Data Sheet 1_Identification of tumor associated neutrophils-related genes in triple-negative breast cancer for predicting prognosis and therapeutic response through integrated single-cell analysis.docx

Tumor-associated neutrophils (TANs) significantly influence tumor development, immune system suppression, and the spread of cancer in triple-negative breast cancer (TNBC). However, their molecular pathways and potential for therapy are not completely understood. We utilized Seurat and Harmony to perform quality control, batch correction, and cell annotation on single-cell RNA-seq data from TNBC patients (GSE222854). Comprehensive bioinformatics approaches—including immune infiltration analysis, GSEA, GSVA, drug sensitivity profiling, and ligand-receptor interaction network analysis were combined with functional validation (colony formation and Transwell assays) and clinical correlation studies via polychromatic immunofluorescence. Four TAN-associated genes (RASGRP4, TIMM10B, TNFRSF13C, and GRAP) with distinct roles in TNBC progression were identified. Functional assays revealed pro-tumorigenic effects of RASGRP4, TIMM10B, and GRAP, whereas TNFRSF13C exhibited tumor-suppressive properties. Clinically, elevated RASGRP4 and TIMM10B expression with reduced TNFRSF13C expression correlated with poor survival and accelerated disease progression, underscoring their prognostic significance. Our study revealed RASGRP4, TIMM10B, and TNFRSF13C as promising therapeutic targets in TNBC. Targeting these TAN-associated genes may disrupt pro-tumor immune responses, suggesting novel strategies to improve patient outcomes.

肿瘤相关中性粒细胞（Tumor-associated neutrophils, TANs）对三阴性乳腺癌（triple-negative breast cancer, TNBC）的肿瘤发生、免疫抑制及癌症扩散均具有显著影响。然而，其分子调控通路及治疗潜力尚未完全阐明。本研究利用Seurat与Harmony工具，对三阴性乳腺癌患者的单细胞RNA测序（single-cell RNA-seq）数据（GSE222854）开展质控、批次校正及细胞注释工作。本研究整合了包括免疫浸润分析、基因集富集分析（Gene Set Enrichment Analysis, GSEA）、基因集变异分析（Gene Set Variation Analysis, GSVA）、药物敏感性分析以及配体-受体相互作用网络分析在内的综合生物信息学方法，并结合功能验证实验（集落形成实验与Transwell实验）以及基于多色免疫荧光的临床关联研究。本研究筛选得到4个与TANs相关的基因（RASGRP4、TIMM10B、TNFRSF13C及GRAP），它们在三阴性乳腺癌进展中发挥着截然不同的作用。功能实验结果显示，RASGRP4、TIMM10B与GRAP具有促肿瘤发生作用，而TNFRSF13C则表现出肿瘤抑制活性。临床层面，RASGRP4与TIMM10B表达上调、TNFRSF13C表达下调与患者不良生存结局及疾病进展加速显著相关，凸显了这三个基因的预后价值。本研究证实RASGRP4、TIMM10B及TNFRSF13C可作为三阴性乳腺癌极具潜力的治疗靶点。靶向这些TANs相关基因或可阻断促肿瘤免疫应答，为改善患者临床结局提供全新策略。