A new mouse model for studying the pathogenesis and immunobiology of intrahepatic cholangiocarcinoma and improving its immunotherapy

摘要 肝内胆管细胞癌（ICC）是一种侵袭性原发性肝癌（PLC）， 治疗，总体5年生存率为8%。像主要的PLC，肝细胞癌（HCC），ICC 由于胆汁淤积性肝病，非酒精性脂肪性肝病， 2型糖尿病和酗酒和吸烟。与HCC不同，ICC通常在晚期被发现， 生存前景很差，这是目前放射学诊断与早期HCC鉴别不准确的并发症 技术.两种PLC的早期鉴别诊断是至关重要的，因为它们的不同管理和缺乏 ICC和HCC共有的靶向致癌驱动因子。这个问题可以通过识别PLC来解决- 类型不可知的治疗方法，如免疫检查点抑制（ICI），适用于两种ICC 和HCC。然而，尽管基于ICI的疗法被批准用于HCC，并且其疗效已经通过以下方式得到改善： 与VEGF抑制剂组合，它们在ICC中表现不佳。这一失败可以部分归因于 对ICC特异性免疫抑制肿瘤微环境（TME）的机制理解不足， 缺乏合适的小鼠模型来评估ICI与能够 解除免疫抑制我们通过开发一种新的ICC模式解决了这个问题， 先前的小鼠模型并不依赖于强致癌驱动因子的强制过表达， 在没有选择性压力的情况下诱导ICC以获得额外的突变。我们的MUP-uPA/NRF 2Act 模型依赖于ER应激与转录因子NRF 2激活的组合， 肝胆祖细胞和成熟肝细胞，并显示出稳健的，高度渗透的，类似人的逐步 向ICC的进展，伴随着表现出CD 8 + T细胞的免疫抑制性TME的积累 细胞排斥我们计划建立MUP-uPA/NRF 2Act小鼠作为研究进化的主要模型 ICC及其免疫抑制性TME的研究，并寻找解除免疫抑制的治疗方法， 增强对现有PD-（L）1抑制剂的反应。为了实现这一目标，我们将充分描述内务部- uPA/NRF 2Act小鼠，定义ICC各阶段的转录组学、表观遗传学和遗传学改变 进展，包括胆管反应、胆管上皮内瘤变和确诊的促纤维增生性癌。 我们还将定义肿瘤浸润淋巴细胞在每个阶段的T细胞受体（TCR）库。 恶性进展，并挖掘癌细胞产生的介导 结缔组织增生、免疫抑制和CD 8 + T细胞排斥。这些研究将得到以下方面的补充： 免疫表型和免疫耗竭实验，其目标是鉴定靶分子 负责建立ICC特异性免疫抑制性TME的开关。我们将使用以下信息 确定临床批准的治疗方法，可以克服免疫抑制，大大提高疗效， 基于PD-（L）1抑制剂的ICI治疗。

ABSTRACT Intrahepatic cholangiocarcinoma (ICC) is an aggressive primary liver cancer (PLC) that is particularly hard to treat, having an overall 5-year survival rate of 8%. Like the major PLC, hepatocellular carcinoma (HCC), ICC incidence is on the rise due to the ongoing increase in cholestatic liver diseases, non-alcoholic fatty liver diseases, type 2 diabetes and alcohol and tobacco abuse. Unlike HCC, ICC is often detected at an advanced stage when survival prospects are poor, a complication of its imprecise differentiation from early HCC by current radiological techniques. Early differential diagnosis of two PLCs is critical due to their distinct management and the absence of targetable oncogenic drivers shared by ICC and HCC. This problem could be solved by identification of a PLC- type agnostic therapeutic approach, such as immune checkpoint inhibition (ICI), that is applicable to both ICC and HCC. However, while ICI based therapies were approved for HCC and their efficacy has been improved by combination with VEGF inhibitors, they have performed poorly in ICC. This failure can be attributed, in part, to poor mechanistic understanding of the ICC-specific immunosuppressive tumor microenvironment (TME) and lack of suitable mouse models that allow the evaluation of ICI in combination with therapeutics capable of dismantling immunosuppression. We have solved this problem by developing a new ICC model that unlike previous mouse models does not depend on forced overexpression of potent oncogenic drivers that rapidly induce ICC in the absence of selective pressure for acquisition of additional mutations. Our MUP-uPA/NRF2Act model depends on combination of ER stress with activation of transcription factor NRF2 in bipotential hepatobiliary progenitors and mature hepatocytes and shows robust, highly penetrant, human-like stepwise progression towards ICC that is accompanied by the buildup of an immunosuppressive TME exhibiting CD8+ T cell exclusion. We plan to establish the MUP-uPA/NRF2Act mouse as the leading model for studying the evolution of ICC and its immunosuppressive TME and for finding treatments that will dismantle immunosuppression and boost the response to existing PD-(L)1 inhibitors. To accomplish this goal, we will fully characterize the MUP- uPA/NRF2Act mouse, defining the transcriptomic, epigenetic, and genetic alterations at each stage of ICC progression, including ductular reactions, biliary intraepithelial neoplasia and established desmoplastic cancer. We will also define the T cell receptor (TCR) repertoire of tumor infiltrating lymphocytes at each stage of malignant progression and mine the transcriptomic data for cancer cell produced factors that mediate desmoplasia, immunosuppression and CD8+ T cell exclusion. These studies will be complemented by immunophenotyping and immunodepletion experiments whose goal is the identification of targetable molecular switches responsible for establishment of the ICC-specific immunosuppressive TME. We will use this information to identify clinically approved treatments that can overcome immunosuppression and vastly improve the efficacy of ICI therapy based on PD-(L)1 inhibitors.