巨噬细胞M1/M2型极性转化对肝转移瘤微循环构建的作用与机制

Liver metastases are most often derived from cancer derived from the digestive tract. Immune tolerance is often induced via portal vein inoculation in the liver. Tissue macrophages, which are characterized as M1/inhibit or M2/heal in varied situations, are the most important line of defense of the innate immunity. In the previous research, we found that disseminated colorectal cancer (CRC) cells via spleen injection into the C57BL/6 mouse liver formed tube-like structures around liver sinusoid and central veins, where they entered the organ; the metastatic niche gradually establish their microcirculation system, and the metastases progress rapidly in a replacement growth pattern. The liver macrophages are accumulated in situ. Based on these, we hypothesize that the liver macrophages function as M2/heal to facilitate microcirculation establishment of the liver metastases. The rate-limiting steps include the establishment of Vasculogenic Mimicry (VM) network lined by metastatic CRC cells; integration of the liver arterial circulations with the VM network, phagocytosis on unnecessary liver tissue to provided space for rapidly growing metastases, etc. The methods include selective deletion of macrophages at different stages of liver metastases by i.p. injection of Clodroante liposomes ® in the mouse model; casting molds of the liver arterial circulation structures after perfusion and scanned electronic microscopy; apply in specialized tissue staining; analysis of the differentiation of GFP+bone marrow-derived stem cell into liver macrophages, and targeted inhibition/activation of a specific stem cells factor molecule on the metastatic CRC cells or systemically with anti-metastasis and anti-angiogenesis antibodies, among others. We aim to elucidate mechanisms and regulations of liver macrophages on the microcirculation establishment and growth of the metastases and promote understanding on the interaction of innate immunity and metastatic cancer, and provide suitable therapeutic strategies for patients with metastatic CRC in future.

肝脏有门脉耐受特征也是消化道转移癌好发器官。组织巨噬细胞是机体固有免疫防线，存在M1/抑制型和M2/修复型。我们前期发现，经门静脉进入鼠肝脏的结肠癌细胞沿肝脉管形成转移巢并逐步构建其管状结构、转移瘤以替换式生长模式快速进展、管周巨噬细胞聚集。该特征类似相关临床标本。我们假设在转移瘤进展期，肝巨噬细胞适时进行M1/M2型极化辅助其构建脉管系统：参与血管生成拟态通路并促进其与肝固有动脉拟合连接（M2型）、吞噬清除作用以提供瘤肝内生长空间（M1型）等。拟用基因敲除模式动物或脂质体等在不同阶段选择性去除巨噬细胞及诱导其M1极化、血管灌注铸型与电镜观察、特殊组织染色、3D结构重建、肝内活体细胞示踪；收集M2型细胞进行基因分型与分子标记；靶分子和/或系统性干预等策略，明确肿瘤相关巨噬细胞亚群分化对肝转移瘤血管新生与重构的动态作用和调控机制，阐明肝固有免疫与转移瘤交互作用规律，为相关临床治疗提供新依据。

背景：肝脏是结直肠癌血行转移最主要的靶器官，约50%病人合并或术后发生肝转移，是病人最主要的死亡原因。肝转移瘤除手术治疗外，可进行化疗、靶向治疗、免疫治疗，以及联合治疗方案。其中，分子靶向药多为靶向异常激活的酪氨酸激酶受体，包括靶向血管生长因子及其受体（VEGFR）的贝伐珠单抗、瑞格菲尼等。此外，肝动脉和结直肠肿瘤区域灌注化疗也取得一定疗效，说明肝转移瘤血供是非常重要的肿瘤生长途径也是治疗途径。.主要研究内容：以免疫适能小鼠结直肠癌肝转移瘤模型为研究对象，对转移瘤微环境中巨噬细胞的来源、分化及其功能作用，特别是促进转移瘤血管生成及肿瘤生长的作用进行了研究探索。通过构建不同时期小鼠肝转移瘤模型，免疫组织化学、免疫荧光、流式细胞术、电镜，以及连续切片免疫组化染色进行3-D重构等，特异性敲除巨噬细胞的动物实验等；通过转录组数据，系统性分析（标本留取）、进一步明确巨噬细胞与肿瘤及微环境互作、临床数据分析及其治疗，提出最佳治疗方案。.重要结果：1）结肠癌细胞肝内转移瘤体系动力学；血管生成拟态及其与肝固有动脉连接在瘤微循环体系构建中的关键环节；2）应用转基因模式动物在不同阶段去除肝巨噬细胞，明确肝巨噬细胞 M1/M2 型极化对于转移瘤微循环体系建立的辅助作用。并对比观察肝微环境中其它基质细胞的作用；3）明确瘤微循环体系构建的关键阶段，肿瘤血管相关巨噬细胞亚群变化、分布、基因型和分子标记；4）通过降低转移性结肠癌细胞侵袭性以及靶向干预巨噬细胞 M2 分化，系统性和局部标本数据分析对瘤微循环构建的作用与关键靶点。.关键数据及其科学意义：本研究项目获得系列性的数据证实肝转移瘤微环境巨噬细胞及血管生成相关的重要作用，通过留取系统性和肿瘤微环境中系列性标本数据（GEO206211），并紧密联系相关临床数据，已发表研究论文，进一步对存留数据的分析将揭示巨噬细胞分群及靶向治疗相关策略，以控制转移瘤肝内生长，具有重要的理论和实际应用意义。