Key Genetic Determinants Driving Esophageal Squamous Cell Carcinoma Initiation and Immune Evasion

Gastroenterology. 2023 Sep;165(3):613-628.e20. doi: 10.1053/j.gastro.2023.05.030. Epub 2023 May 29.


Background & aims: Despite recent progress in identifying aberrant genetic and epigenetic alterations in esophageal squamous cell carcinoma (ESCC), the mechanism of ESCC initiation remains unknown.

Methods: Using CRISPR/Cas 9-based genetic ablation, we targeted 9 genes (TP53, CDKN2A, NOTCH1, NOTCH3, KMT2D, KMT2C, FAT1, FAT4, and AJUBA) in murine esophageal organoids. Transcriptomic phenotypes of organoids and chemokine released by organoids were analyzed by single-cell RNA sequencing. Tumorigenicity and immune evasion of organoids were monitored by allograft transplantation. Human ESCC single-cell RNA sequencing data sets were analyzed to classify patients and find subsets relevant to organoid models and immune evasion.

Results: We established 32 genetically engineered esophageal organoids and identified key genetic determinants that drive ESCC initiation. A single-cell transcriptomic analysis uncovered that Trp53, Cdkn2a, and Notch1 (PCN) triple-knockout induces neoplastic features of ESCC by generating cell lineage heterogeneity and high cell plasticity. PCN knockout also generates an immunosuppressive niche enriched with exhausted T cells and M2 macrophages via the CCL2-CCR2 axis. Mechanistically, CDKN2A inactivation transactivates CCL2 via nuclear factor-κB. Moreover, comparative single-cell transcriptomic analyses stratified patients with ESCC and identified a specific subtype recapitulating the PCN-type ESCC signatures, including the high expression of CCL2 and CD274/PD-L1.

Conclusions: Our study unveils that loss of TP53, CDKN2A, and NOTCH1 induces esophageal neoplasia and immune evasion for ESCC initiation and proposes the CCL2 blockade as a viable option for targeting PCN-type ESCC.

Keywords: CCL2; CDKN2A; Esophageal Squamous Cell Cancer; Immune Evasion; Immunotherapy; NOTCH; Organoids; Patient Stratification; Single-Cell RNA-Sequencing; TP53.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Carcinoma, Squamous Cell* / genetics
  • Carcinoma, Squamous Cell* / pathology
  • Esophageal Neoplasms* / pathology
  • Esophageal Squamous Cell Carcinoma* / genetics
  • Esophageal Squamous Cell Carcinoma* / pathology
  • Humans
  • Immune Evasion / genetics
  • LIM Domain Proteins / genetics
  • Mice
  • Mutation


  • AJUBA protein, human
  • LIM Domain Proteins