肠道细菌经代谢产物短链脂肪酸介导黑色素瘤肝转移对PD-1抗体耐药的机制研究

The application of PD-1 immune checkpoint inhibitor significantly improves the prognosis of patients with advanced melanoma; however, the patients with liver metastases have a poor prognosis. It was found that the enrichment of gut bacteria Bacteroidetes leads to no response to ant-PD-1 treatment. But there is no study about the role of Bacteroidetes in the poor prognosis of liver metastases. And it was only known that Bacteroidetes may mediate extraintestinal immunity through sugar metabolites. Our sequencing study of gut microbiota in other solid tumors also suggested that some bacteria existed in the patients with liver metastases were involved in the production of carbohydrate metabolites, short chain fatty acids. The content of short chain fatty acid, propionate, in peripheral blood was meaused to be significantly higher in melanoma patients with liver metastases. The animal model confirmed that supplementation of propionate could promote the growth of liver metastases. This study intends to use gene sequencing to explore the composition of the gut microbiota of melanoma patients with or without liver metastases. We will perform fecal transplantation in an animal model of liver metastases, to verify the Bacteroidetes enrichment lead to poor response to anti-PD-1 treatment, and use single-cell sequencing to identify the functional cells. We also plan to perform both in vivo and in vitro studies to observe the impact of short chain fatty acids on anti-tumor effect of PD-1 antibody and the changes of biological characters of functional immune cells; and to verify short chain fatty acids act as a histone deacetylase inhibitor to produce these effects. This study will provide an experimental basis for revealing the mechanism of PD-1 antibody resistance and guiding clinical treatment.

免疫检查点抑制剂PD-1抗体改善了晚期黑色素瘤患者预后，但有肝转移者疗效差。研究发现肠道细菌Bacteroidetes富集导致治疗无效，但与肝转移瘤治疗预后的关系不详；且仅知其可能经糖代谢产物介导肠道外调控作用。我们在其他实体瘤的肠道菌群研究发现，肝转移者疗效差可能与糖代谢产物短链脂肪酸相关；外周血短链脂肪酸检测显示黑色素瘤肝转移患者的丙酸含量较高；且动物模型证实其可促进肝转移瘤生长和Treg细胞浸润。本研究拟利用基因测序鉴定有/无肝转移的黑色素瘤患者的肠道菌群，筛选符合预测模型的粪便，移植到肝转移动物模型中，观察PD-1抗体抑瘤效应；经单细胞转录组测序肿瘤浸润免疫细胞，筛选关键细胞；在此动物模型基础上，补充短链脂肪酸，观察其对PD-1抗体抑瘤效果和关键免疫细胞功能的影响；以及其组蛋白去乙酰化酶抑制剂的效应，并在体外实验中验证。本研究将为揭示PD-1抗体耐药机制和指导临床治疗提供实验依据。

微生物对肿瘤发生发展产生的影响日益引起关注。在本项目资助下，我们对接受PD-1抗体治疗的晚期黑色素瘤患者的肠道菌群进行分析，宏基因组测序和功能分析；通过比较其中的治疗有效患者和治疗无效患者的肠道微生物，我们发现了33种丰度差异细菌。其中治疗有效组富集的Faecalibacterium prausnitzii也曾经在其他的文章中被报道。另外治疗有效组还富有与丁酸代谢相关的Clostridium butyricum，Ruthenibacterium和 lactatiformans菌在治疗无效组有明显富集。这些差异菌为后续深入研究挖掘肠道微生物对黑色素瘤的PD-1治疗影响提供了基础。此外，我们对接受PD-1联合靶向药物治疗或者联合化疗的病人进行了肠道微生物进行分析，我们首次报道了联合治疗相对单药治疗组具有更低的肠道微生物多样性以及更低丰度的拟杆菌属，真细菌属和梭菌属。我们的发现对于临床开展联合治疗具有指导意义。我们从55个肠道细菌DNA检测靶点中筛选出在获得不同临床疗效的患者中具表达差异的DNA检测靶点，构建与治疗疗效相关模型，探索性地提出用于预测免疫治疗疗效的中国患者人群的肠道菌群标志物。但在研究中我们发现中国患者的肠道菌群特征与既往文献中欧美人群存在差异性，因此最初的研究假设基于欧美人群肠道菌群特征，并不能用于指导进一步的研究工作开展。因此，在后期研究工作中，我们将研究方向转向与肠道菌密切相关且与黑色素瘤疾病发展高度相关的黑色素瘤瘤内细菌研究，检测了黑色素瘤瘤内细菌的分布，发现瘤内菌与患者临床预后存在相关性，并在黑色素瘤病灶中检测到细菌分泌的胞外囊泡，后续研究将进一步探讨胞外囊泡的生物学功能。同时，我们在该项目资助下还进行了以下研究：1、收集透明细胞肉瘤组织标本，进行全外显子测序分析，并对筛选出的药物靶点进行功能验证；2、回顾性分析了在本中心接受PD-1抗体联合白蛋白紫杉醇方案治疗的晚期黑色素瘤患者的临床基线资料、预后及基因突变情况等，筛选出获益人群。