Ma, L.; Liu, M.; Piskareva, O.

Extracellular vesicles (EVs) are emerging mediators of oncogenic communication within the neuroblastoma (NB) tumour microenvironment (TEM). Here, we investigated how constitutive MYCN overexpression influences the proteomic and functional properties of small EVs (sEVs) derived from SKNAS-MYCN-GFP (SK-M) cells and assessed their impact on non-cancerous immune cells. SK-M cells exhibited robust MYCN upregulation at both the mRNA and protein levels and produced sEVs that were selectively enriched in MYCN. Transwell co-culture revealed transfer of MYCN-GFP to recipient DC2.4 nuclei, indicating intercellular transport of functional transcription factor cargo. LC-MS/MS profiling showed that SK-M sEVs incorporated oncogenic cargo non-randomly, displaying significant enrichment of metabolic and MYC/MYCN-regulated pathways, including glycolysis, mTORC1 signalling, and suppression of oxidative phosphorylation (OXPHOS). These observations are consistent with emerging evidence that MYC family proteins can regulate metabolism through vesicular transfer of glycolytic kinases to neighbouring cells. Functionally, SK-M cells displayed elevated lactate secretion and reduced acetyl-CoA, and their sEVs induced a glycolytic shift in recipient immune cells, increasing lactate output in DC2.4, RAW264.7, BMDCs, and splenocytes. sEV-treated BMDCs and splenocytes acquired immunoregulatory phenotypes characterised by increased IL-10, reduced IL-12, expansion of regulatory T cells (Tregs), and macrophage polarization toward an M2-like state. These findings demonstrate that MYCN-driven NB cells disseminate metabolic and immunosuppressive cues via sEVs, reshaping the local immune landscape to favour tumour tolerance. This study provides mechanistic insight into how MYCN-amplified NB cells exploit EV-based communication to coordinate metabolic rewiring and immune escape.