Uncovering functional divergence and cellular clusters with specific gene signatures in HNSCC clonal spheroids.

Head and neck squamous cell carcinoma (HNSCC) exhibits substantial genetic and functional heterogeneities. We generated clonal spheroids (CS) from HNSCC cell lines and patient tumor cells, uncovering distinct functional differences. Hyperproliferative spheroids (HRPS) showed increased proliferation and tumorigenic potential, whereas hypoproliferative spheroids (HOPS) demonstrated enhanced migration and resistance to cisplatin and radiation. In patient tumor-derived spheroids, spheroid size distribution analysis across various TNM stages indicated that early-stage tumors (T2N0M0) predominantly formed HRPS, while later-stage tumors (T4N2M0) exhibited a higher prevalence of HOPS. Single-cell RNA sequencing (scRNA-seq) identified six clusters in the HOPS and four in the HRPS, each defined by unique gene expression profiles and pathway enrichment. HOPS were enriched with fibroblast, mesenchymal, and keratinizing epithelial cells, indicating a metastatic phenotype. They also expressed genes associated with stemness (KRT15 and ALDH3A1). Conversely, HRPS was characterized by proliferative genes, including TOP2A, AURKA, CKS2, AREG, and ANLN, suggesting an advanced HNSCC proliferative signature. Despite being generated from only two cells/well, the various clusters in both spheroid types mirrored the diverse tumor microenvironment in HNSCC, including neoplastic, epithelial, basal, fibroblast, and mesenchymal cells. Notably, the gene expression profiles of Cluster 3 in the HOPS and Cluster 1 in the HRPS closely matched the expression patterns observed in the HNSCC-TCGA dataset. These clusters also displayed a high transcriptional correlation between patient tumors and their xenografts, reinforcing the clonal nature of HNSCC's genetic and functional diversity of HNSCC.