Multiparameter imaging reveals clinically relevant cancer cell – stroma interaction dynamics in head and neck cancer

Epithelial tumors are characterized by abundant inter- and intra-tumor heterogeneity, which complicates diagnostics and treatment. The contribution of cancer-stroma interactions to this heterogeneity is poorly understood. Here we report a paradigm to quantify phenotypic diversity in head and neck squamous cell carcinoma (HNSCC) with single cell resolution. By combining cell state markers with morphological features, we identify phenotypic signatures that correlate with clinical features, including metastasis and recurrence. Integration of tumor and stromal signatures reveals that partial epithelial-mesenchymal transition (pEMT) renders disease outcome highly sensitive to stromal composition, generating a strong prognostic and predictive signature. Spatial transcriptomics and subsequent analyses of cancer spheroid dynamics identifies cancer-associated fibroblast–pEMT axis as a nexus for intercompartment signaling that reprograms pEMT cells into an invasive phenotype. Taken together, we establish a paradigm to identify clinically relevant tumor phenotypes, and discover a cell state-dependent interplay between stromal and epithelial compartments that drives cancer aggression. Human tongue squamous cell carcinoma cells UTSCC74a (isolated from a 31-year-old male with T3N1M0 G1-2 primary tumor) and UTSCC76a (isolated from a 52-year old male with T3N0M0 G2 primary tumor) were obtained from University of Turku. CAFs were isolated from a T2N2bM0 G1 primary tumor of the buccal mucosa. Cells were plated as single-cell suspensions into U-bottom ultra-low attachment 96-well plates (Corning 4520) in in DMEM (4500, mg/L glucose, 1x GlutaMAX, Gibco) supplemented with 1x non-essential amino acids (Gibco), 1x penicillin/streptomycin (Gibco) and 10% fetal bovine serum (Sigma-Aldrich). Cancer cells were cultured either alone or as co-cultures with CAFs. Cells were harvested after 48h.