Actin cytoskeleton modulation reprograms resistant GBM spheroids and GSCs, enhancing response to IR and temozolomide in vitro and in vivo

Actin cytoskeleton regulate essential processes in cancer progression, including cell division, migration, invasion, and genomic stability. Nuclear F-actin has emerged as a critical regulator of DNA repair, chromatin organization, and transcription, representing a potential therapeutic target. Glioblastoma (GBM), a highly aggressive tumor with strong resistance to ionizing radiation (IR) and temozolomide (TMZ), frequently relies on GBM stem-like cells (GSCs) to sustain recurrence. GSCs exhibit enhanced DNA repair, and their stemness is influenced by pathways linked to actin dynamics. We investigated whether targeting actin polymerization sensitizes GBM to therapy by modulating DNA damage response (DDR) and GSC phenotypes. IR- and TMZ-resistant U87-MG cells generated through cyclic treatments were grown as 3D spheroids. Resistant spheroids showed increased proliferation, faster cell-cycle progression, elevated cell death within the core, and markedly enhanced self-renewal, supporting a stem-like tumorigenic profile. Interestingly, resistant cells exhibited distinct invasion patterns: IR-resistant were more invasive, whereas TMZ-resistant were more migratory, consistent with pFAK and vimentin immunoblotting, indicating distinct GSC-like subtypes within each resistant population. Strikingly, spheroid assembly in the presence of F-actin-disrupting drugs (cytochalasin D/B or latrunculin B) reduced therapy resistance and diminished tumorigenicity. Zebrafish xenotransplantation confirmed the aggressive proliferation, mortality rates, and metastatic dissemination of resistant cells, all of which were significantly reduced upon actin-targeting treatments, resulting in increased animal survival. RNA-seq, qPCR, immunoblotting, and immunofluorescence revealed upregulation of GSC markers in resistant spheroids, while actin disruption markedly reduced their expression. We propose that nuclear F-actin regulates transcriptional circuits that maintain stemness and therapy resistance. Consistently, resistant cells exhibited elevated nuclear actin which was dynamically modulated by genotoxic stress or pharmacological actin disruption. Ongoing chromatin-fraction proteomics aim to identify nuclear F-actin-associated proteins under damage conditions. Finally, in PDX GSC23 cells, inhibition of F-actin polymerization reduced stemness markers under self-renewal conditions and prevented serum-induced differentiation. Cytochalasin D enhanced sensitivity of GSC23 to IR and TMZ, reducing proliferation, viability, and tumorigenicity. Collectively, these findings reveal actin cytoskeleton regulation, particularly nuclear F-actin, as a vulnerability that can be therapeutically exploited to overcome GBM resistance, supporting actin-modulating compounds as promising adjuvants for GBM therapy.