肿瘤相关巨噬细胞来源外泌体激活IFN通路促进胃癌细胞免疫逃逸

Tumor-associated macrophages-derived exosomes promote immune escape of gastric cancer cells via IFN pathway

为探讨肿瘤相关巨噬细胞(tumor associated macrophage,TAM)外泌体促进胃癌细胞发生免疫逃逸的机制,采用体外诱导M2型巨噬细胞的方法模拟TAM,通过超速离心法提取M2型巨噬细胞外泌体(M2 macrophages’exosome,M2e),并与胃癌细胞共培养以获得摄取了M2e的胃癌细胞,再与CD8^(+)T细胞进行共培养。使用天冬氨酸半胱氨酸特异性蛋白酶(cysteinyl aspartate specific proteinase,Caspase)-3/7的荧光底物和AnnexinV-7-氨基放线菌素D(7-amino-actinomycin D,7-AAD)试剂检测胃癌细胞的凋亡情况,以评估T细胞的杀伤作用。利用MicroArray技术深入探索M2e影响胃癌细胞的信号通路,并用RT-PCR、Western blotting和流式细胞术对这一信号通路进行进一步的验证。结果显示,胃癌细胞能大量摄取M2e。相比未摄取M2e的胃癌细胞,摄取了M2e的胃癌细胞与活化T细胞共培养后,凋亡比例下降,说明其能减弱CD8^(+)T细胞的杀伤作用。MicroArray结果显示,摄取了M2e的胃癌细胞IFN通路被广泛激活。IFN通路激活能促进胃癌细胞表达PD-L1,进而使活化的CD8+T细胞的杀伤功能减弱,因此胃癌细胞产生了免疫逃逸。该研究揭示,巨噬细胞与胃癌细胞的信息交互作用使胃癌细胞产生了抗免疫杀伤作用,胃癌细胞免疫逃逸可能与TAM外泌体有潜在联系。

To explore the mechanism of immune escape of gastric cancer cells promoted by tumor-associated macrophage(TAM)-derived exosomes,M2 macrophages were induced in vitro and M2 macrophages’exosomes(M2 e)were extracted by ultracentrifugation.The M2 e thus produced were co-cultured with gastric cancer cells,and those that having taken M2 e(M2 e-containing gastric cancer cells)were further co-cultured with CD8^(+)T cells.The killing effect of CD8^(+)T cells was evaluated by detection of apoptotic gastric cancer cells by fluorescent substrate of cysteinyl aspartate specific proteinase(Caspase)-3/7 and AnnexinV-7-amino-actinomycin D(7-AAD)reagent.MicroArray technology was used to further explore the signal pathway that may be involved.Finally,RT-PCR,Western blotting and flow cytometry were used to verify the signaling pathway involved.The results showed that gastric cancer cells could uptake a large amount of M2 e.Compared with the original gastric cancer cells,the apoptosis rate of M2 e-uptaking gastric cancer cells decreased after co-culture with activated T cells.This indicated that the exosome could weaken the killing effect of CD8~+T cells.MicroArray results showed that IFN signaling was activated in M2 e-containing gastric cancer cells.The activation of IFN pathway could promote the expression of PD-L1 on gastric cancer cells,which impaired the killing function of activated CD8~+T cells in turn,leading to the immune escape of gastric cancer cells.This study revealed the anti-immune killing effect of gastric cancer cells caused by the exosomes interaction between M2 macrophages and gastric cancer cells,suggesting a connection between gastric cancer immune escape and TAM exosomes.