The molecular and functional contributions of intratumoral nerves to disease remain largely unknown. We localized synaptic markers within tumors suggesting that these nerves form functional connections. Consistent with this, electrophysiological analysis shows that malignancies harbor significantly higher electrical activity than benign disease or normal tissues. We also demonstrate pharmacologic silencing of tumoral electrical activity. Tumors implanted in transgenic animals lacking nociceptor neurons show reduced electrical activity. These data suggest that intratumoral nerves remain functional at the tumor bed. Immunohistochemical staining demonstrates the presence of the neuropeptide, Substance P (SP), within the tumor space. We show that tumor cells express the SP receptor, NK1R, and that ligand/receptor engagement promotes cellular proliferation and migration. Our findings identify a mechanism whereby intratumoral nerves promote cancer progression.

Cancer neuroscience has become an increasing area of focus in cancer research as our understanding of the presence and function of intra-tumoral neurons continues to emerge. Various publications indicate that tumor infiltrating neurons impact components of the tumor microenvironment promoting cancer initiation and progression. We have previously published that head and neck squamous cell carcinomas are innervated. We now focus on defining the contribution(s) of intra-tumoral neurons to disease progression in this cancer. Tumor implantation in a double transgenic mouse (TRPV1cre::DTAfl/wt ) that lacks the cancer-recruited TRPV1-expressing sensory neurons allows us to study the consequences of this neuronal loss on the local immune response. Thus, wildtype and TRPV1cre::DTAfl/wt mice were orthotopically implanted with tumor and tumoral immune cell populations identified by flow cytometry. Cytokine and peptide concentrations were measured by ELISA. In vitro assays were preformed to molecularly define the neuronal signals and immune responses. Here, co-culture experiments of immortalized head and neck cancer cell lines and dorsal root ganglia from wildtype mice were used to model tumor innervation in vitro. The conditioned media generated was applied to bone marrow derived (BMD) immune cells, or BMD myeloid derived suppressor cells and the impact on immune cell phenotypes assayed by flow cytometry. Utilizing a 12-color flow cytometry panel, we show that tumors from TRPV1cre::DTAfl/wt mice harbor a significantly smaller population of myeloid derived suppressor cells (MDSCs) as compared to those from wildtype animals. Further flow cytometry analysis demonstrates that this population shift occurs primarily due to a decrease in the CD11b+ Ly6G+/Ly6Clo population. In vitro studies using BMD immune cells show that tumor cell-DRG co-culture conditioned media induces an expansion of the MDSC population compared to condition media from DRG or tumor cells alone. Additionally, transwell migration assays show that co-culture condition media also increases cellular migration compared to condition media from DRG or tumor cells alone. Moreover, ELISA analysis indicates that co-culture of tumor cells and DRG increases neuronal release of the neuropeptide, Substance P (SP), which in turn potentiates IL-6 release from tumor cells. Immuno-depletion of IL-6 attenuates the in vitro generation of MDSCs. In summary, we identified several mechanisms by which TRPV1-expressing intra-tumoral neurons promote MDSC infiltration at the tumor bed and promote disease progression. These mechanisms define targets for therapeutic intervention including SP and IL-6 signaling. Future studies will test the clinical utility of these targets. Together, our data suggests that intra-tumoral neurons contribute to cancer progression through generation of a tumor supportive microenvironment. Citation Format: Anthony C Restaino, Austin Walz, Jeffrey Barr, Paola Vermeer. TRPV1+ sensory neurons provide a tumor supportive environment through recruitment of MDSCs [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A14.