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Physiologically relevant 3D tumor models incorporating extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs) are essential for studying tumor progression and drug resistance yet often suffer from hydrogel contraction and instability – especially in microfluidic formats, where ECM deformation hampers long-term culture and quantitative imaging. Here, we present a microfluidic tumor-stroma co-culture platform for head and neck squamous cell carcinoma (HNSCC) that overcomes these limitations through a dual-material strategy: APTES-mediated surface silanization anchors the ECM to the chip, while Genipin-based crosslinking enhances matrix stiffness without compromising cell viability. This approach stabilizes collagen-rich hydrogels for over 10 days, preserving 3D architecture, sustaining >85% viability, and supporting active proliferation. Fourier-transform infrared spectroscopy (FTIR) confirmed successful collagen crosslinking, combining covalent modification of biomaterials with improved mechanical performance. The platform further integrates AI-assisted, high-content imaging to quantify dynamic phenotypic drug responses at both single-cell and higher multicellular/tissue level resolution. Drug chemosensitivity assays, including the co-culture of tumor cells with patient-derived CAFs, demonstrated the quantitative assessment of clinically relevant chemoprotective effects. By combining biomaterial engineering with functional microfluidic design, this system enables reproducible, physiologically relevant modeling of tumor-stroma interactions, offering a scalable tool for preclinical drug screening and personalized medicine or precision oncology applications.