Abstract 4089: Immunosuppressive cellular topography and genomic adaptations sustain colorectal cancer metastasis to the brain

Abstract Colorectal cancer metastases to the brain (CRC-BMets) are lethal and resistant to immunotherapy and radiation. The cellular and molecular adaptations that sustain CRC-BMets remain poorly defined. The objective of our study was to identify the tumor-intrinsic and microenvironmental programs that support metastatic growth in the brain. To understand these mechanisms as they operate in the patient’s metastatic niche, we performed sequencing and imaging-based spatial transcriptomics on 51 patients, including a subset with paired primary tumors and longitudinal radioresistant tumors. Using patient-derived three-dimensional co-culture systems, microfluidic assays and single-cell sequencing, we experimentally perturbed the intercellular interactions that promote tumor growth in the metastatic niche.Metastatic tumor cells showed significantly elevated chromosomal instability and activation of RNA-processing, stress-response, and junctional remodeling pathways compared with paired primary tumors. After radiation therapy, resistant clones retained their copy-number alterations and displayed increased epithelial-mesenchymal transition and transcriptional plasticity. Spatial mapping revealed that tumor cells proliferated preferentially in endothelial-rich regions. Tumor cells acted as multicellular ligand hubs (MIF, GDF15, PRSS3, SEMA3C) reinforcing tumor-supportive interactions with multiple surrounding microenvironmental cell types in the metastatic niche. Adjacent brain parenchyma expressed astrocytic and glial activation, together with angiogenic and matrix-associated pro-tumor features. CRC-BMets reconstructed a stromal microenvironment dominated by macrophages and fibroblasts in proximity to tumor cells, with sparse lymphocytes. This conserved macrophage-fibroblast neighborhood promoted angiogenesis and extracellular matrix remodeling, with increased regulatory T cell and exhaustion gene signatures. Macrophages expressed high levels of SPP1, predicted to orchestrate a matrix remodeling immunosuppressive program via interactions with corresponding receptors on neighboring cells. In a microfluidic device, CRISPR deletion of SPP1 reduced macrophage mobility in the presence of tumor cells. When co-cultured with a CRC-BMet patient-derived spheroid and fibroblasts, SPP1 knockout led to reduced expression of lipid-metabolism related genes in macrophages and disrupted tumor-promoting interactions. Together, these findings indicate that CRC-BMets are maintained by spatially organized tumor-intrinsic adaptations and multicellular stromal programs that persist after radiotherapy. These tumor genomic adaptations, multicellular ligand hubs, and an SPP1-dependent macrophage-fibroblast axis define targetable vulnerabilities in this aggressive metastatic site. Citation Format: Anuja Sathe, Mengrui Zhang, Xiangqi Bai, Ji In Kang, Rithika Meka, Huiyun Sun, Mouhita Humayun, Xun Wang, Susan M. Grimes, Aparajita Khan, Mingen Liu, Andrew S. Luksik, Michael Lim, Claudia K. Petrisch, Christopher M. Jackson, Hannes Vogel, Jeanne Shen, Roger D. Kamm, Melanie Gephart, Summer Han, Hanlee P. Ji. Immunosuppressive cellular topography and genomic adaptations sustain colorectal cancer metastasis to the brain abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4089.