Co‐Assembling 3D In Vitro Model to Recreate the Colorectal Tumor Microenvironment

Current three-dimensional (3D) in vitro models fail to recapitulate the complexity and heterogeneity of the tumor microenvironment (TME), particularly the dynamic architecture of the extracellular matrix (ECM) and cell-matrix interactions critical to tumor progression. To address this, we have designed nanofibrous composite hydrogels by co-assembling peptide amphiphiles (PAs) with tumor-specific ECM macromolecules, including collagen type I, hyaluronan, laminin alpha 4, and fibronectin, enabling control over composition and nanofibre organization, including either randomly oriented (rPA) or aligned (aPA) nanofibers. These hydrogels recapitulate key biochemical composition, fibrillar architecture, and mechanical properties of the colorectal cancer TME (CRC-TME). Using colorectal cancer cell lines and patient-derived organoids (PDOs), we demonstrate that rPA-based hydrogels support long-term organoid growth (21 days), preserving tumor-specific morphology (compact, cystic structures) and transcriptional profiles characteristic of cell-ECM interactions compared to aPA hydrogels. Transcriptomic analysis revealed rPA-ECM hydrogels upregulate ECM remodeling genes while maintaining lineage-specific differentiation, enabling precise interrogation of matrix-driven signaling. By integrating tumor-specific ECM macromolecules with architecturally defined nanofibers, this platform bridges the gap between simplistic in vitro models and clinical tumor complexity. This approach offers a complex, yet defined and reproducible, platform with significant potential to recapitulate TMEs for personalized medicine.