Abstract C124: MCAT™: A 3D Co-Culture Platform for Genome-Wide CRISPR Screening to Identify Stromal-Driven Vulnerabilities in CMS4 Colorectal Cancer

Abstract INTRODUCTION: Cancer-associated fibroblasts (CAFs) are key drivers of the mesenchymal, therapy-resistant phenotype observed in the CMS4 subtype of colorectal cancer (CRC). CAFs are abundant in the tumor stroma, where they orchestrate extracellular matrix (ECM) remodeling, activate pro-tumorigenic signaling (e.g., TGF-β, PDGF), and suppress anti-tumor immunity. These interactions are difficult to model in conventional 2D cultures, which lack the complexity of the tumor microenvironment (TME). To overcome this limitation, we utilized MCAT™ (MEDiC’s Casual and Actionable Target)—a physiologically relevant and scalable 3D platform—to model CMS4 tumor biology and identify cancer cell dependencies influenced by stroma. Using MCAT™, we performed genome-wide CRISPR knockout screens to uncover tumor vulnerabilities and resistance mechanisms emerging through tumor–stroma interactions. METHODS: Two CMS4 CRC cell lines (HCT116, SW620) and human skin fibroblasts (BJ) were co-cultured at defined ratios and encapsulated in ECM ladden alginate hydrogel microcapsules to form 3D spheroids. Fibroblast-induced changes in cancer cell proliferation were tracked via live-cell imaging. Transcriptomic profiling was performed to assess fibroblast-driven changes. For genome-wide CRISPR screening, cancer cells expressing Cas9 were transduced with a pooled sgRNA library (∼60,000 sgRNAs targeting ∼20,000 genes). Co-cultures were maintained for four weeks to allow for functional selection under stromal influence. RESULTS: Fibroblast co-culture at a 1:3 (cancer:fibroblast) ratio significantly enhanced proliferation of both HCT116 and SW620 cells, whereas a 3:1 ratio showed no effect. qPCR analysis revealed upregulation of CMS4 markers (PDGFRA, c-KIT) in 3:1 co-cultures. Transcriptomic analysis identified ∼2,000 differentially expressed genes (DEGs) between 2D and 3D conditions. 3D co-culture significantly enriched mesenchymal traits, with upregulation of EMT markers (TWIST, VIM) and pathways related to ECM organization (COL5A1, BGN), ECM degradation (MMP2, TIMP3), and PDGF signaling—hallmarks of invasion and metastasis. Genome-wide CRISPR screening revealed 27 genes with a negative-growth phenotype and 92 genes with a positive-growth phenotype in the presence of fibroblasts. Notably, several TGF-β pathway regulators showed negative-growth phenotype only in co-culture, highlighting the stroma-specific role of this pathway. Genes involved in metabolic processes were enriched among negative hits, which could be potentially targeted for therapeutics. CONCLUSION: The MCAT™ platform enables physiologically relevant modeling of tumor–stroma interactions in CMS4 CRC. It supports high-throughput functional genomics to uncover context-specific vulnerabilities, providing a powerful tool for discovering novel therapeutic targets in aggressive, stroma-rich cancers. REFERENCES: [1] Han, K., Pierce, S.E., Li, A. et al. CRISPR screens in cancer spheroids identify 3D growth-specific vulnerabilities.Nature 580, 136–141 (2020). https://doi.org/10.1038/s41586-020-2099-x Citation Format: Pranay Agarwal, Maya Tureez, Jojo Baek, Aashka Mehta, Kriti Bhardwaj, Ella Liu, Jina Song, Kyuho Han, Hong-Pyo Lee. MCAT™: A 3D Co-Culture Platform for Genome-Wide CRISPR Screening to Identify Stromal-Driven Vulnerabilities in CMS4 Colorectal Cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C124.