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

Three-dimensional (3D) in vitro cell culture models are invaluable tools for investigating the tumor microenvironment. However, analyzing the impact of critical stromal elements, such as extracellular matrix (ECM), remains a challenge. In this study, 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 MatriSpheres actively incorporated microgram quantities of decellularized small intestine submucosa ECM, which proteomically mimicked colorectal cancer tumor ECM compared with traditional formulations like Matrigel. Solubilized ECM, at subgelation concentrations, was organized by colorectal cancer cells into intercellular stroma-like regions within 5 days, displaying morphologic similarity to colorectal cancer clinical pathology. MatriSpheres featured ECM-dependent transcriptional and cytokine profiles associated with malignancy, lipid metabolism, and immunoregulation. Model benchmarking with single-cell RNA 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. Significance: MatriSpheres provide a hydrogel-free 3D platform for decoupling the influence of heterogeneous extracellular matrix components on tumor biology and can broadly facilitate high-throughput drug discovery and screening applications. See related commentary by Ernst and De Wever, p. 1568.