Resumo:
INTRODUCTION: Oral squamous cell carcinoma (OSCC) is an aggressive tumor whose progression is influenced by tumor microenvironment interactions. Cancer-associated fibroblasts (CAFs) and cell-in-cell (CIC) structures have emerged as critical elements in modulating tumor plasticity, tissue remodeling, and immune evasion. However, the spatial influence of CAFs on tumor gene expression, particularly regarding CIC formation, remains poorly understood. AIM: To investigate the tumor transcripts associated with key biological processes in tissue remodeling, cell adhesion, and tumor plasticity (including the formation of CIC structures), as well as the influence of CAFs and the tumor microenvironment on the spatial transcriptome of OSCC spheroids and tissue. MATERIAL AND METHODS: An in silico analysis was performed using TCGA data to identify highly variable tumor transcripts and to establish gene correlation and functional enrichment networks. Gene expression analyses were also conducted on OSCC cell lines. Three-dimensional tumor spheroids were generated via magnetic bioprinting (NanoShuttle-PL™, Greiner Bio-One) using OSCC cell lines (SCC-4 and H357) and normal or activated fibroblasts (NHOF and NHOF-MYO). After fixation, processing, and TMA construction, spatial transcriptomics analysis was performed using the Xenium™ platform (10x Genomics). Additionally, a clinical OSCC case was analyzed through Visium™ spatial transcriptomics and histology for translational evaluation. RESULTS: The in silico analysis identified the most variable tumor transcripts associated with extracellular matrix remodeling, tumor plasticity, and cell adhesion, as well as functional enrichment networks. Spatial transcriptomics revealed gene expression patterns associated with the presence of activated fibroblasts in tumor spheroids and in the clinical case, involving genes linked to more aggressive phenotypes and the formation of CIC structures. CONCLUSIONS: The findings suggest that cancer-associated fibroblasts may influence tumor gene expression in OSCC in a spatially organized manner, favoring profiles associated with tissue remodeling, plasticity, and CIC formation. The integrative approach, combining in silico analyses, 3D models, and clinical samples, reinforces the translational relevance of the study and contributes to a deeper understanding of tumor-stroma interactions in OSCC.
