目的：从肿瘤相关巨噬细胞（TAM）在化疗前后的表型变化入手，寻找通过调控肿瘤免疫微环境（TIME）从而影响肿瘤治疗效果和预后的功能分子。方法：利用欧洲核苷酸数据库（ENA）的PRJEB45598 数据集，分析进展期胃癌患者化疗前后活检肿瘤组织单细胞测序数据，采用主成分分析（PCA）和一致流形近似与投影（UMAP）降维，获得31 个亚群细胞，并进一步进行TAM亚型分析、差异基因筛选，寻找化疗后M2型TAM中高表达的基因。通过黑色素瘤B16-F10 细胞皮下移植瘤模型验证化疗前后特定基因mRNA和蛋白水平表达变化，并通过Incucyte 体外分析该蛋白是否调控化疗药物诱导的肿瘤细胞死亡。结果：聚焦单细胞测序数据中M2型TAM的特征表达基因，发现半乳糖凝集素3（LGALS3）在胃癌化疗后mRNA水平显著升高（P<0.01），在多种肿瘤中LGALS3 高表达且与患者生存期呈负相关（P<0.05 或P<0.01）。黑色素瘤 B16-F10 细胞移植瘤模型中，LGALS3在M2型TAM中高表达（P<0.01），且奥沙利铂化疗后表达进一步升高（P<0.05）。体外对肿瘤细胞给予重组LGALS3蛋白可抑制化疗药物奥沙利铂诱导的肿瘤细胞死亡（P<0.01）。结论：奥沙利铂化疗后的M2型TAM通过合成和分泌LGALS3促进黑色素瘤细胞的化疗抵抗，因此通过免疫治疗的方法靶向LGALS3分子可能有效提高肿瘤的治疗效果。

Objective: To find functional molecules that regulate tumor immune microenvironment (TIME) and influence therapeutic effect and prognosis by focusing on phenotype changes of tumor-associated macrophages (TAM) before and after chemotherapy.Methods: The data set PRJEB45598 from ENA (European Nucleotide Archive) was used to analyze the single-cell sequencing data of biopsy tumor tissues from patients with advanced gastric cancer before and after chemotherapy. 31 subpopulations of cells were obtained using PCA (principal component analysis) and UMAP (uniform manifold approximation and projection). Further TAM subtype analysis and differential gene screening were performed to find the genes highly expressed in M2-like TAM cells after chemotherapy. The mRNA and protein expression changes of the specific gene before and after chemotherapy were verified by subcutaneous melanoma B16-F10 cell transplanted xenograft model. Whether the protein regulates chemotherapy-induced tumor cell death was analyzed in vitro by Incucyte. Results: Focusing on the characteristic expression genes in M2-like TAM in single-cell sequencing data, we found that the mRNA level of galactose-specific lectin 3 (LGALS3) was significantly increased after chemotherapy (P<0.01), and its high expression in various tumors was negatively related to the survival of patients (P<0.05 or P<0.01). LGALS3 washighly expressed in M2-like TAM in melanoma B16-F10 cell transplanted xenograft model in vivo (P<0.01), and its expression was further increased after oxaliplatin chemotherapy (P<0.05). Recombinant LGALS3 protein inhibited oxaliplatin-induced tumor cell deathin vitro. Conclusion: M2-like TAM promotes chemoresistance of tumor cells through synthesis and secretion of LGALS3 after chemotherapy. Therefore, targeting LGALS3 through immunotherapy may effectively improve the therapeutic effect of tumors.

国家自然科学基金（No.82071762）