ABSTRACT: Canine acanthomatous ameloblastoma (CAA) is a locally invasive benign oral neoplasm. Clinically, differentiating CAA from canine oral squamous cell carcinoma (COSCC) has proven to be difficult as they share many clinical, radiological, and histological features. Molecularly, even though they both have an overactive MAPK pathway, their mutational landscape is distinct. Furthermore, previous studies using bulk RNA sequencing (RNA-seq) have demonstrated pronounced differences in programs related to, among others, hypoxia, PI3K-AKT signaling, and cell proliferation. However, thesee bulk studies lacked the resolution to elucidate the cellular heterogeneity of CAA relative to OSCC. To address this, we employed single nucleus RNA-seq to characterize the gene expression architecture of CAA, COSCC, and healthy gingiva at high resolution. Across ~205,000 nuclei, we identified major epithelial, immune, endothelial, and mesenchymal populations, as well as two epithelial subtypes uniquely enriched in CAA. The CAA-specific keratinocytes exhibited a neuronal-like expression program defined by synaptic regulators, KRAS-associated signaling pathways, and markedly elevated expression of PEG3, ERBB4, GABRB1, MAGI2, and CASK. We corroborated these findings using both bulk RNA-seq and qPCR. At the protein level, using immunohistochemistry, the transcription factor PEG3 was strongly detected and shown to localize to the nucleus of CAA epithelial cells but not COSCC or healthy gingiva. Together, these findings reveal a previously unrecognized neuroepithelial cell state that defines CAA, distinguishes it from COSCC, and may reflect unique cell signaling circuitry with potential diagnostic and therapeutic relevance.
