Effects of secretome and EVs from neural crest-derived stem cells on glioblastoma multiforme cells

Glioblastoma multiforme (GBM), an incurable primary brain cancer, is a very heterogeneous, and aggressive type of cancer, with poor patient outcome despite multimodal therapy. The secretomes of GBM cells contribute to enhanced tumour sustainability, tumour stemness, and immune evasion mainly due to activation of very potent signalling pathways such as NF-κB, enhancing tumour cell proliferation, as well as resistance to cancer drugs. The secretome, rich with adhesion molecules, proteases, as well as growth factors, mediates the extracellular matrix, thereby creating a microenvironment, a safe niche, as well as a strong inducer of aggressive tumour. The cancer secretome, consisting of the total array of both biomolecules and extracellular vesicles (EVs), released from cancer cells as well as their microenvironment, remains one of the strongest predictors of therapeutic resistance. Secretomes and sEVs (small EVs), derived from human neural crest-derived stem cells (NCSCs), were characterised and systematically tested for their potential to condition to inhibit the proliferation of GBM. Secretomes and EVs were derived from NCSCs characterised from three different donors, whose molecular identities were also confirmed. A series of functional assays were performed, including viability assays, proliferation, migration, tumour-forming colony assays, as well as sphere assays on U251, U737 and U87 GBM cells. Secretome from the NCSCs significantly inhibited the survival as well as the migratory properties of these cells. Signalling studies conducted utilizing NF-κB and IRF3 reporter assays, a cytokine array, and immunocytochemistry, demonstrated significant inhibition of NF-κB activation even in the context of secretome from GBM cells as well as inflammatory challenge, while suggesting a nuclear enrichment and activation of IRF3. Secretomes from NCSCs not only inhibit these inflammatory processes, but they also specifically regulate the cytokine secretion, leading to reduced activation of microglia, as well as the normalisation of an anti-tumour immune environment. The ex vivo results from organotypic brain slices demonstrated a remarkable decrease in the tumour colony, as well as persistence in the combined setting of temozolomide (TMZ), without any toxicity to normal tissue. By integrating the cancer cell secretome data, the following has been confirmed: the secretomes and EVs from glioblastoma propagate autocrine and paracrine signalling in proliferation, immune evasion, and resistance to chemotherapeutic drugs. Safety and translation challenges were considered in light of the current market conditions for treatment, as described in the study (Asadpour et al. 2023). Even while being hopeful about preclinical adaptation, the use of EVs and secretomes for a preclinical setting is faced with some serious challenges, such as the lack of standardised production, variability, lack of knowledge about the mode of action, as well as limited regulation. This paper highlights the need for common standards and stricter rules to ensure safe application, as mentioned in the study. This thesis provides the first conclusive proof that secretome/EVs derived from NCSCs represent a novel approach to overcome GBM resistance based on modulation of cell signalling at a cellular, and molecular levels. By studying immunomodulation, enhancing chemotherapy responses, and focusing on safe use, this approach offers a new and valuable tool for therapy development.