A Patient-Derived Scaffold-Based 3D Culture Platform for Head and Neck Cancer: Preserving Tumor Heterogeneity for Personalized Drug Testing

Standard 3-D models for head-and-neck cancer (HNC) often lose stromal partners that influence drug response or never include them. We developed a patient-derived cell culture system that maintains tumor cells, cancer-associated fibroblasts (CAFs), and cells undergoing partial epithelial-to-mesenchymal transition (pEMT) for rapid sensitivity testing. Biopsies from four HNC patients were enzymatically dissociated. CAFs were directly cultured, and their conditioned medium (CAF-CM) was collected. Cryopreserved tumour cell suspensions were later revived, screened in five different growth media in 2-D conditions, and the most heterogeneous cultures were re-embedded in 3-D hydrogels with varied gel mix, medium, and seeding geometry. A perimeter-derived complexity index was used to quantify tumoroid morphology and viability after exposure to cisplatin or the Notch modulators RIN-1 (activator) and FLI-06 (inhibitor), which were assessed by live imaging and WST-8 assays. ECM-2 medium alone produced compact CAF-free spheroids, whereas ECM-2 supplemented with CAF medium generated invasive aggregates that deposited endogenous matrix; Matrigel plus this medium and single-point seeding yielded the highest complexity scores. Two of the three patient tumoroids were cisplatin-sensitive, and all showed significant growth inhibition with the FLI-06 inhibitor, while RIN-1 induced minimal change. The optimised scaffold retains tumour–stroma cross-talk and affords patient-specific drug-response data within days, supporting personalised treatment selection in HNC.