Background: Ameloblastoma is a benign odontogenic tumor known for its high local invasiveness and recurrence. Hypoxia is a critical component of the tumor microenvironment in many solid tumors and may contribute to invasiveness and recurrence features, particularly in BRAF-mutated cases. Objectives: This study aimed to investigate the role of hypoxia and autophagy in modulating key biological processes within the ameloblastoma tumor microenviroement, including stromal remodeling, autophagy, and cell migration, using a combination of immunohistochemistry, 3D functional assays, and in vivo models.

Materials and Methods: Immunohistochemistry was performed on 22 formalin-fixed, paraffin-embedded human ameloblastoma samples (14 BRAF-positive, 8 BRAF-negative), with BRAF mutation status pre-determined by immunohistochemical staining. Samples were obtained from institutions across the United States and Africa under IRB-approved protocols. Expression of HIF-1α (hypoxia), GLUT1 (metabolism), ATG5 (autophagy), α-SMA (mesenchymal/invasiveness), and CD34 (stromal progenitors/vascularity) was evaluated in the epithelial, stellate reticulum-like, and stromal compartments using QuPath software. To model the functional impact of hypoxia, AM-1 ameloblastoma cells were cultured as three-dimensional Matrigel spheroids under normoxic, transient hypoxic, and continuous hypoxic conditions. Western blotting was performed to assess LC3 (autophagosome formation), p62 (autophagic flux), and Ki-67 (proliferation). Scratch wound assays evaluated cell migration, and xenograft models were used to assess the persistence of hypoxia-induced phenotypes in vivo.

Results: BRAF⁺ tumors showed increased HIF-1α (hypoxia response), α-SMA (mesenchymal marker), GLUT1 (glucose metabolism), and ATG5 (autophagy-related protein), along with a higher α-SMA/CD34 ratio, indicating stromal remodeling. ATG5 correlated positively with α-SMA and inversely with GLUT1, linking autophagy to epithelial–mesenchymal transition and metabolic reprogramming. In vitro, continuous hypoxia increased HIF-1α (hypoxia marker), LC3 (autophagosome formation), and reduced p62 (autophagic flux indicator), while Ki-67 (proliferation marker) was decreased—suggesting dormancy through activation of autophagy and autophagic flux. Continuous hypoxia also enhanced AM-1 cell migration in scratch assays.In xenografts, tumor size positively correlated with epithelial α-SMA and negatively with stromal CD34, supporting the role of stromal remodeling in tumor expansion.

Conclusion: Hypoxia mediated through HIF-1α signaling promotes epithelial–stromal crosstalk, autophagy, and migration in BRAF⁺ ameloblastoma. These hypoxia-driven adaptations apparently support ameloblastoma invasive growth phenotype, recurrence and resistance to therapy. Targeting hypoxia-autophagic crosstalk may offer novel strategies to improve treatment outcomes in ameloblastoma.