补体C3调控巨噬细胞自噬促进肺癌转移的作用和机制

Our preliminary data shown that the serum level of complement component 3 (C3) and the infiltration of macrophages were associated with distant metastasis and poor prognosis in patients with lung cancer. The phenotype of macrophages changed to M2 after treated by supernatant of lung cancer cells and complement C3. Further experiment found that macrophages expressed C3 receptors and the macrophages autophagy is reduced after treated by tumor supernatant or complement C3. We also observed that these macrophages could promote the invasion ability of lung cancer cells in vitro. We thus supposed that complement C3 might promote the invasion and metastasis of lung cancer by regulating macrophages autophagy. To test this hypothesis, this project will at first observe the serum level of complement C3 and the expression of C3, C3R, and macrophages in lung cancer tissues and its relationship with clinical pathological characteristics and prognosis to clarify the predictive effects of complement C3 on the prognosis and curative effect of patients with lung cancer. Then we will detect the effects and mechanism of complement C3 on the phenotypes and autophagy of macrophages. Finally the effects and mechanisms of autophagy on the phenotypes and functions of macrophages and its influence on the invasion and metastasis of lung cancer cells will be observed. It will be expected to illuminate the possible ways to promote lung cancer metastasis by complement C3 through regulating macrophages autophagy and to provide a new method for the treatment of lung cancer.

我们前期发现肺癌患者外周血补体C3含量及组织中浸润的巨噬细胞与远处转移和不良预后有关。进一步实验表明，用肺癌细胞培养液或补体C3处理后的巨噬细胞向M2型转化，且后者C3受体C3R的表达增加；同时经上述处理后的巨噬细胞表达LC3-II/I减少，P62升高，提示其自噬水平降低，并可增强肺癌细胞的体外侵袭能力。据此推测补体C3可能通过调控巨噬细胞自噬继而促进肺癌的侵袭和转移。为证实这一假说，本项目拟首先通过大样本资料深入阐述肺癌患者血清补体C3含量及C3、C3R、巨噬细胞在肺癌组织中的表达及其与临床病理特征和预后的关系，明确C3对肺癌患者预后及疗效的预测作用；继而检测补体C3对巨噬细胞表型和自噬的调控作用并探讨其相关机制；最后观察巨噬细胞自噬对其表型和功能及肺癌细胞侵袭转移的影响并明确其作用机理。有望初步阐明补体C3通过调控巨噬细胞自噬继而促进肺癌转移的可能途径，为肺癌的治疗提供新的思路和手段。

研究表明，免疫微环境在恶性肿瘤的发生发展中起重要作用。本研究中我们从临床数据与基础研究等方面探讨了体液免疫和细胞免疫在肺癌侵袭和转移中的作用，以期为肺癌患者提供新的预后指标和潜在治疗策略。.首先，我们收集了308例晚期转移性NSCLC患者，发现AGR（一种基于炎症的指数）与PNI、LMR、PLR和NLR等实验室参数及患者的预后显著相关。AGR低预示OS和PFS差。另一方面，我们将患者按晚期肺癌炎症指数（ALI）分为两组，结果显示高ALI组的OS优于低ALI组。提示AGR和ALI可作为晚期NSCLC患者的有效预后指标。.另外，我们利用癌症基因组图谱（TCGA）数据库中的生存图数据分析了补体对33种不同癌症生存率的影响。发现补体基因C1QA、C1QB、C3、C3AR1等在肿瘤中的表达与病理分期及CD4+T细胞、巨噬细胞、中性粒细胞和DC等免疫细胞的浸润水平呈正相关。提示补体与恶性肿瘤的发生发展密切相关。.进一步研究发现，肺癌患者外周血单核细胞的C3分泌显著增加，免疫荧光染色法检测显示肺癌组织中可检测到C3表达。对肿瘤组织中浸润的巨噬细胞进行荧光共染色，观察到巨噬细胞为双阳性细胞。继而体外提取小鼠骨髓单核巨噬细胞并诱导分化培养，发现补体C3主要由M1型巨噬细胞分泌，同时采用mC3处理巨噬细胞，发现C3可促进后者表达iNOS，IL-6和IL-1β，抑制Arg-1，TGF-β和TNF-α的表达，同时其自噬水平也发生了改变。体内采用氯膦酸盐脂质体去除巨噬细胞后，发现补体C3的分泌显著减少。这些结果共同提示，肺癌患者的肿瘤组织巨噬细胞和外周血单核细胞均可分泌C3且后者可调节巨噬细胞的自噬和分化。.接下来，我们观察到在肺癌细胞系LLC和A549细胞及其分泌的外泌体中，MALAT1的表达显著增加。采用siMALAT1和DC-siMALAT1干扰其表达后，肿瘤细胞增殖和肿瘤体积显著减少；且DC吞噬功能、炎症反应、共刺激分子表达和T细胞增殖显著增强，而抑制DC自噬，同时p-AKT、AKT、p-mTOR和mTOR水平显著降低。提示肺癌细胞来源的外泌体中MALAT1可抑制DC功能和T细胞增殖、增强DC自噬，提示抑制MALAT1可能是肺癌临床治疗的潜在策略。