Calcitonin Related Polypeptide Alpha Mediates Oral Cancer Pain

Abstract

Oral cancer patients suffer pain at the site of the cancer. Calcitonin gene related polypeptide (CGRP), a neuropeptide expressed by a subset of primary afferent neurons, promotes oral cancer growth. CGRP also mediates trigeminal pain (migraine) and neurogenic inflammation. The contribution of CGRP to oral cancer pain is investigated in the present study. The findings demonstrate that CGRP-immunoreactive (-ir) neurons and neurites innervate orthotopic oral cancer xenograft tumors in mice. Cancer increases anterograde transport of CGRP in axons innervating the tumor, supporting neurogenic secretion as the source of CGRP in the oral cancer microenvironment. CGRP antagonism reverses oral cancer nociception in preclinical oral cancer pain models. Single-cell RNA-sequencing is used to identify cell types in the cancer microenvironment expressing the CGRP receptor components, receptor activity modifying protein 1 Ramp1 and calcitonin receptor like receptor (CLR, encoded by Calcrl). Ramp1 and Calcrl transcripts are detected in cells expressing marker genes for Schwann cells, endothelial cells, fibroblasts and immune cells. Ramp1 and Calcrl transcripts are more frequently detected in cells expressing fibroblast and immune cell markers. This work identifies CGRP as mediator of oral cancer pain and suggests the antagonism of CGRP to alleviate oral cancer pain.

Keywords: CALCRL/RAMP1; CGRP; cancer innervation; oral cancer; pain; peptidergic neurons.

Figure 1

CGRP-expressing neurons innervating oral cancer xenograft tumors in the tongue. (a) HSC-3 tongue xenograft. The boxes indicate the area shown in (c,e). (b) OSC-20 tongue xenograft. The boxes indicate the area shown in (d,f). (c,d) Higher magnification images. Cancer cells (white, TRP63 isoforms, p63 + p40-ir) are innervated by bundles of neurons and neurites (yellow arrows). The neurons and neurites are identified by expression of GAP43-ir (teal), SOX10-ir (red, Schwann cells) and CGRP-ir (yellow). The nerve bundles run in close proximity to blood vessels (orange, PECAM-ir). Lymph vessels (green, LYVE1-ir) are also present. (e,f) Tongue dorsum. CGRP-ir and GAP43-ir nerves running with blood vessels below the epithelium innervate the fungiform (arrowhead in (e)) and filiform (arrowheads in (f)) papillae.

Figure 2

Accumulation of CGRP-ir in nerve fibers innervating oral cancer inoculated into the paw of mice. (a) The sciatic nerve was ligated distal to convergence of spinal nerves L4–L6 in mice 3 weeks after inoculation of HSC-3 cancer cells in the paw. The sciatic nerve sample was collected 24 h after ligation (red arrow). (b) Co-localization of CGRP-ir (green) inside the axons (PGP9.5-ir, red) of the sciatic nerve segment. Arrows indicate the colocalization of CGRP-ir (green) inside the axons (red). Scale bar in (b,c) = 10 µm. (c) CGRP-ir in the sciatic axons from sham operated mice (no SNL, n = 3 mice, n = 1829 axons) compared to sciatic nerve ligation mice (SNL, n = 4 mice, n = 2949 axons), t₍₄₇₇₆₎ = 4.88, ** p < 0.01, unpaired Student’s t-test.

Figure 3

The CGRP receptor antagonist, olcegepant (OCP), reduces oral cancer nociception evoked by inoculation of oral cancer cells in the paw or tongue. (a) Experiment timeline indicating the baseline nociception measurement (BL) and the time at which HSC-3 or MOC2 cancer cells (red arrows) were inoculated into the left hind paw of NU/J mice or tongue of C57BL/6J mice, administration of olcegepant (OCP) or vehicle (blue arrows) and times of testing with the paw von Frey filament or facial nociception assay. (b) Paw cancer model. At 2 and 3 weeks after cancer cell inoculation (red arrow), the mice had developed cancer nociception. They were given OCP or vehicle (blue arrow) and tested for nociception with the paw von Frey filament assay. The direction indicating less nociception is indicated to the right of the plot (c) Orthotopic tongue cancer model. At 2 weeks after cancer cell inoculation (red arrow) the mice had developed nociception. They were given OCP or vehicle (blue arrow) and tested for nociception with the facial von Frey filament assay. The direction indicating less nociception is indicated to the right of the plot. In (b) F_(8,72) = 2.89, * p = 0.03, n = 5 mice in each group. In (c) F_(7,114) = 134.1, ** p < 0.0001, n = 10 mice in each group, OCP versus control at indicated time points by Two-way ANOVA.

Figure 4

Expression of RAMP1 and CALCRL in cell lines measured via qRT-PCR. (a) Expression of RAMP1 relative to GAPDH was greater in dysplastic and cancer cell lines compared to non-tumorigenic skin keratinocyte line, HaCaT (b). The relative expression levels of CALCRL relative to GAPDH in HaCaT were greater than in DOK and OSC-20. Data are represented as mean ± SD. A one-way ANOVA, followed by Tukey’s multiple comparisons test, was carried out to evaluate statistical differences between groups.

Figure 5

A greater number of HSC-3 and OSC-20 cells express RAMP1 than CALCRL. Shown are the percent of cells expressing human CGRP receptor components and the epithelial marker gene, COL17A1 in HSC-3 and OSC-20 cells grown (a) on tissue culture plastic and (b) as tongue xenografts. The relative frequencies of cells expressing human RAMP1 and CALCRL were similar in tissue culture and in xenograft samples.

Figure 6

Cells expressing the CGRP receptor components were more frequent in fibroblast and immune cells in the HSC-3 and OSC-20 xenograft microenvironments. (a) Composition of the xenograft microenvironment for selected tissue types as defined by the expression of the marker genes for immune cells (Ptprc, encoding CD45), fibroblasts (Pdgfrb), lymphatic endothelial cells (Prox1, Lyve1), blood endothelial cells (Vwf) and Schwann cells (Plp1). (b,c) Expression of Ramp1 and Calcrl in selected tissue types of the xenograft microenvironment as determined by co-expression with tissue marker genes. (d) t-SNE representation of cell barcodes co-expressing Ramp1 and Calcrl—Ramp1 + Calcrl cells. (e) Tissue type distribution of Ramp1 + Calcrl cells in the xenograft microenvironment as defined by co-expression with tissue marker genes.