The role of apoptotic neuroblastoma-derived extracellular vesicles on mesenchymal stem cell and their regulation of T cells

Abstract

Neuroblastoma is the second most common solid tumour found in paediatrics, even with a combination of therapies including the state of the art anti- disialoganglioside 2 (GD2) immunotherapy, only 50 to 60% of the patients with metastatic disease survived. One of the contributory factors is the unfavourable tumour microenvironement (TME) for the immune cells. Within the TME, mesenchymal stem cells (MSCs) can be found for they can migrate through the blood vessels. They are known for their immunosuppressive function hence they may become a major hindrance to the effective immunotherapy treatment. Nevertheless, studies have shown that MSCs function can be manipulated by the environment. Under certain stimuli, MSCs can convert to a pro-inflammatory phenotype. In neuroblastoma patients who are receiving chemotherapy, an accumulation of apoptotic cells (AC) is expected. The aim of this study was to investigate how apoptotic neuroblastoma cells may affect MSCs T cell modulatory function. By priming the immortalised MSC (hTMSC) with neuroblastoma-derived apoptotic cells, we found an increase of T cell proliferation and activation. Moreover the T cells were induced to differentiate to pro-inflammatory phenotype. We also found a reduction in regulatory T cells (Treg) differentiation. Moreover these T cells that were co-cultured with AC-primed hTMSC has a greater cancer killing function. We later discovered that this activation of hTMSC may be due to the dying neuroblastoma cells after cisplatin treatment. Not only does cisplatin induce apoptosis in neuroblastoma, we also discovered signs of immunogenic cell death pathway necroptosis - a programmed necrosis cell death occurred. As immunogenic molecules were not found in the supernatant of the dying neuroblastoma cells, we explored the possible involvement of extracellular vesicles (EVs). EVs-derived from neuroblastoma cell line was highly heterogeneous, we also compared the EVs derived from both cisplatin treated or untreated neuroblastoma cells. We found that each population of EVs had their unique characteristic, interestingly apoptotic neuroblastoma-derived EVs contains the HMGB1 protein which is often associated with pro-immunostimulatory function. The apoptotic microvesicle was the most effective group in inducing the hTMC to decreased Treg differentiation and increase T cell proliferation. To further confirm the importance of HMGB1, we studied the downstream of HMGB1. There was a slight up-regulation of the HMGB1 receptor, RAGE and TLR2 on the hTMSC. This eventually led to an activation of the NF-kB signalling pathway and increased the secretion of pro-inflammatory cytokines IL-6, IL-8 and IL-33. In conclusion, we proposed that with chemotherapy, abundant ACs will be released. This accumulation of ACs from the TME may alter the immune response. Apoptotic neuroblastoma can release EVs containing immunogenic proteins such as HMGB1 to convert the hTMSC in switching from immunosuppressive to pro-inflammatory phenotype. Our findings suggest a paradigm shift in anti-cancer concept. This is to improve the efficacy of immunotherapy by manipulating the TME towards a more favourable condition for immune surveillance.