肝癌干细胞特异性分泌蛋白SPINK1在调控肿瘤相关巨噬细胞的作用和机制研究

肝癌是我国五大恶性肿瘤之一。靶向及免疫疗法近年发展迅猛，但肝癌细胞异质性及复杂微环境使其在肝癌治疗中收效甚微。肝癌干细胞（TICs）和肿瘤相关巨噬细胞（TAM）在肝癌发展中起重要作用。我们发现体内条件敲除CD133+肝癌细胞能有效减缓肿瘤生长，并显著降低TAM富集。CD133+肝癌细胞显著高表达SPINK1蛋白，SPINK1在体敲低后小鼠存活时间延长，肿瘤内M2-TAM减少，说明肝癌TICs可能通过分泌SPINK1蛋白富集TAM以促进肝癌发展。临床肝癌样品中SPINK1与CD68+及CD163+TAM数量呈正相关，SPINK1蛋白表达水平与肝癌患者生存率呈负相关，表明SPINK1可能成为肝癌治疗的潜在靶点。本课题旨在研究肝癌TICs表达分泌的SPNK1蛋白与TAM的相互关系，揭示SPINK1对调控TAM及促进肝癌发展的机制，明确SPINK1单克隆抗体作为靶向肝癌TICs治疗的新方案。

Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies in China and the prognosis of HCC is dismal. The development of novel targeted therapies and immunotherapies are increasing nowadays. However, effective treatments for HCC remain extremely limited, largely contributed by HCC cells heterogeneity and the intricate tumor microenvironment. Tumor-associated macrophages (TAMs) are correlated with poor prognosis of HCC by providing trophic support to tumor cells and suppressing immune clearance. It is still not clear how macrophages are recruited to the tumor immunity. There is now clear evidence to show that tumor-initiating cells (TICs) represent an important subset responsible for HCC formation and progression. We and others have previously shown the functional role of liver TICs that are phenotypically marked by CD24, CD133, EpCAM and Side Population. Recently we found that in vivo conditional depletion of CD133+ (Prominin1+) HCC cells not only significantly impeded tumor growth, but also reduced TAMs accumulation in the tumor immunity. These results indicate the potential role of HCC TICs in recruiting TAMs for further tumor progression. Targeting this regulatory mechanisms of liver TICs may be a novel treatment option for HCC patients. Our preliminary data suggests secretory SPINK1 to play a critical role in TIC-associated TAM recruitment. Transcriptome sequencing profiling reveals that in HCC mouse models, SPINK1 is preferentially expressed by CD133+ tumor cells. SPINK1 expression in HCC tumors of clinical patients is correlated with poor overall survival and tumor-infiltrating CD68+ and CD163+ macrophages. hSPINK1 recombinant protein is a mediator of macrophage chemotaxis in vitro. Lenti-viral mediated in vivo sh-knockdown of SPINK1 prolonges survival of HCC-bearing mice, reduces TAMs content and M2 macrophage ratio. Based on these findings, we hypothesize (i) that TAMs are preferentially recruited by liver TICs-derived SPINK1 proteins; (ii) that secreted SPINK1 proteins have chemotactic and activating role on TAM; and (iii) that SPINK1 monoclonal antibody may possibly represent a novel targeted therapy of HCC. To test this hypothesis, functional cell / molecular biological studies on HCC and TAM cell lines, lentiviral based in vivo mouse models, as well as analysis of clinical expression data will be performed. Findings from this study will shed lights on how SPINK1 is correlated with TICs and its role in recruiting TICs-elicited TAM, and potentially lead to the establishment of a novel treatment option for HCC by circumventing tumor‐friendly microenvironment.