate tumor immune escape,and provide potential targets for clinical immunotherapy.
作者
尚靖
路畅
侯阳波
袁沁
李炜
王海静
陈进宝
Shang Jing;Lu Chang;Hou Yangbo;Yuan Qin;Li Wei;Wang Haijing;Chen Jinbao(Dept of Traditional Chinese Medicine Oncology,Putuo Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200062;Dept of Radiology,Putuo Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200062;Dept of General Surgery,Putuo Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200062;Dept of Neurology,Putuo Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200062;Dept of Pharmacy,Putuo Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200062)
出处
《安徽医科大学学报》
北大核心
2025年第6期1036-1042,共7页
Acta Universitatis Medicinalis Anhui
基金
上海市启明星基金(扬帆专项)(编号:22YF1441400)
上海市普陀区卫生健康系统科技创新项目(编号:ptkwws202409)
上海市第六人民医院联合共建科学研究基金(编号:23-LY-03)
上海中医药大学附属普陀医院百人计划(编号:2022-RCLH-03)
上海中医药大学预算内项目(编号:2021LK055)
上海市卫生健康系统重点学科计划(编号:2024ZDXK0046)。
