Extended Data Fig. 3: Histopathological analysis of CIN gastric cancer EPO-GEMMs.

(A–I) Histological characterization of tumor progression in MYC-p53^−/− tumors. (A-C) Initially, MYC-p53^−/− tumors were moderately well-differentiated adenocarcinomas: (a) Areas of tubular gland formation (arrows) (bar = 200 μm). Representative image of n = 3 mice. (b) Higher magnification of adenomatous proliferation (arrow) in a typical early tumor. The tumor cells formed single and multiple layers around the lumens (bar = 100 µm). Representative image of n = 3 mice. (c) Neoplastic glands (arrows) with cell debris (asterisks) in lumen. Neoplastic glands were lined by tightly adherent pleomorphic columnar and stratified epithelial cells with irregular large nuclei, clumped chromatin, and prominent nucleoli (bar = 100 µm). Representative image of n = 3 mice. (d) As MYC-p53^−/− tumors progressed, the area of glandular formation (arrows) decreased and blended with larger sheets of tumor cells (arrowheads) typical of a diffuse tumor phenotype (bar = 200 µm). Representative image of n = 3 mice. (e) When MYC-p53^−/− tumors transitioned from adenomatous to diffuse, the tumors occasionally developed focal areas of micro-lobules (arrows) separated by prominent stroma (arrowheads) (bar = 250 µm). Representative image of n = 3 mice. (f–i) Eventually, MYC-p53^−/− tumors lost all adenomatous features and became diffuse tumors with solid sheets of neoplastic cells. Representative image of n = 3 mice. (F) These late-stage tumors featured small monomorphic epithelial tumor cells with small basophilic nuclei and scant cytoplasm. The neoplastic cells had a high nuclear/cytoplasm ratio consistent with malignancy (bar = 200 µm). Representative image of n = 3 mice. (G) High magnification of an MYC-p53^−/− tumor characterized by sheets of tumor cells with minimal stroma and a diffuse tumor phenotype. There were numerous mitotic figures (arrows) and apoptotic/necrotic cells (arrowheads) (bar = 100 µm). Representative image of n = 3 mice. (H) The tumors were highly invasive and penetrated the submucosa and muscle layers of the stomach (arrows) and spread into the sub-serosa (arrowhead) (bar = 500 µm). Representative image of n = 3 mice. (I) The invasive tumors (arrows) penetrated all layers of gastric muscle (arrowhead) (bar = 200 µm). Representative image of n = 3 mice. (j-l) Representative immunohistochemistry staining of a MYC-p53 EPO-GEMM gastric tumor for MYC (J), hydrogen/potassium ATPase (H+K) (K), and MUC6 (L). Representative images of n = 3 mice. (M) Gross pathology image of a mMyc-p53^−/− tumor-bearing EPO-GEMM stomach. Representative image of n = 2 mice. (n-p) Representative hematoxylin & eosin (H&E) staining (N) and immunohistochemical staining for MYC (O) and E-cadherin (P) of a mMyc-p53^−/− tumor. Representative images of n = 2 mice. (q) Gross pathology image of a MYC-p53Q97* tumor-bearing EPO-GEMM stomach. Representative image of n = 1 mouse. (R) H&E staining of a MYC-p53Q97* tumor. Representative image of n = 1 mouse. (S) Sanger sequencing results confirming CRISPR base editing of the Trp53 locus targeted by the indicated CRISPR/Cas9-sgRNA in a MYC-p53Q97* EPO-GEMM gastric tumor.

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