Synergistic Upregulation of Extracellular Vesicles and Cell-Free Nucleic Acids by Chloroquine and Temozolomide in Glioma Cell Cultures

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Abstract
Extracellular vesicles (EVs) secreted by glioblastoma multiforme and other types of cancer cells are key factors contributing to the aggressiveness of the disease and its resistance to therapy. Chloroquine (CHQ), a lysosomal inhibitor, has shown potential as an enhancer of temozolomide (TMZ) cytotoxicity against glioblastoma cells. Since both CHQ and TMZ are known to modulate EV secretion, we sought to investigate their potential interplay in this process. Simultaneous treatment of TMZ-sensitive (U87-MG) and TMZ-resistant (U138-MG) glioblastoma cells with TMZ and CHQ led to a synergistic upregulation of EV secretion. Although CHQ did not enhance the TMZ cytotoxicity in U87-MG cells, it synergized with the latter to upregulate the release of extracellular nucleic acids implicating activation of unconventional secretory pathways. Synergistic upregulation of the autophagy markers LC3B-II and p62 by CHQ and TMZ in both cells and EVs indicates secretory autophagy is likely involved in the observed unconventional secretion. Moreover, a significant enrichment of caveolin-1 in small EVs highlights their potential role in modulating tumor aggressiveness. The synergy in EV upregulation was not confined to the specific biological activity of TMZ and CHQ; similar effects were observed upon co-treatments with CHQ and etoposide (a topoisomerase inhibitor) and TMZ and Bafilomycin A1 (another lysosomal inhibitor). Heightened EV release was also observed in THP-1 monocytes and macrophages treated with Bafilomycin and TMZ, highlighting a broader, cell type-independent mechanism. These findings indicate that combined DNA damage and lysosomal inhibition synergistically stimulate secretory autophagy and EV release, potentially impacting the tumor microenvironment and driving disease progression.

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