Background
The unique immune landscape of the brain tumor microenvironment, shaped in part by the blood–brain barrier, presents a major obstacle to the success of immunotherapy in patients with melanoma brain metastases (MBM). Emerging evidence suggests that tumor-derived small extracellular vesicles (td-sEVs) play a pivotal role in modulating immune response and remodelling of the pre- metastatic niche. However, the molecular mechanisms underlying how td-sEVs affects the immune response of MBM remain unclear.
Methods
Size exclusion chromatography was used to enrich sEVs from cell lines established from spontaneously developing primary cutaneous melanoma (CM), lymph node (LN) and brain metastases (BM) from the MT ret-transgenic melanoma model.Characterization of sEVs was performed using the bicinchoninic acid (BCA) assay, nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and western blot. Neutrophil plasticity and function was assessed by flow cytometry. Proteome was used to compare the cytoki- nes associated with td-sEVs cargo in comparison to sEVs free supernatant group.
Results
BM-sEVs significantly enhance reactive oxygen species (ROS) production and neutrophil extracellular traps (NETs) formation implicated in promoting immunosuppression and impairing anti-tumor immunity. Specifically, melanoma-derived sEVs reprogram neutrophils from an N1 anti-tumor phenotype toward an N2 pro-tumor, which shown much more significantly in MBM derived sEVs. Proteome profiler revealed that sEVs are the primary carriers of proteins linked to angiogenesis and tumor progression.
Conclusion
Our findings uncover a previously unrecognized phenomenon whereby BM-derived sEVs promote neutrophil survival and enhance ROS/NETs production, which may also moderate neutrophil reprogramming. This process contributes to tumor immune eva- sion, immunosuppression and therapy resistance. These findings provide a basis for novel therapeutic strategies aimed at enhancing the efficacy of immunotherapy in patients with metastatic melanoma of the brain.
Keywords
Melanoma; small extracellular vesicles; neutrophil; immunotherapy
Funding/Acknowledgments
This work was partially financed by the the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) – Project-ID 418179183 – KFO 337 (HE 5294/2-1 (IH).
