Bone Marrow-Derived Extracellular Vesicles Carry RNAs Shaping The Tumor Microenvironment in High-Risk Neuroblastoma Patients

Background Despite the intensive treatment, the survival rate of high-risk neuroblastoma (HR-NB) patients remains low. Over 10% of patients have tumors that lack the expression of GD2, a NB-associated ganglioside, making them ineligible for anti-GD2 antibody immunotherapy. Therefore, the development of novel therapeutic strategies to enhance immunotherapy efficacy and mitigate side effects is urgently needed. Circulating small extracellular vesicles (sEVs) can contribute to tumor progression by promoting metastasis, immunosuppression, and resistance to treatment, ultimately interfering with antibody-dependent cellular cytotoxicity. Aims We aimed at profiling the RNA content of bone marrow (BM)-derived circulating sEVs to investigate their ability to shape the tumor microenvironment (TME), identifying new therapeutic targets to improve immunotherapy outcomes. Methods We isolated sEVs from BM plasma samples of HR-NB patients at diagnosis with or without BM infiltration (n=27). We assessed quality and quantity of isolated EVs and performed total RNA sequencing. Results We implemented a novel protocol for the efficient isolation of sEVs from 0.5 ml of plasma derived from BM. The average quantity of RNA obtained ranged from 300 to 600 ng. We observed that sEVs carry the transcripts of genes (mRNAs) microRNAs (miRNAs) and long non-coding RNAs (lncRNAs) involved in cell metabolism, cell signaling and immune response, such as Natural Killer-cell mediated cytotoxicity and chemokine signaling. sEV-miRNAs and lncRNAs showed differential expression between infiltrated and non-infiltrated BM samples. Three sEV-miRNA downregulated in infiltrated BM samples were involved in the regulation of PD1/PDL1 interaction and signaling. Moreover, bioinformatic integration analysis of mRNAs, lncRNAs and miRNAs stratified HR-NB patients into three clusters, identifying a signature associated with the hypoxic status and the poor prognosis. These results suggest that BM-derived sEVs carry RNAs contributing to an immunosuppressive TME, negatively affecting treatment response and clinical outcome. Conclusions BM-derived sEVs carry RNA molecules that regulate immune pathways, hindering the response of HR-NB patient to immunotherapy. These findings provide insights into the role of sEVs in TME modulation and identify potential targets for improving immunotherapy.