Oral Squamous Cell Carcinoma (OSCC) remains a significant public health concern. Despite advances in treatments, including surgery, and chemotherapy, choices remain limited, underscoring the need for innovative therapeutic strategies. Developing new drugs is both costly and time-intensive. Drug repurposing offers a compelling solution by leveraging existing, well-characterized medications that are already approved and toxicologically safe. In this context, aiming to identify promising candidates for repositioning in OSCC, we combined integrated in silico analyses tools based on molecular and genetic insights with in vitro cytotoxicity screening based on bi- (2D) and three-dimensional (3D) models.

A total of 237 drug candidates were retrieved from DrugBank to identify druggable targets. Ranking was based on cancer-related pathways and protein-protein interaction centrality. The in vitro cytotoxicity of selected compounds was evaluated in OSCC cell lines (HSC-3, H357) following the National Cancer Institute (NCI) Developmental Therapeutics Program (DTP) guidelines, with CellTiter-Glo® as the viability assay. Homotypic OSCC spheroids were generated using optimized magnetic 3D bioprinting. After ranking, 21 compounds were selected and underwent single-dose screening (10 μM). H357 cells were more sensitive than HSC3, showing lower viability across multiple compounds. Three most promising candidates (Daunorubicin, Erlotinib, and Romidepsin), which displayed growth inhibition properties (at least a 35% reduction), advanced to five-dose screening assays in both 2D and 3D models. In 2D cultures, Romidepsin exhibited the strongest cytotoxic effect, with the lowest IC50 values in both cell lines. In 3D assays, all three drugs demonstrated cytotoxic activity, with Romidepsin once again emerging as the most potent compound.

The ranking strategy accelerated candidate selection and increased the chances of success by focusing on targets that exert greater influence on pathways relevant to OSCC. HSC-3 cells, known for higher tumorigenic potential, exhibited greater drug resistance than H357. Different mechanisms of action that maximize the therapeutic applications of the selected drugs were found. They include epidermal growth factor receptor (EGFR) inhibition, topoisomerase II inhibition, and a potent class I histone deacetylase (HDAC) blocker. Differences in 2D and 3D responses highlight the need for physiologically relevant models to improve translational relevance.

These findings reinforce the value of drug repurposing as an efficient strategy for expanding OSCC treatment options. By integrating computational approaches with physiologically relevant 3D models, this approach paves the way for more precise and personalized cancer therapies.