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  • 张冰冰,唐海双,张晶*,郑建明*.肿瘤相关成纤维细胞通过IL-6/STAT3通路介导胰腺癌中髓源性抑制细胞的分化[J].第二军医大学学报,2019,40(5):520-527    [点击复制]
  • ZHANG Bing-bing,TANG Hai-shuang,ZHANG Jing*,ZHENG Jian-ming*.Cancer-associated fibroblasts mediate differentiation of myeloid-derived suppressor cells in pancreatic cancer through IL-6/STAT3 pathway[J].Acad J Sec Mil Med Univ,2019,40(5):520-527   [点击复制]
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肿瘤相关成纤维细胞通过IL-6/STAT3通路介导胰腺癌中髓源性抑制细胞的分化
张冰冰,唐海双,张晶*,郑建明*
0
(海军军医大学(第二军医大学)长海医院病理科, 上海 200433
*通信作者)
摘要:
目的 探讨胰腺导管腺癌(PDAC)中肿瘤相关成纤维细胞(CAF)介导髓源性抑制细胞(MDSC)分化相关作用机制及生物学意义,为揭示CAF和MDSC通过重塑胰腺癌微环境促进胰腺癌进展提供理论基础和实验依据。方法 分离纯化PDAC肿瘤组织中原代CAF,以人包皮成纤维细胞(HFF)作为对照,通过实时荧光定量PCR、酶联免疫吸附试验检测筛选CAF中表达上调的细胞因子。分别使用CAF和HFF培养上清培养人外周血单个核细胞(PBMC),观察PBMC的分化情况,研究上述细胞因子参与调节MDSC分化、发挥募集作用的具体机制。结果 分离的原代CAF表达活化标志物α-平滑肌肌动蛋白(α-SMA)和成纤维细胞激活蛋白a(FAPa),对照细胞HFF不表达α-SMA和FAPa。CAF培养上清中白细胞介素6(IL-6)、基质细胞衍生因子1(SDF-1)、单核细胞趋化蛋白1(MCP-1)的表达量均高于HFF培养上清(P均<0.01),而且IL-6、SDF-1、MCP-1的表达量随培养时间的延长而增加(P均<0.01)。与HFF培养上清相比,CAF培养上清能够促进更多的PBMC分化成CD13高表达的中性粒细胞样MDSC(CD13hi-nMDSC;P<0.01)。在培养体系中单独加入人重组IL-6蛋白可以诱导PBMC向CD13hi-nMDSC分化(P<0.01),单独加入人重组SDF-1或MCP-1蛋白不能诱导CD13hi-nMDSC亚群的增加;加入IL-6中和抗体或信号转导与转录激活因子3(STAT3)抑制剂FLLL32后能够明显减少由CAF培养上清诱导的分化(P<0.05)。结论 CAF可通过IL-6/STAT3通路促进PBMC分化为CD13hi-nMDSC。
关键词:  胰腺肿瘤  肿瘤微环境  肿瘤相关成纤维细胞  髓源性抑制细胞  细胞分化
DOI:10.16781/j.0258-879x.2019.05.0520
投稿时间:2019-03-18修订日期:2019-05-15
基金项目:国家自然科学基金(81502466),海军军医大学(第二军医大学)校级教学研究和改革项目(JYC2016028).
Cancer-associated fibroblasts mediate differentiation of myeloid-derived suppressor cells in pancreatic cancer through IL-6/STAT3 pathway
ZHANG Bing-bing,TANG Hai-shuang,ZHANG Jing*,ZHENG Jian-ming*
(Department of Pathology, Changhai Hospital, Naval Medical University(Second Military Medical University), Shanghai 200433, China
*Corresponding authors)
Abstract:
Objective To explore the mechanism and biological significance of differentiation of myeloid-derived suppressor cells (MDSCs) mediated by cancer-associated fibroblasts (CAFs) in the pancreatic ductal adenocarcinoma (PDAC), so as to provide theoretical and experimental basis for revealing the roles of CAFs and MDSCs in promoting the progression of pancreatic cancer by remodeling the pancreatic cancer microenvironment. Methods We isolated and purified primary CAFs from PDAC tumor tissues, and screened the up-regulated cytokines in CAFs by quantitative real-time PCR and enzyme-like immunosorbent assay. The human foreskin fibroblasts (HFFs) were used as controls. Human peripheral blood mononuclear cells (PBMCs) were cultured with supernatant of CAFs and HFFs, respectively. The differentiation of PBMCs was observed and the mechanisms of the above cytokines in regulating the differentiation and recruitment of MDSCs were studied. Results The biomarkers (α-smooth muscle actin[α-SMA] and fibroblast activation protein a[FAPa]) were detected in the isolated primary CAFs, but not found in the HFFs. The expression levels of interleukin 6 (IL-6), stromal cell-derived factor 1 (SDF-1) and monocyte chemotactic protein 1 (MCP-1) in the culture supernatant were significantly gradually increased in the CAFs than those in the HFFs (all P<0.01). Compared with culture supernatant of HFFs, the culture supernatant of CAFs promoted more PBMCs to differentiate into CD13-high expression neutrophil-like MDSCs (CD13hi-nMDSCs; P<0.01). IL-6 human recombinant protein alone in the co-culture system could induce the differentiation of PBMCs into CD13hi-nMDSCs (P<0.01). SDF-1 or MCP-1 human recombinant protein alone could not induce the increase of CD13hi-nMDSCs subpopulation. IL-6 neutralizing antibody or signal transducer and activator of transcription 3 (STAT3) blocker FLLL32 could significantly inhibit the differentiation induced by the culture supernatant of CAFs (P<0.05). Conclusion CAFs can promote the differentiation of PBMCs into CD13hi-nMDSCs via the IL-6/STAT3 pathway.
Key words:  pancreatic neoplasms  tumor microenvironment  tumor-associated fibroblasts  myeloid-derived suppressor cells  differentiation