BNIP3+成纤维细胞与缺氧和炎症相关，预测胰腺导管腺癌的预后和免疫治疗反应

胰腺导管腺癌(PDAC)是最具侵袭性的肿瘤之一，缺乏有效的治疗手段。癌症相关成纤维细胞(CAFs)作为肿瘤微环境的重要组成部分，参与肿瘤进展、预后和治疗反应。本研究旨在探索新型CAF相关靶点，以改善PDAC的治疗策略。下载并整合了GSA数据库中的PDAC单细胞测序数据（CRA001160，n=35），对成纤维细胞进行细分类别。利用功能富集分析和共表达调控网络分析，鉴定不同成纤维细胞亚型的功能表型和生物学特性。通过伪时间分析构建成纤维细胞的分化轨迹，识别初始和终末分化亚型。比较对免疫治疗响应良好和不良的患者中不同成纤维亚型的比例变化，以及基于GSA和GEO数据库分析成纤维亚型与免疫治疗反应的关系。采用分子生物学方法验证BNIP3在CAF中的缺氧和炎症作用。将CAF与胰腺癌细胞共培养，检测其对细胞迁移和侵袭的影响。单细胞分析将成纤维细胞划分为六个亚型，分化轨迹显示BNIP3+成纤维亚型表现出终末分化，其相关的缺氧和炎症反应基因表达逐渐增加。BNIP3+成纤维亚型的特异性过表达基因与PDAC患者的总生存期和无病生存期显著相关。有趣的是，BNIP3+成纤维亚型比例越高，患者对免疫治疗的反应越差，CRTL治疗方案能有效降低BNIP3+成纤维亚型的比例。敲除BNIP3后，CAF的缺氧标志物和炎症因子表达受到抑制。CAF与胰腺癌细胞共培养能增强癌细胞的迁移和侵袭能力，但通过敲除BNIP3可以逆转这一效果。本研究揭示了与缺氧和炎症反应相关的BNIP3+成纤维亚型，密切关联PDAC患者的不良预后，并鉴定出能预测免疫治疗反应的特征基因。

Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant tumors that lacks effective treatment options. Cancer-associated fibroblasts (CAFs), an important component of the tumor microenvironment, associated with tumor progression, prognosis, and treatment response. This work aimed to explore the novel CAFs-associated target to improve treatment strategies in PDAC.The PDAC single-cell sequencing data (CRA001160, n = 35) were downloaded and integrated based on GSA databases to classify fibroblasts into fine subtypes. Functional enrichment analysis and coexpression regulatory network analysis were used to identify the functional phenotypes and biological properties of the different fibroblast subtypes. Fibroblast differentiation trajectories were constructed using pseudochronological analysis to identify initial and terminally differentiated subtypes of fibroblasts. The changes in the proportions of different fibroblast subtypes before and after PDAC immunotherapy were compared in responsive and nonresponding patients, and the relationships between fibroblast subtypes and PDAC immunotherapy responsiveness were determined based on GSA and GEO database. Using molecular biology methods to confirm the effects of BNIP3 on hypoxia and inflammation in CAFs. CAFs were co cultured with pancreatic cancer cells to detect their effects on migration and invasion of pancreatic cancer.Single-cell data analysis divided fibroblasts into six subtypes. The differentiation trajectory suggested that BNIP3+ Fibro subtype exhibited terminal differentiation, and the expression of genes related to hypoxia and the inflammatory response increased gradually with differentiation time. The specific overexpressed genes in the BNIP3+ Fibro subtype were significantly associated with overall and disease progression-free survival in the patients with PDAC. Interestingly, the greater the proportion of the BNIP3+ Fibro subtype was, the worse the response of PDAC patients to immunotherapy, and the CRTL treatment regimen effectively reduced the proportion of the BNIP3+ Fibro subtype. After knocking out BNIP3, the hypoxia markers and inflammatory factors of CAFs were inhibited. Co-culture of CAFs with pancreatic cancer cells can increase the migration and invasion of pancreatic cancer, but this could be reversed by knocking out BNIP3.This study revealed the BNIP3+ Fibro subtype associated with hypoxia and inflammatory responses, which was closely related to the poor prognosis of patients with PDAC, and identified signature genes that predict the immunotherapy response in PDAC.