背景:结直肠癌(CRC)通常表现出对顺铂治疗的耐受性,但潜在的机制仍不清楚。在肿瘤微环境中,巨噬细胞在抵抗化疗的细胞毒性作用中发挥作用,通过参与细胞凋亡来清除化疗药物诱导的凋亡细胞。细胞外囊泡(EV)的参与,肿瘤微环境中的细胞间通信器,在调节有效细胞增殖以促进耐药性方面尚未得到彻底研究。
方法:我们构建了表达GFP荧光的CRC细胞系(包括GFP-CT26和GFP-MC38),以通过流式细胞术分析检测巨噬细胞的胞出量。我们使用多步超速离心方法分离和纯化CRC分泌的EV,并通过电子显微镜和纳米流式细胞术鉴定它们。进行蛋白质组学分析以鉴定CRC-EV携带的蛋白质分子。使用CRISPR-Cas9构建MFGE8敲除CRC细胞系,并通过使用Western印迹的体外和体内实验验证其效果,免疫荧光,和流式细胞仪分析,证实这些电动汽车激活巨噬细胞αvβ3-Src-FAK-STAT3信号通路,从而促进红细胞增多。
结果:在这项研究中,我们发现CRC衍生的EVs(CRC-EVs)可增强顺铂诱导的凋亡CRC细胞的巨噬细胞胞作用.癌症基因组图谱(TCGA)数据库的分析显示,在CRC患者中,efferocytosis相关基因MFGE8的高表达,提示预后较差。此外,基于质谱的蛋白质组分析在CRC-EV中鉴定出高丰度的MFGE8蛋白。利用CRISPR-Cas9基因编辑系统,我们产生了MFGE8敲除的CRC细胞,证明他们的电动汽车不能在体外和体内上调巨噬细胞的有效细胞增殖。此外,我们证明了在CRC-EVs中的MFGE8通过增加细胞表面αvβ3的表达来刺激巨噬细胞的细胞增殖,从而激活细胞内Src-FAK-STAT3信号通路。
结论:因此,这项研究强调了携带MFGE8的CRC-EV激活巨噬细胞有效胞吞作用的机制.这种激活促进了顺铂诱导的凋亡CRC细胞的清除,促进CRC对顺铂的耐药。这些发现为化疗药物的潜在协同应用提供了新的见解,电动汽车抑制剂,和Efferocytosis拮抗剂用于CRC治疗。
BACKGROUND: Colorectal cancer (CRC) commonly exhibits tolerance to cisplatin treatment, but the underlying mechanisms remain unclear. Within the tumor microenvironment, macrophages play a role in resisting the cytotoxic effects of chemotherapy by engaging in efferocytosis to clear apoptotic cells induced by chemotherapeutic agents. The involvement of extracellular vesicles (EVs), an intercellular communicator within the tumor microenvironment, in regulating the efferocytosis for the promotion of drug resistance has not been thoroughly investigated.
METHODS: We constructed GFP fluorescent-expressing CRC cell lines (including GFP-CT26 and GFP-MC38) to detect macrophage efferocytosis through flow cytometric analysis. We isolated and purified CRC-secreted EVs using a multi-step ultracentrifugation method and identified them through electron microscopy and nanoflow cytometry. Proteomic analysis was conducted to identify the protein molecules carried by CRC-EVs. MFGE8 knockout CRC cell lines were constructed using CRISPR-Cas9, and their effects were validated through in vitro and in vivo experiments using Western blotting, immunofluorescence, and flow cytometric analysis, confirming that these EVs activate the macrophage αvβ3-Src-FAK-STAT3 signaling pathway, thereby promoting efferocytosis.
RESULTS: In this study, we found that CRC-derived EVs (CRC-EVs) enhanced macrophage efferocytosis of cisplatin-induced apoptotic CRC cells. Analysis of The Cancer Genome Atlas (TCGA) database revealed a high expression of the efferocytosis-associated gene MFGE8 in CRC patients, suggesting a poorer prognosis. Additionally, mass spectrometry-based proteomic analysis identified a high abundance of MFGE8 protein in CRC-EVs. Utilizing CRISPR-Cas9 gene edition system, we generated MFGE8-knockout CRC cells, demonstrating that their EVs fail to upregulate macrophage efferocytosis in vitro and in vivo. Furthermore, we demonstrated that MFGE8 in CRC-EVs stimulated macrophage efferocytosis by increasing the expression of αvβ3 on the cell surface, thereby activating the intracellular Src-FAK-STAT3 signaling pathway.
CONCLUSIONS: Therefore, this study highlighted a mechanism in CRC-EVs carrying MFGE8 activated the macrophage efferocytosis. This activation promoted the clearance of cisplatin-induced apoptotic CRC cells, contributing to CRC resistance against cisplatin. These findings provide novel insights into the potential synergistic application of chemotherapy drugs, EVs inhibitors, and efferocytosis antagonists for CRC treatment.