Novel strategies for glioblastoma: integrating repurposed drugs with differentiation treatment

GBM represents the most aggressive primary malignant tumor of the central nervous system in adults. Despite the adoption of the Stupp protocol, consisting of maximal surgical resection followed by radiotherapy with concomitant and adjuvant TMZ, clinical outcomes remain dismal, with a median overall survival of approximately 12-15 months and a 5-years survival rate of less than 10%. Tumor recurrence is very frequent and is largely attributed to the persistence of GSCs, a subpopulation endowed with self-renewal capacity and intrinsic resistance to cytotoxic therapies. The selective survival of these cells following standard treatment plays a central role in therapeutic failure and represents a critical obstacle in GBM management. In recent years, differentiation therapy has emerged as a promising strategy to counteract stemness-associated resistance by inducing GSCs to exit their quiescent, tumorigenic state and undergo lineage commitment. Among available differentiating agents, ATRA has shown the ability to modulate gene expression and promote astrocytic and neuronal differentiation in glioma models. In parallel, drug repurposing offers a cost-effective and time-efficient avenue for anticancer drug discovery. Curcumin, disulfiram, and metformin are among the repurposed agents that have demonstrated activity against various cancer models, including evidence of selective targeting of cancer stem-like populations. Based on this evidence, we hypothesized that combining an ATRA-based differentiative treatment with selected cytotoxic drugs (TMZ, Curc, DSF and Met) would enhance the therapeutic vulnerability of GSCs and possibly represent a novel treatment strategy to fight GBM. To this end, a 3D culture system based on GL261 murine glioma spheroids was employed. The GL261 murine cell line was chosen because of its ability to closely mimic the aggressive and invasive features of human GBM. Moreover, they represent an optimized model for the investigation of tumor development following orthotopic injections into syngeneic immunocompetent C57BL/6J mice. The specific aim of this thesis was to determine whether ATRA-induced differentiation alters the responsiveness of glioma spheroids to these pharmacological treatments when delivered either sequentially, following a preconditioning phase with ATRA 5 μM, or simultaneously. Treatment with ATRA successfully induced morphological differentiation, evidenced by appearance of spindle-like cells and spheroid disaggregation, confirming its use as a priming agent. While simultaneous treatments showed limited synergy for all tested drugs, the sequential treatment strategy offered profound mechanistic insights. Notably, the sequential combination of ATRA followed by Met emerged as the most promising treatment strategy. This regimen resulted in a dramatic, time-dependent reduction in cell viability, suggesting that ATRA-induced differentiation creates a metabolic change that sensitizes cells to metformin, allowing induction of energy depletion and its long-term accumulation even at low millimolar concentrations. Conversely, the efficacy loss for curcumin and DSF following ATRA pretreatment confirmed their specificity for stem-like populations. On the other hand, ATRA pretreatment appeared to induce a temporary desensitization to TMZ, possibly suggesting a transient reduction in cell proliferation after differentiation. Moving forward, next steps will involve molecular validation of differentiation markers and statistical consolidation of ATRA-metformin data, paving the way for in vivo experimentation on syngeneic immunocompetent mouse models to confirm the potential of this metabolic targeting strategy while also expanding in vitro experimentation on primary cell cultures that can properly replicate tumor complexity.