胰腺星状细胞分泌GCP-2募集的肿瘤相关中性粒细胞促进胰腺癌进展的分子机制

Pancreatic cancer(pancreatic ductal adenocarcinoma, PDAC) is a lethal disease. Despite improvements in the clinical management, the prognosis of PDAC remains dismal. The presence of an intense firbo-inflammatory reaction, composed of immune cells and pancreatic stellate cell (PSC, also known as cancer-associated fibroblasts), is a prominent pathologic feature of PDAC. These components of tumor microenvironment promote the progression of PDAC. However, the relationship between tumor associated neutrophils(TAN) and PSC, and the role of TAN in PDAC, are still unkonwn. Our preliminary study showed that PSC promoted chemotaxis and migration of TAN by secreting granulocyte chemoattractant protein-2(GCP-2); PDAC patients exhibiting high expression of TAN showed a poorer survival rate; TAN could secret high level of APRIL. Thus, we speculated that PSC recruited TAN by secreting GCP-2, then TAN promoted progression of PDAC by secreting APRIL. To confirm this hypothesis, we try to explore the relationship between TAN and PSC, and the effects of TAN on malignant phenotype in PDAC according to in vitro, and in vivo study. To elucidate the mechanism of PSC recruiting TAN by secreting GCP-2, and focus on p38MAPK signaling pathway mediated by GCP-2; To clarify the mechanism of TAN affecting proliferation, migration and invasion of PDAC cells by secreting a proliferation-inducing ligand (APRIL), and focus on PI3K/AKT signaling pathway mediated by APRIL; To test whether targeting TAN is a potential novel strategy for inhibiting the progression of PDAC. The present study provides the foundation for improving treatment of PDAC.

胰腺癌预后差，治疗效果不佳，其显著病理学特征：丰富间质存在明显纤维化反应、免疫细胞和PSC，这些成分构成了促进胰腺癌进展的肿瘤微环境。然而，肿瘤相关中性粒细胞（TAN）与PSC和胰腺癌细胞的作用关系尚未清楚。我们前期研究发现：PSC可分泌GCP-2促进TAN趋化迁移；高表达TAN的胰腺癌患者生存时间更短；TAN分泌高含量的APRIL。因此我们提出假设：PSC可通过分泌GCP-2募集TAN；TAN分泌APRIL参与促进胰腺癌进展。为了证实这一假设，我们通过体外和体内实验，探讨PSC通过GCP-2募集TAN的分子机制，检测TAN的p38MAPK信号通路中基因和蛋白表达变化；探讨TAN分泌APRIL对胰腺癌细胞增殖、迁移和侵袭的影响，重点检测在APRIL影响下，胰腺癌细胞PI3K/AKT信号通路中基因和蛋白表达变化；验证干预TAN的策略是否有助抑制胰腺癌进展，为改善胰腺癌治疗效果提供新思路。

胰腺癌是一种进展迅速、死亡率高的恶性肿瘤，5年生存率不超过10%。胰腺癌显著的病理学特点表现为间质成分较多，纤维增生反应明显。针对胰腺癌间质的研究和治疗策略，有可能提高胰腺癌的综合治疗效果，改善胰腺癌患者的预后。胰腺星状细胞（PSC）是胰腺癌间质的重要组成部分。研究表明，胰腺癌细胞可诱导PSC活化，活化的PSC分泌各种效应因子从而影响胰腺癌细胞在增殖、侵袭、转移、血管生成和抗原调变等生物学行为。同时，肿瘤间质中表达成纤维激活蛋白α (FAPα)的肌成纤维样细胞可促进肿瘤细胞增殖和侵袭、影响肿瘤细胞凋亡、并与恶性肿瘤病人的不良预后密切相关。胰腺癌间质中肌成纤维样细胞的主要来源为胰腺星状细胞，然而针对表达FAPα的胰腺星状细胞在胰腺癌的进展过程中所发挥作用及其相关机制尚无研究报道。此外，目前研究报道，处于肿瘤微环境的肿瘤相关中性粒细胞（TAN）发挥着促肿瘤作用，TAN可通过分泌增殖诱导配体（APRIL）促进肺癌、胃癌、乳腺癌等多种恶性肿瘤的进展，然而目前对于TAN与胰腺癌细胞相互作用及对胰腺癌进展的影响尚未见文献报道。.本研究发现，胰腺癌微环境中FAPα主要由PSC表达。与癌旁组织相比，胰腺癌间质组织中FAPα的表达明显升高。此外，表达FAPα阳性的PSCs（FAPα+ PSCs）数量与淋巴结转移和生存时间相关。信号通路机制上，验证了胰腺癌细胞通过释放TGFβ1，诱导PSCs表达FAPα；FAPα+ PSCs通过释放趋化因子CXCL1促进胰腺癌细胞中酪氨酸激酶受体EphB1和EphB3的磷酸化，CXCL1，EphrinB1和EphrinB3共同通过Akt磷酸化促进胰腺癌细胞的迁移和侵袭。因此，FAPα+ PSCs可通过Akt信号通路促进胰腺癌的迁移、侵袭和转移。 FAPα+ PSCs与胰腺癌细胞的这种相互作用可能为胰腺癌的综合治疗方案提供新的策略。另一方面，胰腺癌微环境下促进PSC释放GCP-2，且有显著募集TAN的作用。另外，TAN培养基中APRIL水平显著高于中性粒细胞组，并且胰腺癌细胞表达APRIL的受体BCMA 和TACI。通过验证，TAN释放的APRIL促进了胰腺癌细胞的增殖。因此，胰腺癌微环境下PSC释放GCP-2募集TAN，TAN释放APRIL增加，进而促进胰腺癌细胞PANC1的增殖活性，为胰腺癌进展的机制研究和治疗方法的探索提供了新的思路。