肝内胆管癌（ICC）是一种预后不良且治疗选择有限的疾病。我们研究了肿瘤免疫微环境 (TIME)，以确定疾病结果的预测因素并探索治疗调节的目标。肝组织样本是在 2008 年至 2019 年期间从诊断为 ICC 的患者 (n = 139) 中收集的，这些患者在未接受新辅助化疗的情况下接受了治疗性手术。在组织微阵列 (TMA) 上对来自发现队列 (n = 86) 的样本进行免疫组织化学分析，以了解肿瘤核心和基质区域中 CD68、CD3、CD4、CD8、Foxp3、PD-L1、STAT1 和 p-STAT1 的表达。使用 QuPath 软件对结果进行数字分析，并与临床病理特征相关。为了验证 TIME 相关生物标志物，我们在验证队列中进行了多重成像质谱流式分析 (IMC) (n = 53)。CD68 细胞是 ICC TIME 中的主要免疫细胞类型。 CD4 高 T 细胞密度与更好的总生存 (OS) 相关。预测模型与验证队列一起证实了 CD4 细胞、基质中免疫细胞的 PD-L1 表达和 N 期与总体疾病结果的相关性。反过来，IMC 分析显示沉默的 CD3 CD4 簇对生存产生负面影响。在注释的免疫细胞簇中，PD-L1 与 CD4 FoxP3 细胞的表达最为相关。具有高密度免疫细胞（“热”簇）的肿瘤子集与 PD-L1 表达相关，并且可以识别一组免疫检查点抑制 (ICI) 的候选者。最终，较高水平的 STAT1 表达与较高的淋巴细胞浸润和 PD-L1 表达相关。这些结果强调了 CD4 T 细胞在针对 ICC 的免疫反应中的重要性。其次，具有“热”TIME 的肿瘤子集代表了 ICI 的潜在候选者，而刺激 STAT1 通路可能是 ICC 中将“冷”TIME 转变为“热”TIME 的潜在目标。© 2024 作者。经泰勒许可出版

Intrahepatic cholangiocarcinoma (ICC) is a disease with poor prognosis and limited therapeutic options. We investigated the tumor immune microenvironment (TIME) to identify predictors of disease outcome and to explore targets for therapeutic modulation.Liver tissue samples were collected during 2008-2019 from patients (n = 139) diagnosed with ICC who underwent curative intent surgery without neoadjuvant chemotherapy. Samples from the discovery cohort (n = 86) were immunohistochemically analyzed on tissue microarrays (TMAs) for the expression of CD68, CD3, CD4, CD8, Foxp3, PD-L1, STAT1, and p-STAT1 in tumor core and stroma areas. Results were digitally analyzed using QuPath software and correlated with clinicopathological characteristics. For validation of TIME-related biomarkers, we performed multiplex imaging mass cytometry (IMC) in a validation cohort (n = 53).CD68+ cells were the predominant immune cell type in the TIME of ICC. CD4+high T cell density correlated with better overall survival (OS). Prediction modeling together with validation cohort confirmed relevance of CD4+ cells, PD-L1 expression by immune cells in the stroma and N-stage on overall disease outcome. In turn, IMC analyses revealed that silent CD3+CD4+ clusters inversely impacted survival. Among annotated immune cell clusters, PD-L1 was most relevantly expressed by CD4+FoxP3+ cells. A subset of tumors with high density of immune cells ("hot" cluster) correlated with PD-L1 expression and could identify a group of candidates for immune checkpoint inhibition (ICI). Ultimately, higher levels of STAT1 expression were associated with higher lymphocyte infiltration and PD-L1 expression.These results highlight the importance of CD4+ T cells in immune response against ICC. Secondly, a subset of tumors with "hot" TIME represents potential candidates for ICI, while stimulation of STAT1 pathway could be a potential target to turn "cold" into "hot" TIME in ICC.© 2024 The Author(s). Published with license by Taylor & Francis Group, LLC.