Chromosomal instability shapes the tumor microenvironment of esophageal adenocarcinoma via a cGAS–chemokine–myeloid axis

Abstract Chromosomal instability (CIN), a characteristic feature of esophageal adenocarcinoma (EAC), drives tumor aggressiveness and therapy resistance, presenting an intractable problem in cancer treatment. CIN leads to constitutive stimulation of the innate immune cGAS–STING pathway, which has been typically linked to anti-tumor immunity. However, despite the high CIN burden in EAC, the cGAS– STING pathway remains largely intact. To address this paradox, we developed novel esophageal cancer models, including a CIN-isogenic model, discovering myeloid-attracting chemokines – with the chemokine CXCL8 (IL-8) as a prominent hit – as conserved CIN-driven targets in EAC. Using high-resolution multiplexed immunofluorescence microscopy, we quantified the extent of ongoing cGAS-activating CIN in human EAC tumors by measuring cGAS-positive micronuclei in tumor cells, validated by orthogonal whole-genome sequencing-based CIN metrics. By coupling in situ CIN assessment with single-nucleus RNA sequencing and multiplex immunophenotypic profiling, we found tumor cell-intrinsic innate immune activation and intratumoral myeloid cell inflammation as phenotypic consequences of CIN in EAC. Additionally, we identified increased tumor cell-intrinsic CXCL8 expression in CINhigh EAC, accounting for the inflammatory tumor microenvironment. Using a novel signature of CIN, termed CINMN, which captures ongoing CIN-associated gene expression, we confirm poor patient outcomes in CINhigh tumors with signs of aberrantly rewired cGAS–STING pathway signaling. Together, our findings help explain the counterintuitive maintenance and expression of cGAS–STING pathway components in aggressive, CINhigh tumors and emphasize the need to understand the contribution of CIN to the shaping of a pro-tumor immune landscape. Therapeutic strategies aimed at disrupting the cGAS-driven inflammation axis may be instrumental in improving patient outcomes in this aggressive cancer. Competing Interest Statement EEP has served on advisory boards and received fees from companies including Boehringer Ingelheim, Curadev, InhaTarget and AkamisBio. She is an employee of the University of Oxford which has received funding or other support for research work from AstraZeneca and STIpe Therapeutics. SRL has served on advisory boards and received fees or expenses from companies including Eisai, Prosigna, Roche, Pfizer, Novartis, Sanofi, Shionogi, Rejuversen, Oxford Biodynamics, ExScientia, Synthon, and Piqur Therapeutics. He is an employee of the University of Oxford and has received funding or other support for research work from the World Cancer Research Fund, CRUK, NIHR, Against Breast Cancer, and Pathios Therapeutics. IM declares grants from AstraZeneca, Roche, Genmab, Catalym, Bristol Myers and consultancy fees from Roche, Genmab, F_STAR, Catalym, Highlight Therapeutics, Light chain, Curon, Pioneers, AbbVie and Bright Peaks. BI is a consultant for or received honoraria from Volastra Therapeutics, Johnson & Johnson/Janssen, Novartis, GSK, Eisai, AstraZeneca and Merck, and has received research funding to Columbia University from Agenus, Alkermes, Arcus Biosciences, Checkmate Pharmaceuticals, Compugen, Immunocore, Regeneron, and Synthekine. BI is a scientific founder of Basima Therapeutics, Inc. EEP, IM, TC and SRL have been the CI/PI of industry-sponsored clinical trials. The remaining authors have no declarations. None of these relationships influence the results or discussion of this work.