Abstract 943: Modeling and dissecting of esophageal squamous cell carcinoma initiation

Abstract Esophageal cancer is the 7th common cancer with the 6th highest cancer mortality rate worldwide. The 5-year survival rate of esophageal cancer is 10 ~ 25%. Esophageal cancer is mainly divided into esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma. ESCC accounts for 80% of all esophageal cancer cases and shows a poor prognosis due to a lack of symptoms at early stages. Given that early diagnosis of ESCC may lead to better outcomes, understanding the biology of ESCC initiation is imperative. To recapitulate the early events of ESCC, we employed the murine esophageal organoid (EO) system recently developed. Based on in silico analyses, we selected nine candidate genes (TP53, CDKN2A, NOTCH1, NOTCH3, KMT2D, KMT2C, FAT1, FAT4, and AJUBA) frequently mutated in ESCC patients. The EOs were then genetically manipulated with the combinatorial ablation of nine genes by CRISPR/Cas9 system. The EOs carrying PN (Trp53 and Notch1 KO), PC (Trp53 and Cdkn2a KO), and PCN (Trp53, Cdkn2a, and Notch1 KO) showed hyperplastic and neoplastic properties with impaired squamous cell differentiation and organoid integrity, compared to the wild-type (WT) EOs. Single-cell RNA-seq (scRNA-seq) analyses of WT, PC, PN, and PCN organoids showed the highest enrichment of ESCC cancer stem cell markers in PCN organoids. Unlike the cell lineage in WT EOs, neoplastic EOs (PC, PN, and PCN) exhibited distinct cell lineage along with multiple root cell clusters. While WT EOs failed to become neoplastic, PN, PC, and PCN EOs displayed the transforming activity in vitro and in vivo xenograft transplantation assays. Intriguingly, in allograft mouse models, while PN organoids-derived cells barely developed tumors, PC and PCN organoids-derived cells developed tumors with marked downregulation of genes related to antigen processing and presentation. These results suggest that combinatorial loss of P53, CDKN2A, and NOTCH1 play a pivotal role in ESCC neoplasia, stemness, and immune checkpoint, unveiling the biology of ESCC initiation. Citation Format: Kyung Pil Ko, Gengyi Zou, Yuanjian Huang, Bongjun Kim, Jie Zhang, Sohee Jun, Jae-Il Park. Modeling and dissecting of esophageal squamous cell carcinoma initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 943.