Rab3D在肝细胞癌耐药复发中通过微环境调控肿瘤干性和药物代谢的机制研究

It is well-known that liver cancer stem cells (LCSCs) and LCSCs-associated microenvironments are critical for chemoresistance and recurrence of hepatocellular carcinoma (HCC) via various secreting pathways. However, due to the complexity of the secretions in tumor microenvironment, targeting a sole secreting factor is hardly capable to improve HCC chemoresistance. Instead of focusing on some secreting factors, this study will investigate the functions of a regulator for secretion, Rab3D, in HCC chemotherapy. Our previous results suggested that Rab3D might function in liver cancer stem cells (LCSCs), and might be involved in immune response and drug metabolism. To verify the function of Rab3D in HCC, we will further analyze the clinical chemotherapeutic samples, chemoresistant cell lines and chemotherapeutic mouse in this study. We will also perform antibody chips analysis on secretions, sequencing and metabolite analysis on chemotherapeutic samples, in vitro co-culture of tumor cells and microenvironment immune cells (especially tumor-associated macrophage and T cells), and related in vitro or in in vivo verifying experiments to investigate the mechanisms of how Rab3D regulates the stemness of LCSCs via specific secretion, how Rab3D modulates the recruitment and activation of immune cells, and how Rab3D functions in immune cells-mediated drug metabolism changes. This study is helpful to clarify the mechanisms of the interaction between LCSCs and tumor microenvironment and their mutual promotion on HCC chemoresistance, providing theoretical basis for improving HCC chemotherapy.

大量研究证实肝癌干细胞（LCSCs）及其所处微环境通过分泌途径影响肝细胞癌（HCC）的耐药和复发。然而由于微环境中分泌谱的复杂性，靶向单种分泌因子并不足以解决化疗耐受的困境。本课题将独辟蹊径，研究负责整个分泌通路的调控蛋白Rab3D，在HCC耐药复发中的作用。本课题前期结果提示Rab3D能影响LCSCs的功能，并参与免疫反应和药物代谢的调控。在此基础上，本课题将分析临床化疗样本、处理HCC耐药株、并构建小鼠化疗模型，以证实Rab3D在耐药过程中的作用。之后，本课题将通过分析分泌蛋白的抗体芯片、化疗耐药样本的测序和代谢结果、以及免疫细胞的体外共培养上清等，验证Rab3D在HCC耐药复发中促进LCSCs干性、影响免疫细胞的招募活化、以及介导免疫细胞调控肿瘤药物代谢的功能，并揭示相关机制。本课题将有助于理解LCSCs和肿瘤微环境共同促进耐药的机理，为改善化疗技术提供坚实的理论依据。

近期有研究发现上皮样肿瘤细胞内部存在共生菌，然而肝细胞癌中是否也存在胞内共生菌，这些菌的作用及相关分子机制仍是未知。我们通过组织芯片、免疫染色、电镜和菌群测序等技术在患者组织和体外共培养体系中证实HCC癌细胞内部能够共存细菌。这些菌通过促干性增加复发风险，其中埃希菌属与复发关系最大。通过体外和体内进一步的研究我们发现，埃希菌通过与Rab3D互作，一方面直接诱导肿瘤细胞增加wnt受体蛋白FZD7的表达，另一方面间接促进微环境中CAFs等其他细胞内对应配体Wnt7b的合成和分泌，最终大幅促进肿瘤细胞的干性。最后，我们在小鼠模型中，对与胞内菌共生的肿瘤进行Rab3D的干扰、抗生素的使用、以及wnt信号的抑制都能显著降低体外成球和体内成瘤能力，提示术前穿刺样本的共生菌检测以及术前后抗生素的使用能够有助于优化治疗方案，改善患者的治疗效果。