Sensory Neurotransmitter Calcitonin Gene‐Related Peptide Modulates Tumor Growth and Lymphocyte Infiltration in Oral Squamous Cell Carcinoma

Abstract

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

The impact of tumor immunogenicity on cancer pain phenotype using syngeneic oral cancer mouse models

“…For investigation into the syngeneic allograft transplant model, we chose to focus on three nociception-related proteins expressed on primary afferent sensory neurons, TRPV1, CGRP, and ATF3 and their associated genes, Trpv1, Calca, Atf3 . Cancer-induced increases in TRPV1 and CGRP protein expression in the TG neurons have been previously reported in association of nociception in both rat ( 47 ) and mouse ( 19 , 22 ) oral cancer models. We found, within the MOC1-and MOC2- models, that Calca gene expression and CGRP protein in tongue-innervating neurons are increased in primarily female mice, an effect which occurs along with the sex differences in nociceptive behavior.…”

“…Therefore, we also assessed relative gene expression in specifically tongue-innervating primary afferent neurons using retrograde labeling and single cell PCR. We have previously identified a role for the sensory neuropeptide, calcitonin gene-related peptide (CGRP), and chemosensitivity receptor, transient receptor vanilloid channel 1 (TRPV1), in oral cancer pain ( 19 , 22 ). Additionally, given the differences in immunogenicity and tumor growth in vivo , we sought to understand the potential contribution of nerve injury and subsequent neuropathic pain ( 9 ) for each model.…”

“…To generate the syngeneic orthotopic oral cancer mouse model, adult male and female mice under 3–5% isoflurane anesthesia were inoculated into the anterior lateral portion of the tongue with either 7.5 × 10 5 MOC1 cells or 2 × 10 4 MOC2 in 30 μL of a mixture of DMEM (Life Technologies) and Matrigel (Corning) at a 1:1 ratio as previously described ( 19 ). Injection of DMEM and Matrigel alone was used as control (i.e., sham).…”

“…Reverse transcription was performed with Quantitect Reverse Transcription Kit (Qiagen Inc.) according to the manufacturer's instructions. For single cell analysis, DiI-positive single neurons were dissociated as previously described ( 19 , 21 , 22 ), identified using fluorescence microscopy, and limited to ≤ 25 μm in diameter. Cells were then picked up using glass capillaries (World Precision Instruments) which were held by micromanipulator (Sutter Instruments), headstage (EPC10 HEKA) and electrode holder under brightfield optics.…”

Nociceptor neurons affect cancer immunosurveillance

“…In head and neck tumours, a loss of TP53 drives the reprogramming of tumourinnervating sensory nerves into adrenergic neurons that promote tumour growth 1 . The presence of such neo-innervation in cancer, together with the diverse actions of neuropeptides on immune cells [11][12][13][14][15][16][17][18] , led us to examine whether the local release of neuropeptides from activated nociceptors could favour cancer growth by suppressing immune surveillance.…”