Objective Elucidating complex ecosystems and molecular features of gallbladder cancer (GBC) and benign gallbladder diseases is pivotal to proactive cancer prevention and optimal therapeutic intervention. Design We performed single-cell transcriptome analysis on 230 737 cells from 15 GBCs, 4 cholecystitis samples, 3 gallbladder polyps, 5 gallbladder adenomas and 16 adjacent normal tissues. Findings were validated through large-scale histological assays, digital spatial profiler multiplexed immunofluorescence (GeoMx), etc. Further molecular mechanism was demonstrated with in vitro and in vivo studies. Results The cell atlas unveiled an altered immune landscape across different pathological states of gallbladder diseases. GBC featured a more suppressive immune microenvironment with distinct T-cell proliferation patterns and macrophage attributions in different GBC subtypes. Notably, mutual exclusivity between stromal and immune cells was identified and remarkable stromal ecosystem (SC) heterogeneity during GBC progression was unveiled. Specifically, SC1 demonstrated active interaction between Fibro-iCAF and Endo-Tip cells, correlating with poor prognosis. Moreover, epithelium genetic variations within adenocarcinoma (AC) indicated an evolutionary similarity between adenoma and AC. Importantly, our study identified elevated olfactomedin 4 (OLFM4) in epithelial cells as a central player in GBC progression. OLFM4 was related to T-cell malfunction and tumour-associated macrophage infiltration, leading to a worse prognosis in GBC. Further investigations revealed that OLFM4 upregulated programmed death-ligand 1 (PD-L1) expression through the MAPK-AP1 axis, facilitating tumour cell immune evasion. Conclusion These findings offer a valuable resource for understanding the pathogenesis of gallbladder diseases and indicate OLFM4 as a potential biomarker and therapeutic target for GBC. Data are available upon reasonable request.

中文翻译：

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综合单细胞分析破译了胆囊癌发病机制中的微环境动力学和免疫调节剂 olfactomedin 4

目的 阐明胆囊癌（GBC）和良性胆囊疾病的复杂生态系统和分子特征对于主动癌症预防和最佳治疗干预至关重要。设计 我们对来自 15 个 GBC、4 个胆囊炎样本、3 个胆囊息肉、5 个胆囊腺瘤和 16 个邻近正常组织的 230 737 个细胞进行了单细胞转录组分析。通过大规模组织学测定、数字空间分析仪多重免疫荧光（GeoMx）等验证了研究结果。通过体外和体内研究进一步证明了分子机制。结果细胞图谱揭示了胆囊疾病不同病理状态下免疫景观的改变。 GBC 具有更具抑制性的免疫微环境，不同 GBC 亚型具有不同的 T 细胞增殖模式和巨噬细胞属性。值得注意的是，基质细胞和免疫细胞之间的相互排斥性被确定，并且在 GBC 进展过程中显着的基质生态系统 (SC) 异质性被揭示。具体来说，SC1 表现出 Fibro-iCAF 和 Endo-Tip 细胞之间的积极相互作用，与不良预后相关。此外，腺癌 (AC) 内的上皮遗传变异表明腺瘤和 AC 之间存在进化相似性。重要的是，我们的研究发现上皮细胞中嗅觉素 4 (OLFM4) 升高是 GBC 进展的核心因素。 OLFM4 与 T 细胞功能障碍和肿瘤相关巨噬细胞浸润有关，导致 GBC 预后较差。进一步的研究表明，OLFM4通过MAPK-AP1轴上调程序性死亡配体1（PD-L1）的表达，促进肿瘤细胞免疫逃避。结论 这些发现为了解胆囊疾病的发病机制提供了宝贵的资源，并表明 OLFM4 作为 GBC 的潜在生物标志物和治疗靶点。数据可根据合理要求提供。