背景:结直肠癌(CRC)是全球第三大常见癌症。连接蛋白是参与间隙连接(GJ)形成的跨膜蛋白。我们之前的研究发现连接蛋白37(Cx37),由间隙连接蛋白α4(GJA4)编码,在成纤维细胞上的表达是CRC的启动子,与上皮间质转化(EMT)和肿瘤免疫微环境密切相关。然而,到目前为止,关于GJA4在肿瘤基质中恶性的机制尚未研究。
方法:采用苏木精-伊红(HE)和免疫组织化学(IHC)染色验证GJA4的表达和定位。使用单细胞分析,富集分析,空间转录组学,免疫荧光染色(IF),天狼星红染色,伤口愈合和transwell分析,蛋白质印迹(WB),细胞计数试剂盒-8(CCK8)测定和体内实验,我们研究了GJA4促进CRC的可能机制.
结果:我们发现在CRC中,成纤维细胞上的GJA4参与促进成纤维细胞活化和通过成纤维细胞依赖性途径促进EMT。此外,GJA4可以与M2巨噬细胞协同作用,通过刺激免疫排斥的桥体屏障的形成来限制T细胞浸润。最后,我们发现GJA4与病理分期(P<0.0001)或D2二聚体(R=0.03,P<0.05)之间存在显着相关性。
结论:我们已经确定在成纤维细胞上表达的GJA4实际上是肿瘤间质表型的启动子。我们的发现表明,GJA4成纤维细胞与M2巨噬细胞之间的相互作用可能是增强肿瘤免疫治疗的有效靶标。
BACKGROUND: Colorectal cancer (CRC) is the third most common cancer worldwide. Connexin is a transmembrane protein involved in gap junctions (GJs) formation. Our previous study found that connexin 37 (Cx37), encoded by gap junction protein alpha 4 (
GJA4), expressed on fibroblasts acts as a promoter of CRC and is closely related to epithelial-mesenchymal transition (EMT) and tumor immune microenvironment. However, to date, the mechanism concerning the malignancy of
GJA4 in tumor stroma has not been studied.
METHODS: Hematoxylin-eosin (HE) and immunohistochemical (IHC) staining were used to validate the expression and localization of
GJA4. Using single-cell analysis, enrichment analysis, spatial transcriptomics, immunofluorescence staining (IF), Sirius red staining, wound healing and transwell assays, western blotting (WB), Cell Counting Kit-8 (CCK8) assay and in vivo experiments, we investigated the possible mechanisms of GJA4 in promoting CRC.
RESULTS: We discovered that in CRC,
GJA4 on fibroblasts is involved in promoting fibroblast activation and promoting EMT through a fibroblast-dependent pathway. Furthermore, GJA4 may act synergistically with M2 macrophages to limit T cell infiltration by stimulating the formation of an immune-excluded desmoplasic barrier. Finally, we found a significantly correlation between
GJA4 and pathological staging (P < 0.0001) or D2 dimer (R = 0.03, P < 0.05).
CONCLUSIONS: We have identified GJA4 expressed on fibroblasts is actually a promoter of the tumor mesenchymal phenotype. Our findings suggest that the interaction between GJA4+ fibroblasts and M2 macrophages may be an effective target for enhancing tumor immunotherapy.