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Peripheral neurons comprise a critical component of the tumor microenvironment (TME). The role of the autonomic innervation in cancer has been firmly established. However, the effect of the afferent (sensory) neurons on tumor progression remains unclear. Utilizing surgical and chemical skin sensory denervation methods, we showed that afferent neurons supported the growth of melanoma tumors in vivo and demonstrated that sensory innervation limited the activation of effective anti-tumor immune responses. Specifically, sensory ablation led to improved leukocyte recruitment into tumors, with decreased presence of lymphoid and myeloid immunosuppressive cells and increased activation of T-effector cells within the TME. Cutaneous sensory nerves hindered maturation of intratumoral high endothelial venules (HEVs) and limited formation of mature tertiary lymphoid-like structures containing organized clusters of CD4+ T cells and B cells. Denervation further increased T-cell clonality and expanded the B-cell repertoire in the TME. Importantly, CD8a depletion prevented denervation-dependent anti-tumor effects. Finally, we observed that gene signatures of inflammation and the content of neuron-associated transcripts inversely correlated in human primary cutaneous melanomas, with the latter representing a negative prognostic marker of patient overall survival. Our results suggest that tumor-associated sensory neurons negatively regulate the development of protective anti-tumor immune responses within the TME, thereby defining a novel target for therapeutic intervention in the melanoma setting.

Extracellular vesicles (EVs) such as exosomes (EXOs) and microvesicles (MVs) are promising carriers for the delivery of biologic drugs such as nucleic acids and proteins. We have demonstrated, for the first time, that EVs derived from hCMEC/D3: a human brain endothelial cell (BEC) line transfer polarized mitochondria to recipient BECs in culture and to neurons in mice acute brain cortical and hippocampal slices. This mitochondrial transfer increased ATP levels by 100 to 200-fold (relative to untreated cells) in the recipient BECs exposed to oxygen-glucose deprivation, an in vitro model of cerebral ischemia. Our previous studies suggested that EXOs, the smaller vesicle subpopulation, derived from a macrophage cell line (RAW264.7) load more exogenous plasmid DNA compared to the larger MVs and the RAW-derived EXOs also demonstrated greater transfection in the recipient BECs compared to EXOs derived from the homotypic hCMEC/D3 BECs. Proteomic analysis of EVs indicated that RAW-EVs are preferentially enriched with proteins that are involved in the trafficking of DNA-containing particles from the cytoplasm towards the nucleus. Intriguingly, although the heterotypic macrophage-derived EVs demonstrated increased transfection in the recipient BECs; the homotypic, BEC-derived EVs demonstrated a greater selectivity to transfer polarized mitochondria and increase endothelial cell survival under ischemic conditions.

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Peripheral glia, specifically the Schwann cells (SCs), have been implicated in the formation of the tumor microenvironment (TME) and in cancer progression. However, in vivo and ex vivo analyses of how cancers reprogram SC functions in different organs of tumor-bearing mice are lacking. We generated Plp1-CreERT/tdTomato mice which harbor fluorescently labeled myelinated and non-myelin forming SCs. We show that this model enables the isolation of the SCs with high purity from the skin and multiple other organs. We used this model to study phenotypic and functional reprogramming of the SCs in the skin adjacent to melanoma tumors. Transcriptomic analyses of the peritumoral skin SCs versus skin SCs from tumor-free mice revealed that the former existed in a repair-like state typically activated during nerve and tissue injury. Peritumoral skin SCs also downregulated pro-inflammatory genes and pathways related to protective anti-tumor responses. In vivo and ex vivo functional assays confirmed immunosuppressive activities of the peritumoral skin SCs. Specifically, melanoma-reprogrammed SCs upregulated 12/15-lipoxygenase (12/15-LOX) and cyclooxygenase (COX)-2, and increased production of anti-inflammatory polyunsaturated fatty acid (PUFA) metabolites prostaglandin E2 (PGE2) and lipoxins A4/B4. Inhibition of 12/15-LOX or COX2 in SCs, or EP4 receptor on lymphocytes reversed SC-dependent suppression of anti-tumor T-cell activation. Therefore, SCs within the skin adjacent to melanoma tumors demonstrate functional switching to repair-like immunosuppressive cells with dysregulated lipid oxidation. Our study suggests the involvement of the melanoma-associated repair-like peritumoral SCs in the modulation of locoregional and systemic anti-tumor immune responses.