Abstract
With the rapid advancement of cancer immunotherapy, there is an urgent need for 3D tumor models that authentically replicate the tumor immune microenvironment (TIME) for drug screening. In this study, we engineered an adipose-derived decellularized extracellular matrix (adECM) scaffold functionalized with a novel bifunctional fusion protein, LRELHLNNN-Gal-3, comprising a collagen-binding peptide (LRELHLNNN) and galectin-3 (Gal-3). This design enables stable anchoring of Gal-3 to type I collagen within the scaffold, enhancing tumor cell adhesion and proliferation. Compared to unmodified scaffolds, the LRELHLNNN-Gal-3-modified adECM significantly promoted the proliferation of colorectal cancer cells (MC38, LoVo, and primary ascites-derived tumor cells), with peak proliferation increasing approximately 6-fold by day 7. In chemoresistance assays, tumor cells in the modified scaffold exhibited significantly higher survival rates after 48 h of treatment with 5-FU (77.7% vs 53.0%), oxaliplatin (95.2% vs 67.1%), and capecitabine (69.7% vs 44.8%) compared to unmodified scaffolds and outperformed 2D cultures. Importantly, the modified scaffold also enhanced the survival of CD45+CD4+ and CD45+CD8+ T cells, with CD8+ T cell counts increasing by ∼5.5-fold compared to unmodified scaffolds. In summary, the LRELHLNNN-Gal-3-modified adECM scaffold authentically recapitulates key features of the tumor microenvironment, including tumor cell growth, drug resistance, and immune cell persistence, making it a promising 3D platform for anticancer drug screening and immunotherapy evaluation.