Spatially resolved transcriptomics identifies epithelial-specific oncogenic signaling networks in ameloblastoma.

BACKGROUND/PURPOSE: Ameloblastoma is a benign odontogenic tumor characterized by locally aggressive growth and a high recurrence rate. Although recurrent mutations such as BRAF V600E have been identified, the epithelial-specific molecular mechanisms underlying tumor initiation and invasion remain incompletely defined. Previous transcriptomic studies have been limited by stromal contamination and lack of spatial resolution. MATERIALS AND METHODS: An integrative transcriptomic strategy was applied, combining bulk RNA sequencing of fresh ameloblastoma and dental follicular tissues, RNA sequencing of primary cultured ameloblastoma epithelial cells and dental follicular epithelial cells, and spatial transcriptomic profiling of formalin-fixed, paraffin-embedded ameloblastoma specimens. RESULTS: Differential expression and functional enrichment analyses identified 871 upregulated and 1464 downregulated genes in tumor tissues, and 360 upregulated and 306 downregulated genes in primary epithelial cells. Integration of these datasets defined a high-confidence set of 75 consistently dysregulated epithelial genes, including BEX1, NOS1, and IRS2, which were validated by quantitative RT-PCR. Pathway enrichment analyses revealed coordinated activation of Wnt/β-catenin, PI3K/AKT, MAPK, and Hedgehog signaling pathways. Spatial transcriptomic analysis further demonstrated that BEX1, NOS1, IRS2, Wnt ligands (WNT3A, WNT6, WNT7A), and Hedgehog effectors (GLI1, GLI2) were predominantly upregulated within ameloblastoma tumor epithelial compartments, confirming pathway activation in situ and minimizing stromal confounding. CONCLUSION: Together, these findings provide a refined molecular characterization of ameloblastoma and demonstrate spatially localized activation of multiple oncogenic signaling pathways within tumor epithelium, extending current single-pathway models of ameloblastoma pathogenesis and supporting the potential development of pathway-based or combinatorial therapeutic strategies.