Tumor-Associated Neutrophils Recruit Macrophages and T-Regulatory Cells to Promote Progression of Hepatocellular Carcinoma and Resistance to Sorafenib
Tumor-Associated Neutrophils Recruit Macrophages and T-Regulatory Cells to Promote Progression of Hepatocellular Carcinoma and Resistance to Sorafenib
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肿瘤相关中性粒细胞招募巨噬细胞和 T 调节细胞促进肝细胞癌的进展和索拉非尼耐药
DOI：
10.1053/j.gastro.2016.02.040
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发表时间：
2016-06-01
期刊：
影响因子：
29.4
通讯作者：
Zhou, Jian
中科院分区：
文献类型：
--
作者：
Zhou, Shao-Lai;Zhou, Zheng-Jun;Zhou, Jian
背景与目的：嗜中性粒细胞通过释放细胞因子促进或抑制肿瘤进展，这取决于肿瘤微环境。肿瘤相关中性粒细胞（TAN）产生的因子及其对肿瘤进展的影响均未得到表征。我们使用从患者分离的细胞系和免疫细胞研究了TAN在肝细胞癌（HCC）进展中的作用。方法：我们用HepG 2、PLC/PRF/5、MHCC 97 H和HCCLM 3人和Hepa 1 -6和H22小鼠HCC细胞系进行了研究;用小发夹RNA敲低趋化因子和细胞因子的表达。在趋化性测定中分析细胞，并在小鼠中分析其生长情况。2012年在中国中山医院从20名接受根治性切除术的患者或20名健康个体（对照）收集HCC组织和外周血。TAN和外周血中性粒细胞（PBN）被分离并暴露于HCC细胞系的条件培养基;逆转录聚合酶链反应被用于量化细胞因子和趋化因子的表达。我们从2012年接受HCC根治性切除术的另外60名患者中收集中性粒细胞，以测量C-C基序趋化因子配体2（CCL 2）和CCL 17的产生。患者随访至2014年3月15日。为了进行免疫组化分析，我们收集了2006年至2008年832例接受根治性切除术的HCC患者的HCC组织和配对的、相邻的、非肿瘤性肝组织。所有患者随访至2013年3月15日。为了研究索拉非尼的作用，我们收集了2010年接受根治性切除术的46例患者的临床和病理数据。结果：TANs和HCC细胞激活的PBNs表达最高的细胞因子是CCL 2和CCL 17。TAN中CCL 2和CCL 17信使RNA和蛋白的水平显著高于PBN，并且在HCC复发患者中增加。当PBN暴露于来自HCC细胞系的条件培养基时，CCL 2和CCL 17信使RNA和蛋白质也增加。组织芯片的免疫组化分析显示，CCL 2+和CCL 17+细胞，也表达中性粒细胞标记物CD 66 b，分布在整个HCC基质，但不是在肿瘤细胞或邻近的非肿瘤肝细胞。CCL 2+或CCL 17 + TAN的数量与肿瘤大小、微血管浸润、肿瘤包膜、肿瘤分化和分期相关。肿瘤中CCL 2+或CCL 17+细胞水平较低的患者比这些细胞数量较高的患者生存时间更长。TAN条件培养基以及重组CCL 2和CCL 17在更大程度上增加了来自HCC患者或小鼠的巨噬细胞和T调节（Treg）细胞的迁移活性。针对CCL 2和CCL 17或其受体C-C趋化因子受体2和C-C趋化因子受体4的中和抗体降低了巨噬细胞和Treg细胞的迁移活性。注射到小鼠中的HCC细胞系在与TAN共注射时形成更大的肿瘤，并形成更多的肺转移;这些肿瘤被Ly 6 G+细胞、F4/80+巨噬细胞和Foxp 3 + Treg细胞浸润。在人PBN的磷酸激酶阵列中，暴露于来自所有4种HCC细胞类型的条件培养基后，磷酸化AKT和P38的水平增加。AKT和P38的药理学抑制剂抑制这些PBN分泌CCL 2和CCL 17。在荷瘤小鼠中，索拉非尼增加了肿瘤中TAN的数量以及CCL 2和CCL 17的水平。手术前接受索拉非尼治疗的患者的HCC组织比未接受索拉非尼治疗的患者的组织含有更多的TAN。在敲除细胞中，HCC细胞衍生的CXCL 5是在缺氧条件下中性粒细胞迁移的最强效应子。在小鼠中，索拉非尼和TAN消耗的组合比单独索拉非尼更大程度地抑制肿瘤生长和新血管形成。结论：TANs募集巨噬细胞和Treg细胞到HCC中，以促进其生长、进展和对索拉非尼的耐药性。
BACKGROUND & AIMS: Neutrophils can either promote or inhibit tumor progression, depending on the tumor microenvironment, via release of cytokines. Neither the factors produced by tumor-associated neutrophils (TANs) nor their effects on tumor progression have been characterized. We investigated the roles of TANs in progression of hepatocellular carcinoma (HCC) using cell lines and immune cells isolated from patients. METHODS: We performed studies with HepG2, PLC/PRF/5, MHCC97H, and HCCLM3 human and Hepa1-6 and H22 mouse HCC cell lines; expression of chemokines and cytokines were knocked down with small hairpin RNAs. Cells were analyzed in chemotaxis assays and as growth as tumors in mice. HCC tissues and peripheral blood were collected from 20 patients undergoing curative resection or 20 healthy individuals (controls) in 2012 at Zhongshan Hospital in China. TANs and peripheral blood neutrophils (PBNs) were isolated and exposed to conditioned media from HCC cell lines; reverse-transcription polymerase chain reaction was used to quantify the expression of cytokines and chemokines. We collected neutrophils from another 60 patients undergoing curative resection for HCC in 2012 to measure the production of C-C motif chemokine ligand 2(CCL2) and CCL17. Patients were followed up until March 15, 2014. For immunohistochemical analyses, we collected HCC tissues and paired, adjacent, nontumor cirrhotic liver tissues from 832 HCC patients undergoing curative resection from 2006 through 2008. All patients were followed up until March 15, 2013. To study the effects of sorafenib, we collected clinical and pathology data from 46 patients who underwent curative resection in 2010. RESULTS: CCL2 and CCL17 were the cytokines most highly expressed by TANs and HCC cell-activated PBNs. Levels of CCL2 and CCL17 messenger RNAs and proteins were significantly higher in TANs than in PBNs, and increased in patients with HCC recurrence. CCL2 and CCL17 messenger RNA and proteins also increased when PBNs were exposed to conditioned media from HCC cell lines. Immunohistochemical analysis of a tissue microarray showed that CCL2+ and CCL17+ cells, which also expressed the neutrophil marker CD66b, were distributed throughout the HCC stroma, but not in tumor cells or the adjacent nontumor liver cells. The number of CCL2+ or CCL17+ TANs correlated with tumor size, microvascular invasion, tumor encapsulation, tumor differentiation, and stage. Patients whose tumors had lower levels of CCL2+ or CCL17+ cells had longer survival times than those with higher numbers of these cells. TAN-conditioned media, as well as recombinant CCL2 and CCL17, increased the migratory activity of the macrophages and T-regulatory (Treg) cells from patients or mice with HCC to a greater extent that PBN-conditioned media. Neutralizing antibodies against CCL2 and CCL17, or their receptors C-C chemokine receptor 2 and C-C chemokine receptor 4, reduced the migratory activities of macrophage and Treg cells. HCC cell lines injected into mice formed larger tumors when they were co-injected with TANs and formed more pulmonary metastases; these tumors were infiltrated by Ly6G+ cells, F4/80+ macrophages, and Foxp3+ Treg cells. In a phosphokinase array of human PBNs, levels of phosphorylated AKT and P38 increased after exposure to conditioned media from all 4 HCC cell types. Pharmacologic inhibitors of AKT and P38 inhibited secretion of CCL2 and CCL17 by these PBNs. In tumor-bearing mice, sorafenib increased the numbers of TANs and levels of CCL2 and CCL17 in tumors.HCC tissues from patients who received sorafenib before surgery contained more TANs than tissues from patients who did not receive sorafenib. In knockdown cells, HCC cell-derived CXCL5 was the strongest effector of neutrophil migration under hypoxic conditions. In mice, the combination of sorafenib and TAN depletion inhibited tumor growth and neovascularization to a greater extent than sorafenib alone. CONCLUSIONS: TANs recruit macrophages and Treg cells to HCCs to promote their growth, progression, and resistance to sorafenib.