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.