Inflammatory processes in the development of cholangiocarcinoma in Primary Sclerosing Cholangitis – Do the T cells play a role?

肝外胆管癌是原发性硬化性胆管炎（PSC）最可怕的合并症。预后很差，发病机制尚不清楚，但有证据表明免疫系统和微生物群有助于促进这种疾病。一种认为PSC相关的CCA （PSC-CCA）发生癌变的先决条件是慢性胆道炎症与典型的PSC胆道化生和不典型增生密切相关。此外，PSC患者肠道和胆道微生物群的组成也存在差异。最后，微生物群对T细胞功能的影响，通常由细胞因子的分泌和共抑制受体的表达决定，最近被认为与癌变有关。在此基础上，我们假设PSC特异性微生物群有利于T细胞的致瘤功能，最终导致PSC患者的癌变。本研究的第一个目的是通过单细胞RNA/TCR测序，结合转录组和表位的细胞索引测序（CITE-seq）和总组织的RNA测序，确定PSC-CCA中人类肿瘤浸润T细胞的功能表型。结果将使我们能够识别新的细胞群及其与促或抗肿瘤功能表型的细胞关系。此外，通过定义这些人群的共抑制受体谱，我们将为设计特设免疫疗法（例如抗pd1）奠定基础。最后，我们将通过将单细胞RNA分析中鉴定的差异表达基因与周围组织中已知相互作用伙伴（如细胞因子-受体配对）的表达模式（通过组织总RNA测序鉴定）进行匹配，推断细胞与组织之间的潜在相互作用。第二个目标是鉴定能够塑造T细胞反应的特定PSC-CCA微生物群。因此，我们将首先对PSC-CCA患者的粪便和胆道微生物群进行测序，然后通过将人类微生物群转移到无菌小鼠中来检测微生物群- T细胞的相互作用。第三个目标是开发一个特定的小鼠模型，显示PSC表型和发展肝外CCA。我们将通过使用胆管注射技术将致癌物质直接引入Mdr2-/-小鼠的胆道树来实现这一目标。此外，利用cre - lox系统，我们将开发一种转基因小鼠模型，该模型具有胆管细胞中癌基因kras的条件过表达和肿瘤抑制基因pten的缺失。总之，本项目的结果将有助于理解psc相关CCA的发病机制。它们将揭示免疫机制，并将有助于临床翻译可能的治疗靶点，从而直接治疗患有这种疾病的患者。

Extrahepatic cholangiocarcinoma (CCA) is the most dreaded comorbidity in Primary Sclerosing Cholangitis (PSC). The prognosis is very poor and as of yet the pathogenesis is unknown, but there is evidence that the immune system and the microbiota contribute towards promoting this disease. One suggested prerequisite for carcinogenesis in PSC-associated CCA (PSC-CCA) is a strong association of chronic biliary inflammation with biliary metaplasia and dysplasia, typical of PSC. In addition, differences in the composition of the intestinal and biliary microbiota of patients with PSC have been shown. Finally, the impact of the microbiota on T cell function, which is often determined by the secretion of cytokines and the expression of co-inhibitory receptors, has recently been implicated in carcinogenesis. On the basis of all this, we hypothesize that the PSC specific microbiota favours the pro-tumorigenic function of T cells, ultimately leading to carcinogenesis in PSC patients. The first objective of our study is to define the functional phenotype of human tumour infiltrating T cells in PSC-CCA by using single-cell RNA/TCR sequencing in combination with Cellular Indexing of Transcriptome and Epitopes by sequencing (CITE-seq) and RNA seq of total tissue. Results will allow us to identify new cell populations and their cell relationships with either a pro- or anti-tumorigenic functional phenotype. In addition, by defining the co-inhibitory receptor profile of these populations, we will set the basis for designing ad hoc immune therapies (e.g. anti-PD1). Finally, we will infer the potential interaction among the cells and the tissue by matching differentially expressed genes identified in the single cell RNA analysis to expression patterns of known interaction partners (e.g. cytokine-receptor pairings) in the surrounding tissue, identified by total tissue RNA sequencing. The second objective is to identify specific PSC-CCA microbiota able to shape the T cell response. Therefore, we will first sequence the faecal and biliary microbiota from PSC-CCA patients, and then test the microbiota - T cell interaction by transferring human microbiota into germ-free mice. The third objective is to develop a specific mouse model that exhibits a PSC phenotype and develops extrahepatic CCA. We will achieve this by using the bile duct injection technique to introduce carcinogenic substances directly into the biliary tree of Mdr2-/- mice. Additionally, using the Cre-Lox-system, we will develop a transgenic mouse model with a conditional overexpression of the oncogene kras and deletion of the tumour suppressor gene pten in cholangiocytes. In summary, the results of this project will contribute towards the understanding of the pathogenesis of PSC-associated CCA. They will reveal immunological mechanisms and will help the clinical translation of possible therapeutic targets in order to directly treat patients suffering from this disease.