Table2_Circulating neutrophils activated by cancer cells and M2 macrophages promote gastric cancer progression during PD-1 antibody-based immunotherapy.XLS

Aims: To analyze the correlation between the neutrophil-to-lymphocyte ratio (NLR) and prognosis of advanced gastric cancer (AGC) patients treated by PD-1 antibody-based therapy and to delineate molecular characteristics of circulating neutrophils by single-cell RNA sequencing (scRNA-seq).
Methods: The clinicopathological information of 45 AGC patients receiving PD-1 antibody-based regimens at the Department of Oncology, Ruijin Hospital, was reviewed. Treatment outcomes including objective response rate (ORR), progression-free survival (PFS), and overall survival (OS) were recorded. The correlation between NLR and efficacy of PD-1 antibody-based treatment was analyzed. Single-cell RNA sequencing (scRNA-seq) analysis was performed based on multisite biopsy samples from two AGC patients to explore the molecular characteristics of circulating neutrophils and their pro-tumor mechanisms. Tissue samples from 88 gastric cancer patients who underwent radial gastrectomy were collected for immunochemistry staining.
Results: A high posttreatment NLR was associated with poor outcomes of AGC patients receiving PD-1 antibody-based regimens. scRNA-seq analysis showed that an increased number of circulating neutrophils were found in peripheral blood samples after treatment in which neutrophil cluster 1 (NE-1) was the major subcluster. NE-1 was featured with a neutrophil activation phenotype with the high expression of MMP9, S100A8, S100A9, PORK2, and TGF-β1. NE-1 displayed an intermediate state in pseudotime trajectory analysis with gene function enrichment found in neutrophil activation, leukocyte chemotaxis, and negative regulation of MAP kinase activity. Cellular interaction analysis showed that the chemokine signaling pathway is the major interactional pathway of NE-1 between subclusters of malignant epithelial cells (EP-4) and M2 macrophages (M2-1 and M2-2). In turn, the MAPK signaling pathway and Jak-STAT signaling pathway of EP-4, including IL1B/IL1RAP, OSM/OSMR, and TGFB1/TGFBR2 axes, were identified as interacting pathways between EP-4 and NE-1. The high expression of OSMR in tumor cells was closely correlated with lymph node metastasis of gastric cancer.
Conclusion: The posttreatment NLR could be a poor prognostic marker of AGC patients treated with immune checkpoint inhibitors (ICIs). Subclusters of circulating neutrophils activated by tumor cells and M2 macrophages could participate in gastric cancer progression through signaling interactions with tumor cells.

研究目的：分析接受PD-1抗体类治疗的晚期胃癌（advanced gastric cancer, AGC）患者的中性粒细胞与淋巴细胞比值（neutrophil-to-lymphocyte ratio, NLR）与预后的相关性，并通过单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）阐明循环中性粒细胞的分子特征。
研究方法：回顾性收集瑞金医院肿瘤内科45例接受PD-1抗体类方案治疗的晚期胃癌患者的临床病理资料，记录客观缓解率（objective response rate, ORR）、无进展生存期（progression-free survival, PFS）及总生存期（overall survival, OS）等治疗结局，分析NLR与PD-1抗体类治疗疗效的相关性。采集2例晚期胃癌患者多部位活检样本进行单细胞RNA测序分析，以探究循环中性粒细胞的分子特征及其促肿瘤机制。另收集88例行根治性胃切除术的胃癌患者的组织样本，用于免疫组织化学染色检测。
研究结果：治疗后高NLR与接受PD-1抗体类方案治疗的晚期胃癌患者不良预后相关。单细胞RNA测序分析显示，治疗后外周血样本中循环中性粒细胞数量增多，其中中性粒细胞聚类1（neutrophil cluster 1, NE-1）为主要亚群。NE-1呈现中性粒细胞活化表型，高表达MMP9、S100A8、S100A9、PORK2及转化生长因子β1（transforming growth factor-β1, TGF-β1）。拟时轨迹分析显示，NE-1处于中间状态，其基因功能富集于中性粒细胞活化、白细胞趋化及丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）活性负调控过程。细胞互作分析表明，趋化因子信号通路是NE-1与恶性上皮细胞亚群（EP-4）及M2巨噬细胞（M2-1、M2-2）之间的主要互作通路。进一步发现，EP-4的丝裂原活化蛋白激酶信号通路及Janus激酶-信号转导与转录激活因子（Jak-STAT）信号通路（包括IL1B/IL1RAP、OSM/OSMR及TGFB1/TGFBR2轴）为EP-4与NE-1之间的互作通路。肿瘤细胞中OSMR高表达与胃癌淋巴结转移密切相关。
研究结论：治疗后NLR可作为接受免疫检查点抑制剂（immune checkpoint inhibitors, ICIs）治疗的晚期胃癌患者的不良预后标志物。肿瘤细胞与M2巨噬细胞活化的循环中性粒细胞亚群可通过与肿瘤细胞的信号互作参与胃癌进展。