单细胞测序研究CXCL10+中性粒细胞在NSCLC免疫治疗复发中的作用及分子机制

Non-small cell lung cancer (NSCLC) accounts for about 85% of lung cancer, and is the most common malignant tumor with the highest morbidity and mortality. The latest immunotherapy is a breakthrough in the treatment of NSCLC, but some patients will suffer from recurrent drug resistance, and the mechanism is still unclear. Recently, our research group constructed a new orthotopic primary NSCLC model on immune complete mice. We found that this model can better simulate the effect of clinical immunotherapy. Interestingly, after long-term treatment with antibodies to PD-1 / CTLA-4, these mice developed drug resistance to relapse. We preliminarily analyzed the immunocytogram in tumor microenvironment before and after treatment by single-cell transcriptome sequencing. In addition to the known t-cell corresponding changes, we found that tumor-associated neutrophils (TAN), especially CXCL10+ TAN, were associated with immunotherapy resistance. In this study, we will further analyze the subpopulation and molecular characteristics of TAN in the process of tumor immunotherapy, study its function in this process by removing TAN with Ly6G antibody, and finally analyze the correlation between CXCL10+ TAN characteristic gene expression and prognosis in clinical NSCLC samples, especially the effect of immunotherapy. We will further, verify the correlations between these genes and the prognosis and survival of NSCLC tumors, clarify the molecular mechanism of recurrent drug resistance of NSCLC, test potential therapeutic targets of relapse and resistance to immune therapy, and screen the potential therapeutic drugs. This study will help us to understand the regulatory mechanism of the microenvironment of relapse and resistance in NSCLC immunotherapy, and provide ideas for the study of new immunotherapy targets. This study will help to improve the clinical effect of tumor immunotherapy.

非小细胞肺癌（NSCLC）约占肺癌的85%，是发病率和致死率最高的恶性肿瘤。最新的免疫治疗是NSCLC治疗的突破，但部分患者会出现复发耐药，其机制尚不明确。最近我们课题组构建了新型的免疫完全小鼠上的原位原发的NSCLC模型。我们发现该模型能较好的模拟临床免疫治疗的效果。有意思的是长期PD-1/CTLA-4抗体治疗后，这些小鼠会产生耐药复发。通过单细胞转录组测序我们初步分析了治疗前后肿瘤微环境中免疫细胞图谱。除了已知的T细胞对应的变化，我们发现肿瘤相关中性粒细胞（TAN），特别是其中CXCL10+TAN，与免疫治疗耐药相关。本研究中我们将进一步分析TAN在肿瘤免疫治疗过程中的亚群图谱和分子特征，通过Ly6G抗体去除TAN研究其在该过程中的功能，最后分析临床NSCLC样本中CXCL10+TAN特征基因表达与预后，特别是免疫治疗疗效的相关性。本项研究将有助于提高肿瘤免疫治疗的临床效果。

动物模型构建，基于类器官及CRISPR/Cas9基因编辑技术构建了NSCLC腺癌小鼠模型，在该基础上，我们进行免疫治疗（anti-PD-1），该模型很好地模拟了免疫治疗敏感到复发耐药的全过程，多轮治疗后我们成功构建了免疫治疗耐药的小鼠肿瘤模型。完成了免疫治疗敏感及耐药的动物模型构建。构建了体外免疫治疗敏感及耐药的类器官肿瘤模型。NSCLC免疫治疗耐药的机制探究，基于上述构建的免疫治疗敏感及耐药的动物模型，我们对耐药的机制进行研究。通过对未治疗、治疗敏感、复发及复发后耐药的肿瘤单细胞转录组测序数据进行分析,全面描述肿瘤细胞本身及其所在微环境成分的改变，找出免疫治疗耐药的具体分子机制。单细胞转录组测序成功捕获61,572个单细胞，且鉴定出9个细胞群。从肿瘤细胞本身出发，构建从免疫治疗敏感到耐药的肿瘤细胞基因表达的拟时路径，我们发现在免疫治疗耐药的过程中，肿瘤细胞的基因表达发生了较大改变，将差异基因分为4个模块，其中随耐药逐渐上调的基因富集到上皮间质转化（Epithelial-mesenchymal transition, EMT）、血管生成、细胞增殖等通路，表明肿瘤细胞获得了新的分子特征。除了关注肿瘤细胞本身的差异，我们重点关注了耐药肿瘤中微环境的改变。通过单细胞转录组分析，我们发现细胞组分及其差异基因改变较大的是Neutrophil，其中我们发现Neutrophil主要分为4类，其中Lfit1高表达的Neutrophil及Mmp8高表达的Neutrophil在免疫治疗耐药后发生了显著改变，该结果提示这两类Neutrophil可能在耐药肿瘤中发挥重要作用。针对免疫治疗耐药的NSCLC肿瘤的药物筛选。基于我们构建的免疫敏感及耐药的肿瘤类器官体外模型，我们对其进行了初步的药物筛选实验，结果显示，在276个药物中，细胞抑制率大于70%的药物约40个，通过复筛及体内药物治疗实验，我们初步筛选出2个在体内外均能很好抑制免疫治疗耐药的NSCLC肿瘤生长的药物。.