A 3D Self-Assembly Platform Integrating Decellularized Matrix Recapitulates In Vivo Tumor Phenotypes and Heterogeneity

Abstract Three-dimensional (3D) in vitro cell culture models are invaluable tools for investigating the tumor microenvironment (TME). However, analyzing the impact of critical stromal elements, such as extracellular matrix (ECM), remains a challenge. Here, we developed a hydrogel-free self-assembly platform to establish ECM-rich 3D “MatriSpheres” to deconvolute cancer cell-ECM interactions. Mouse and human colorectal cancer (CRC) MatriSpheres actively incorporated microgram quantities of decellularized small intestine submucosa ECM, which proteomically-mimicked CRC tumor ECM compared to traditional formulations like Matrigel. Solubilized ECM, at sub-gelation concentrations, was organized by CRC cells into intercellular stroma-like regions within 5 days, displaying morphological similarity to CRC clinical pathology. MatriSpheres featured ECM-dependent transcriptional and cytokine profiles associated with malignancy, lipid metabolism, and immunoregulation. Model benchmarking with scRNA sequencing demonstrated that MatriSpheres enhanced correlation with in vivo tumor cells over traditional ECM-poor spheroids. This facile approach enables tumor-specific tissue morphogenesis, promoting cell-ECM communication to improve fidelity for disease modeling applications.