A single-cell and spatial transcriptomic analysis of pre- and post- marsupialization conventional ameloblastoma

Ameloblastoma (AM) is the most frequent odontogenic tumor with local invasiveness, and conventional ameloblastoma is the most common type of it. This study aimed to gain an unbiased overview of the cellular and spatial component of conventional ameloblastoma. We applied single-cell RNA sequencing and spatial transcriptomics on pre- and post-marsupialization conventional ameloblastoma slices, followed by cluster, trajectory, network analysis and immunofluorescence validation. We found a continuous cell trajectory from pre-marsupialization epithelium-like cell types to post- marsupialization mesenchyme-like cell types. A comparative analysis with our recently established human embryonic dental cell atlas revealed that epithelium-like cells resembled the most primitive outer enamel progenitors, whereas mesenchyme-like cells did not resemble any embryonic mesenchymal cells, representing a differentiated and abnormal state. Motif enrichment analysis and pseudotime analysis found that transcription factors DLX5 and TWIST1, together with their target genes, showed profound alterations paralleled the epithelium-to-mesenchyme transition (EMT). We verified the existence of EMT by double immunofluorescence of two EMT markers FN1 and VIM, and verified the specific expression of KRT15 in pre-marsupialization tissues. Lastly, signaling network analysis found that mesenchyme-derived proteins MMP13 and THBS2 exert feedback regulatory effects on EMT. Our study constructed the cellular and spatial landscape of conventional ameloblastoma, and highlighted hub genes like DLX5 and MMP13 that could serve as potential therapeutic targets.