larynx, or hypopharynx. [2,3] Risk factors can be behavioral (i.e., tobacco and alcohol use) or infection-associated (i.e., human papillomavirus (HPV)), and these factors vary with geographic location. [4] Head and neck squamous cell carcinoma (SCC) constitutes 90% of cases of HNC and is associated with severe disease and high rates of recurrence despite advances in cancer treatment. [1] The oral cavity is innervated by cranial nerves with a high density of sensory nerves, originating mainly from the trigeminal ganglia (TG). [5] To date, histological patterns of cancer-nerve interaction, defined as perineural invasion (PNI), are identified as the presence of tumor cell clusters within the peripheral nerve sheath or infiltrating the nerves and/or tumor cells encircling one-third of the nerve circumference. [6] PNI was detected in up to 70% of oral SCC in the tongue and/ or floor of the mouth and has been demonstrated as an independent predictor of poor prognosis and an indicator of aggressive tumor behavior. [7][8][9][10] Prior research by Rahima et al. [11] and Laske et al. [12] found that PNI in early stage oral and oropharyngeal carcinomas has a highly negative association with recurrence-free survival and tumor differentiation. Systemic analysis of the neural influences within the cancer microenvironment, including identification of how PNI and other nerve-cancer interactions generally relate to poor prognosis, is crucial given that ongoing research is focusing on the neural invasion for targeted therapies of tumor regression. [13][14][15][16][17][18] Calcitonin gene-related peptide (CGRP) is the most abundant neurotransmitter in trigeminal ganglia neurons (TGN) innervating the tongue [19,20] and serves a prominent role in efferent signaling; upon activation of sensory nerve fibers, CGRP is released from peripheral nerve terminals and exerts paracrine effects on surrounding tissues [21] , including tumor cells. Two isoforms of CGRP exist; αCGRP, derived from the CALCA gene, is the principal form found in the central and peripheral nervous system, whereas βCGRP, derived from the CALCB gene, is found mainly in the enteric nervous system. [21] In a rat oral cancer model, a high percentage of αCGRPimmunoreactive nerve sprouting was found in the tumor microenvironment and orofacial sensitization was accompanied by upregulated αCGRP expression in the maxillary and

Head and neck squamous cell carcinoma (HNSCC) patients report severe function-induced pain at the site of the primary tumor. The current hypothesis is that oral cancer pain is initiated and maintained in the cancer microenvironment due to secretion of algogenic mediators from tumor cells and surrounding immune cells that sensitize the primary sensory neurons innervating the tumor. Immunogenicity, which is the ability to induce an adaptive immune response, has been widely studied using cancer cell transplantation experiments. However, oral cancer pain studies have primarily used xenograft transplant models in which human-derived tumor cells are inoculated in an athymic mouse lacking an adaptive immune response; the role of inflammation in oral cancer-induced nociception is still unknown. Using syngeneic oral cancer mouse models, we investigated the impact of tumor cell immunogenicity and growth on orofacial nociceptive behavior and oral cancer-induced sensory neuron plasticity. We found that an aggressive, weakly immunogenic mouse oral cancer cell line, MOC2, induced rapid orofacial nociceptive behavior in both male and female C57Bl/6 mice. Additionally, MOC2 tumor growth invoked a substantial injury response in the trigeminal ganglia as defined by a significant upregulation of injury response marker ATF3 in tongue-innervating trigeminal neurons. In contrast, using a highly immunogenic mouse oral cancer cell line, MOC1, we found a much slower onset of orofacial nociceptive behavior in female C57Bl/6 mice only as well as sex-specific differences in the tumor-associated immune landscape and gene regulation in tongue innervating sensory neurons. Together, these data suggest that cancer-induced nociceptive behavior and sensory neuron plasticity can greatly depend on the immunogenic phenotype of the cancer cell line and the associated immune response.

Supplemental Digital Content is Available in the Text.Oral cancer mouse models were used to demonstrate that sympathetic neurotransmission modulates oral cancer pain and tumor growth through adrenergic signaling in the tumor microenvironment.

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The impact of peripheral neurons on carcinogenesis is an understudied process in head and neck cancer. Peripheral nerve innervation is increased in preclinical models of oral cancer; sympathectomy resulted in smaller, less invasive cancers. Local neurotransmitter release from peripheral neurons innervating cancer has been linked to cancer growth and immune suppression. We hypothesize that local neurotransmitter release from trigeminal sensory neurons (TGNs) and sympathetic postganglionic neurons promotes cancer proliferation and suppresses the immune response. We used real-time PCR to determine RNA expression in human oral cancer cell lines and a colorimetric cell proliferation assay to quantify cancer cell growth in response to the sympathetic and sensory neurotransmitters. Oral cancer cell lines, HSC-3 and SCC-4, overexpress the norepinephrine (NE) receptor, adrenergic receptor beta 2 (ADRβ2) and sensory neurotransmitter receptors, receptor activity modifying protein1 (RAMP1) and neurokinin1 receptor (NK1R) compared to the non-tumorigenic cell line, HaCaT. Treatment with sympathetic (NE) and sensory (CGRP, substance p) neurotransmitters for 48 hours significantly increased cell proliferation in HSC-3 (>2-fold) and SCC-4 (>1.5-fold) cell lines, but had no effect on HaCaT cell proliferation. Using optical clearing and immunohistochemistry, we observed increased PGP9.5+ neuronal fiber density in the microenvironment using high-resolution microscopy in a tongue oral cancer xenograft mouse model. In vitro we measured a 44.6 ± 1.14% increase in TGN sprouting after 48-hour exposure to conditioned media from the HSC-3, compared to conditioned media from HaCaT. Lastly, HSC-3 cell culture supernatant injection (50 µl) into the tongue of C57Bl/6 female mice resulted in infiltration of CD45+ immune cells (9.10 ± 1.2 % of total live cells). Temporary nerve block with 1% bupivacaine injection into the lingual nerve prior to a 50 µl injection of HSC-3 supernatant into the tongue resulted in significantly more CD45+ immune cell infiltration (21.66 ± 3.4 %) compared to HSC-3 supernatant without pharmacologic nerve block. These results together suggest neurotransmitter-induced increase in oral cancer cell proliferation and neurotransmitter-induced suppression of the cancer-evoked immune response. Understanding the nerve-cancer interaction might improve strategies to treat head ad neck cancer by targeting peripheral neurons in the cancer microenvironment. Citation Format: Megan A. Atherton, Marci L. Nilsen, Nicole N. Scheff. Peripheral neurons invade oral squamous cell carcinoma microenvironment and drive tumorigenesis [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-050.

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