Modeling, dissecting, and subtyping of E-cadherin inactivation-associated diffuse-type gastric adenocarcinoma. Modeling, dissecting, and subtyping of E-cadherin inactivation-associated diffuse-type gastric adenocarcinoma

Diffuse-type gastric adenocarcinoma (DGAC) is lethal cancer that is often diagnosed late and resistant to therapeutics. Although hereditary DGAC is mainly characterized by mutations in the CDH1 gene that encodes E-cadherin, the impact of E-cadherin inactivation in DGAC tumorigenesis remains unclear. To address this, we established and characterized a CDH1 loss-associated DGAC model system with a human DGAC single-cell transcriptome. We genetically engineered murine gastric organoids (GOs; Cdh1 KO, KrasG12D, Trp53 KO [EKP]) that recapitulates human DGAC tumorigenesis. Cdh1 depletion is sufficient to 1. Single-cell transcriptomics of human DGAC datasets classified patients into three subtypes (DGAC1, 2, and 3). By transcriptional signature, EKP GOs belong to the DGAC1 subtype displaying CDH1 downregulation and epithelial-mesenchymal transition. Compared to DGAC2 and DGAC3, the DGAC1 subtype was characterized by T cell exhaustion, PILRA-CD99 enrichment, and potential sensitivity to PD1 inhibitors. Additionally, we identified the EZH2-mediated transcriptional circuit as a key regulon specifically activated in EKP and a therapeutic vulnerability. This study unravels the unexpected role of E-cadherin loss in cell lineage plasticity, transcriptional reprogramming, and immune evasion of DGAC and further stratifies DGAC patients by single-cell transcriptomics, providing novel insights into E-cadherin loss-associated DGAC tumorigenesis. Overall design: Organoids from WT, KP, and EKP were collected 7 days after seeding and digested with 0.05% trypsin-EDTA at 37 ℃ for 30 min. After inactivating trypsin with 10% FBS DMEM, single-cell suspension was collected by passing through the 35 μm cell strainer. cDNA library was prepared with the 10x Genomics 3’ v2 kit, cDNA library was sequenced on an Illumina NovaSeq (Novogene) and mapped to GRCm38/mm10 genome and demultiplexed using Cellranger.

弥漫型胃腺癌（Diffuse-type gastric adenocarcinoma, DGAC）是一类致死性恶性肿瘤，通常确诊时已属晚期，且对治疗方案存在耐药性。尽管遗传性弥漫型胃腺癌主要以编码E-钙粘蛋白（E-cadherin）的CDH1基因突变作为特征性改变，但E-钙粘蛋白失活在弥漫型胃腺癌肿瘤发生过程中的具体作用仍有待阐明。为厘清这一科学问题，我们构建并表征了一套关联CDH1缺失的弥漫型胃腺癌模型系统，该系统整合了人类弥漫型胃腺癌单细胞转录组数据。我们通过基因工程技术改造了小鼠胃类器官（murine gastric organoids, GOs；具体为Cdh1敲除、KrasG12D突变、Trp53敲除，即EKP模型），该模型可复现人类弥漫型胃腺癌的肿瘤发生进程。Cdh1缺失即可诱发相关病理表型，具体如下：1. 对人类弥漫型胃腺癌数据集开展单细胞转录组分析，可将患者划分为三个亚型（DGAC1、DGAC2与DGAC3）。通过转录组特征分析，EKP型胃类器官归属于DGAC1亚型，该亚型表现为CDH1表达下调与上皮-间质转化（epithelial-mesenchymal transition）特征。相较于DGAC2与DGAC3亚型，DGAC1亚型的标志性特征包括T细胞耗竭（T cell exhaustion）、PILRA-CD99信号富集，以及对PD1抑制剂的潜在敏感性。此外，我们鉴定出EZH2介导的转录调控环路是EKP模型中特异性激活的关键调控子，同时也是一处潜在的治疗易感靶点。本研究揭示了E-钙粘蛋白缺失在弥漫型胃腺癌的细胞谱系可塑性、转录重编程与免疫逃逸中此前未被认知的重要作用，并通过单细胞转录组学对弥漫型胃腺癌患者进行分子分层，为关联CDH1缺失的弥漫型胃腺癌肿瘤发生机制提供了全新的研究视角。实验整体设计：将野生型（WT）、KP型及EKP型的胃类器官在接种7天后收集样本，使用0.05%胰蛋白酶-EDTA溶液于37℃下消化30分钟。采用含10%胎牛血清的DMEM培养基终止胰蛋白酶消化反应，随后通过35 μm细胞筛过滤以获取单细胞悬液。使用10x Genomics 3’ v2试剂盒构建cDNA文库，随后在Illumina NovaSeq测序平台（由诺禾致源Novogene完成测序）进行高通量测序；将测序数据比对至GRCm38/mm10参考基因组，并通过Cellranger软件完成解多重分析。