基于肿瘤干细胞免疫微环境TAMs/CCL5通路探讨扶正抑瘤方逆转前列腺癌去势抵抗的分子机制和物质基础

Castration-resistant (CRPC) progression is the main pathological characteristic of advanced prostate cancer. Recent studies have revealed that immune microenvironment of cancer stem cells (CSCs) plays a critical role in the occurrence and development of prostate cancer. Traditional Chinese Medicine (TCM) has unique priority in the treatment of prostate cancer via regulating cancer cell and its microenvironment. Our previous clinical trials have demonstrated that Fuzheng Yiliu formula could significantly prolong the hormone dependent stage and benefit for the overall survival of prostate cancer. However, its underlying bioactive compounds and molecular mechanisms are still remained largely unknown. Our pilot study revealed that Fuzheng Yiliu formula could significantly inhibit prostate cancer cell growth. More importantly, there was a synergistic effect of Fuzheng Yiliu formula and bicalutamide, an anti-androgen hormone therapeutic agent. Moreover, Fuzheng Yiliu formula could significantly inhibit the growth of prostatic CSCs co-cultured with tumor-associated macrophages (TAMs), as well as reduce the CCL5 level of TAMs. Based on above findings, we hypothesized that FuZhengYiLiu formula might prevent the self-renewal ability of prostatic CSCs via inhibiting TAMs to express CCL5 cytokine which can reverse the anti-androgen resistance of prostate cancer. Taken together, our study will comprehensively explore the significance of CCl5 in regulating prostatic CSCs and the bioactive phytochemicals in Fuzheng Yiliu formula by utilizing multiple techniques including clinical specimen detection, CSCs culture, in vitro and in vivo gene modification and bioactivity-guided fractionation, etc. The study will provide novel strategies and powerful tools for molecular elucidation and therapeutic modalities of CRPC from TCM.

去势抵抗性前列腺癌（CRPC）的发生机制尚不明确。最近研究表明，肿瘤干细胞免疫微环境在前列腺癌发生发展中发挥重要作用，中医药在整体调节肿瘤细胞和微环境治疗前列腺癌方面具有独特优势。前期临床研究显示，扶正抑瘤方显著延长前列腺癌患者激素非依赖性转化时间及总体生存期，其物质基础及作用机制有待阐明。我们预实验发现，扶正抑瘤方对CRPC细胞有显著的增殖抑制和凋亡诱导作用，并且对比卡鲁胺有协同增效作用，抑制TAMs共培养的前列腺癌干细胞的生长，降低TAMs分泌的CCL5 水平。由此我们设想扶正抑瘤方可通过调节TAMs/CCL5信号抑制前列腺癌肿瘤干细胞的自我更新，从而逆转前列腺癌的激素抵抗。本研究拟通过临床标本检测、干细胞培养、体内外基因重组及生物活性导向分离等技术，旨在明确扶正抑瘤方靶向TAMs/CCL5信号通路对前列腺癌干细胞的调控机制及物质基础，为CRPC的发生机制和药物开发提供新的思路。

肿瘤微环境中的前列腺癌干细胞在前列腺癌发生发展中发挥重要作用，中医药在整体调节肿瘤细胞和微环境治疗前列腺癌方面具有独特优势，但中医药调控肿瘤干细胞免疫微环境的机制尚不明确。本项目拟通过临床标本检测、干细胞三维培养、体内外基因重组及生物活性导向分离等技术，探讨扶正抑瘤方调控免疫微环境TAMs/CCL5轴对前列腺癌干细胞及转移的影响，并进一步阐明扶正抑瘤方的作用机制及物质基础。本研究首先证实了临床标本中TAMs分泌的CCL5与前列腺癌细胞的干性、上皮-间充质转化密切相关。分子机制证实了CCL5可通过激活CCR5/β-catenin/STAT3信号途径促进PCSCs自我更新和前列腺癌转移。体内研究进一步证实CCL5促进裸鼠移植瘤的PCSCs、生长及骨转移。临床标本和生物信息学分析表明，前列腺癌患者中CCL5的高表达与高恶性程度、预后差、转移和PCSCs活性增高密切相关。在此基础上，本研究进一步证实了扶正抑瘤方通过抑制TAMs的M2表型极化及抑制CCL5的分泌从而抑制前列腺癌细胞干性及转移。体内实验发现扶正抑瘤方通过调控TAMs/CCL5轴抑制前列腺癌骨转移。在此基础上，通过构建CCL5双荧光素酶报告基因体系及生物活性导向分离技术分离鉴定了淫羊藿苷为扶正抑瘤方中靶向CCL5的活性单体。本研究进一步证实了扶正抑瘤方与恩扎卢胺联合能够进一步促进前列腺癌细胞凋亡，表明扶正抑瘤方具有协同增效作用。此外本研究综述了CCL5作为趋化因子在前列腺癌中的研究进展。本研究阐明了前列腺癌细胞及前列腺癌干细胞的在肿瘤免疫微环境中的特征及调控机制。证实了免疫微环境中TAMs/CCL5是潜在的治疗靶点。进一步阐明了扶正抑瘤方靶向TAMs/CCL5肿瘤免疫调控机制及其物质基础。