Seeding of breast cancer cell line (MDA-MB-231LUC+) to the mandible induces overexpression of substance P and CGRP throughout the trigeminal ganglion and widespread peripheral sensory neuropathy throughout all three of its divisions

Abstract

Some types of cancer are commonly associated with intense pain even at the early stages of the disease. The mandible is particularly vulnerable to metastasis from breast cancer, and this process has been studied using a bioluminescent human breast cancer cell line (MDA-MB-231^LUC+). Using this cell line and anatomic and neurophysiologic methods in the trigeminal ganglion (TG), we examined the impact of cancer seeding in the mandible on behavioral evidence of hypersensitivity and on trigeminal sensory neurons. Growth of cancer cells seeded to the mandible after arterial injection of the breast cancer cell line in Foxn1 animals (allogeneic model) induced behavioral hypersensitivity to mechanical stimulation of the whisker pad and desensitization of tactile and sensitization of nociceptive mechanically sensitive afferents. These changes were not restricted to the site of metastasis but extended to sensory afferents in all three divisions of the TG, accompanied by widespread overexpression of substance P and CGRP in neurons through the ganglion. Subcutaneous injection of supernatant from the MDA-MB-231^LUC+ cell culture in normal animals mimicked some of the changes in mechanically responsive afferents observed with mandibular metastasis. We conclude that released products from these cancer cells in the mandible are critical for the development of cancer-induced pain and that the overall response of the system greatly surpasses these local effects, consistent with the widespread distribution of pain in patients. The mechanisms of neuronal plasticity likely occur in the TG itself and are not restricted to afferents exposed to the metastatic cancer microenvironment.

Keywords: CGRP; Cancer; MDA-MB-231LUC+; SP; nociception; pain; sensitization.

Figure 1.

(a) Flowchart, procedures, and classification of the neurons included in the study in wild type (WT: C57BL/6J) and Foxn1 (B6.Cg-Foxn1nu/J) mice with MDA-MB-231^LUC+ tumor in the left mandible (T-M) or elsewhere than the head and neck region (T-E). Neurons were classified by subtype: LTMR: low threshold mechanoreceptors, A-HTMR: A fiber high threshold mechanoreceptor, C-HTMR: C fiber high threshold mechanoreceptor, F-Type: fast action potential (AP) dynamic mechanically unresponsive, S-Type: slow AP dynamic mechanically unresponsive, UNEX: electrically unexcitable cells. (b) Lateral-upper view of the innervation territories of the V1 and V2 divisions of the trigeminal nerve and location of the trigeminal ganglion (TG). (c) Lateral-lower view of the innervation territory of the V3 division of the trigeminal nerve. (d) Locations within the TG where the recordings were performed (yellow). Dotted gray areas (a and b) represent the likelihood of innervation by specific TG branches (V1: ophthalmic nerve; V2, maxillary nerve; V3 mandibular nerve). The green area represents the approximate relative location of the tumor (MDA-MB-231^LUC+) with respect to the TG innervation areas. OpN: optic nerve. BS: brain stem. (e) Sequence of electrophysiologic recordings in the study of subcutaneous injection of the supernatant of cell cultures of MDA-MB-231^LUC+ cells in Wild Type mice (WT/CSn) and foxn1 mice with MDA-MB-231^LUC+ tumors in the mandible or elsewhere beyond the head and neck (T-M/T-E). In all cases cellular baseline properties including receptive field (RF) area and somatic active electrical properties (SAP) obtained during RF characterization occurred first, followed by determining mechanical sensibility by measuring the mechanical stimulation [MT¹] in response to von Frey hair (VFH) application, and in all cases, the last measurement was that of conduction velocity (CV) using an electrical pulse (eP). In the WT/CSn, the time from subcutaneous (s.c.) injection and spontaneous discharge (δt) of HTMRs was recorded, and SAP of these action potentials was measured during a 2 min observation period. Mechanical threshold at the end of these 2 minutes was measured.

Figure 2.

Overlapped image of (a) bioluminescence and (b) X-ray of the mandible of a Foxn1 mouse 3 weeks after intracardiac injection of MDA-MB-231^LUC+ cancer cells (color-coded counts, sidebar). (c) Location in the left mandible in this animal of the tumor-induced bone lesion (dotted white line). (d) Stained section of the lesion for luciferase (pink) and DAPI (blue). (e) Effects of tumor implantation on the reflexive head withdrawal (T-E vs. T-M) (***=p<0.001). (f) Distribution (in %) of TG recorded afferents per animal per treatment (WT vs. T-E vs. T-M). Scale bar: 1 mm, 100 µm.

Figure 3.

Schematic diagram of the receptive field locations of recorded afferents and their presumed trigeminal nerve divisions in (a). Wild type (WT), (b) MDA-MB-231^LUC+ tumor elsewhere from head and neck (T-E), and (c) MDA-MB-231^LUC+ tumor in left mandible (T-M) mice. Data are presented with the location and subtype (right) (●: LTMR; ▲: AHTMR; ■: CHTMR) of recorded afferents. Mechanical threshold (MT) of subtypes of afferents according to trigeminal division: V1/V2 (solid symbol) and V3 (open symbol). Individual data points and medians (horizontal bars, values at the bottom) with boxes representing the 25 and 75 percentiles. The number of afferents per MT is presented aside in parentheses. *=p<0.05, **=p<0.01, ***=p<0.001.

Figure 4.

Representative SP (green), CGRP (red), and their overlay immunoreactivity in sections of trigeminal ganglia (TG) visualized by confocal microscopy. Data ((a) contra and (b) ipsilateral) are presented with the cell count per cell diameter (bin 2, µm, left column) and proportional distribution (pie charts, middle column). (c) Immunoreactivity integrated density (pixels/mm², right column), three weeks after MDA-MB-231^LUC+ tumor left mandible implantation. ** = p<0.01 between SP and CGRP. Scale bar: 100 µm.

Figure 5.

(a) Response of a low, A-high, and C-high threshold mechanoreceptor (LTMR, AHTMR, CHTMR respectively) afferent after subcutaneous injection at the arrow of the supernatant of MDA-MB-231^LUC+ cancer cell cultures (CSn) into their receptor field. Mechanical stimulation is only applied to LTMRs (upper short-dashed bars). Note the delay in the afferent's response (δt in sec, gray) to loss of response to stimulation in the LTMR and onset of spontaneous activity in the HTMRs and the differential modulation on the AP amplitude during and with ongoing discharge in the HTMRs. (b) Effects of CSn injection on mechanical threshold (MT) (●: LTMR; ▲: AHTMR; ■: CHTMR). Numbers are medians of thresholds before (MT¹) and after (MT²), control or CSn injection. The number of afferents per MT is presented aside in parentheses. **=p<0.01, ***=p<0.001. Scale bars: 15 sec, 20 mV