An Organotypic Oral Squamous Cell Carcinoma Model Recapitulates Epithelial-Stromal Complexity at Single-cell Resolution and Reveals Matrix-derived Signalling as a Therapeutic Target.

Three-dimensional (3D) organotypic cultures recapitulate key structural features of oral tumours and provide controlled, ethical, and reproducible research platforms. However, their ability to faithfully recapitulate in vivo tissue composition and their translational relevance require rigorous validation. Here, we characterised a head and neck squamous cell carcinoma (HNSCC) model using single-cell RNA sequencing to assess maturation, cellular heterogeneity, functionality, and inter- and intra-tissue interactions within epithelial and stromal compartments. The epithelial layer of the model differentiated into populations closely resembling the tissue of tumour origin, including dividing and precursor cells, heterogeneous basal layer, suprabasal cells, and metabolically specialised populations. All epithelial lineages emerged from proliferative progenitors, driven by dynamic transcription factor programmes. The collagen-rich stroma contained functionally diverse fibroblasts reflecting the heterogeneity of cancer-associated fibroblasts in vivo, including dividing, matrix-producing, immune-responsive, and tumour-like populations. Importantly, extensive epithelial-stromal communication networks developed, essential for cancer epithelium maintenance and tumour microenvironment regulation. Matrix-derived signals, particularly fibronectin, osteopontin, and laminins, constituted dominant inputs to CD44-expressing cancer cells and are associated with patient survival in HNSCC, highlighting potential therapeutic targets. Overall, this organotypic HNSCC model exhibits high functional fidelity, captures key tumour elements relevant for therapy and resistance and brings confidence in non-animal drug testing.