Interleukin 35 Expression Correlates With Microvessel Density in Pancreatic Ductal Adenocarcinoma, Recruits Monocytes, and Promotes Growth and Angiogenesis of Xenograft Tumors in Mice
Interleukin 35 Expression Correlates With Microvessel Density in Pancreatic Ductal Adenocarcinoma, Recruits Monocytes, and Promotes Growth and Angiogenesis of Xenograft Tumors in Mice
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白介素 35 表达与胰腺导管腺癌中的微血管密度相关，募集单核细胞并促进小鼠异种移植肿瘤的生长和血管生成。
DOI：
10.1053/j.gastro.2017.09.039
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发表时间：
2018-02-01
期刊：
影响因子：
29.4
通讯作者：
Ren, He
中科院分区：
文献类型：
--
作者：
Huang, Chongbiao;Li, Zengxun;Ren, He
背景与目的：单核细胞系细胞参与肿瘤血管生成。白介素35(IL35)是IL12家族的一员，由调节性而非效应性T细胞产生。IL35是包含IL12α和IL27β链的二聚体，分别由IL12A和EBI3编码。与正常胰腺组织相比，胰腺导管腺癌组织中IL35的表达增加，促进肿瘤转移。我们研究了IL35在单核细胞诱导的小鼠PDAC血管生成中的作用。方法：我们检测了2010年在中国医院接受手术的123例动脉导管未闭的序贯组织中IL-35蛋白水平、微血管密度和单核细胞数。我们用人PDAC细胞系CFPAC-1、BxPC3、PANC-1、MIA-PACA-2和小鼠PDAC细胞系Pan02进行了研究。用流式细胞仪从人外周血单核细胞中分离出单核细胞亚群。融合的人或鼠IL12A和EBI3基因在PDAC细胞中过表达或被小发夹RNA击倒。细胞在SCID小鼠体内作为异种移植瘤生长；一些小鼠被注射了IL35中和抗体，并监测了肿瘤的生长。我们进行趋化实验来测量IL35募集单核细胞的能力。我们分析了癌症基因组图谱中179个PDAC的mRNA序列，以确定IL12A和EBI3的表达与单核细胞标志物的相关性。单核细胞与IL35或PDAC细胞上清液孵育后，进行管状形成和内皮迁移实验。结果：在患者的PDAC中，IL35mRNA和蛋白的表达水平与微血管密度和单核细胞系细胞的浸润有关。在异种移植瘤的细胞和小鼠中，IL35增加了单核细胞在PDAC肿瘤中的募集，这需要CCL5。单核细胞暴露于IL35后，其产物促进血管生成的基因(CXCL1和CXCL8)的表达增加。IL35通过IL12RB2、STAT1和STAT4的磷酸化，通过诱导gp130信号通路，激活CCL5、CXCL1和CXCL8的转录。抗IL35的中和抗体和吉西他滨的组合显著降低了小鼠PDAC细胞生长的异种移植瘤的单核细胞浸润率、微血管密度和体积。结论：PDAC细胞产生IL35通过CCL5募集单核细胞，并通过表达CXCL1和CXCL8诱导巨噬细胞促进血管生成。IL35信号通路促进小鼠PDAC细胞异种移植瘤血管生成和生长IL35可能成为胰腺癌患者的治疗靶点。
BACKGROUND & AIMS: Cells of the monocyte lineage contribute to tumor angiogenesis. Interleukin 35 (IL35) is a member of the IL12 family produced by regulatory, but not effector, T cells. IL35 is a dimer comprising the IL12 alpha and IL27 beta chains, encoded by IL12A and EBI3, respectively. Expression of IL35 is increased in pancreatic ductal adenocarcinomas (PDACs) compared with normal pancreatic tissues, and promotes metastasis. We investigated the role of IL35 in monocyte-induced angiogenesis of PDAC in mice. METHODS: We measured levels of IL35 protein, microvessel density, and numbers of monocytes in 123 sequential PDAC tissues from patients who underwent surgery in China in 2010. We performed studies with the human PDAC cell lines CFPAC-1, BxPC3, Panc-1, MIA-PaCa-2, and mouse PDAC cell line Pan02. Monocyte subsets were isolated by flow cytometry from human peripheral blood mononuclear cells. Fused human or mouse IL12A and EBI3 genes were overexpressed in PDAC cells or knocked down using small hairpin RNAs. Cells were grown as xenograft tumors in SCID mice; some mice were given injections of an IL35-neutralizing antibody and tumor growth was monitored. We performed chemotaxis assays to measure the ability of IL35 to recruit monocytes. We analyzed mRNA sequences of 179 PDACs in the Cancer Genome Atlas to identify correlations between expression of IL12A and EBI3 and monocyte markers. Monocytes incubated with IL35 or PDAC cell supernatants were analyzed in tube formation and endothelial migration assays. RESULTS: In PDAC samples from patients, levels of IL35 mRNA and protein correlated with microvessel density and infiltration of monocyte lineage cells. In cells and mice with xenograft tumors, IL35 increased recruitment of monocytes into PDAC tumors, which required CCL5. Upon exposure to IL35, monocytes increased expression of genes whose products promote angiogenesis (CXCL1 and CXCL8). IL35 activated transcription of CCL5, CXCL1, and CXCL8 by inducing GP130 signaling, via IL12RB2 and phosphorylation of STAT1 and STAT4. A combination of a neutralizing antibody against IL35 and gemcitabine significantly decreased monocyte infiltration, microvessel density, and volume of xenograft tumors grown from PDAC cells in mice. CONCLUSIONS: PDAC cells produce IL35 to recruit monocytes via CCL5 and induce macrophage to promote angiogenesis via expression of CXCL1 and CXCL8. IL35 signaling promotes angiogenesis and growth of xenograft tumors from PDAC cells in mice. IL35 might serve as a therapeutic target for patients with pancreatic cancer.