The Role of Peripheral Neuroglia in Oral Squamous Cell Carcinoma Tumor Progression

Abstract

Abstract: Background: Oral squamous cell carcinoma (OSCC) is an aggressive malignancy that accounts for 90% of oral cancers and has a 5-year survival rate of approximately 50% despite the advances in treatment strategies. Recent studies have highlighted the role of the peripheral nervous system in shaping the tumor microenvironment of various cancers, however the role of Peripheral Neuroglia (PNG) in OSCC is largely unknown. Higher neuronal activity is found in advanced OSCC stages; we therefore hypothesize that oral PNG may also have an important role in OSCC progression. Objectives: To address this, we investigated their density, proliferation, and spatial distribution across all stages, prognostic outcomes, and anatomical sites. Methods: Multiplex immunofluorescence was performed on 12 OSCC samples (n=12) across OSCC progressive stages (Premalignant, AJCC TNM 7/8th edition stages1-3, and stage 4 with good and poor prognostic outcomes). SOX10, a key regulator of PNG was used for PNG detection along other markers (S100B, TUBB3, KI67) to assess PNG morphology, axon association, and proliferation. A deep learning imaging analysis pipeline for PNG classification was created for this novel study. Automated image analysis was then used to quantify PNG density, proliferation, and spatial relationships to epithelial-tumor cells based on our customized imaging pipeline analysis. Results: One-way ANOVA analysis of our technical sample (t=33) did not show a statistically significant increase in PNG density across OSCC progression (F(5,27) = 2.068, p = 0.101). However, the small sample size precludes definitive conclusions. Interestingly, we observed site-specific variations in PNG involvement, with higher PNG detection in buccal mucosa OSCC (T2-T3) and stage 4 tumors with good prognosis in the anterior maxillary alveolus more than its counterpart sample in the hard palate. Positive proliferative PNG were more prevalent in later OSCC stages, particularly in stages 2 and 4 with good prognosis. Spatial analysis revealed closer proximity between axon-free PNG and tumor epithelial cells in pre-malignant and early-stage samples compared to advanced stages with the exclusion of stage 4 good prognosis. Conclusion: Our study provides novel insights into tumor-associated PNG cellular dynamics and heterogeneity. These findings suggest a complex role for PNG in OSCC, potentially influencing tumor behavior and prognosis in a stage- and site-dependent manner which confirms our hypothesis. Further large-scale studies are needed to fully determine the functional role of PNG in OSCC progression and explore their potential as prognostic markers or therapeutic targets.