Acute radiotherapy-associated oral pain may promote tumor growth at distant sites

Abstract

Introduction: Patients developing acute radiotherapy induced dermatitis or oral mucositis commonly experience pain. When severe, this radiotherapy-associated pain (RAP) can necessitate treatment breaks; unfortunately, in a variety of cancers, prolongation of the radiotherapy course has been associated with early cancer relapse and/or death. This is often attributed to accelerated repopulation, but it is unknown whether pain or pain signaling constituents might alter tumor behavior and hasten metastatic disease progression. We studied this by testing the hypothesis that severe acute RAP at one site can hasten tumor growth at a distant site.

Methods: Mice underwent single fraction tongue irradiation (27 Gy, or 0 Gy "sham" control) to induce severe glossitis. At the time of maximal oral RAP, one of three luciferase-transfected tumor cell lines were injected via tail vein (4T1, B16F10, MOC2; each paired to their syngeneic host: BALB/c or C57BL/6); tumor burden was assessed via in vivo transthoracic bioluminescence imaging and ex vivo pulmonary nodule quantification. Survival was compared using Kaplan-Meier statistics.

Results: Tongue irradiation and resultant RAP promoted lung tumor growth of 4T1-Luc2 cells in BALB/c mice. This effect was not a result of off-target radiation, nor an artefact of environmental stress caused by standard (subthermoneutral) housing temperatures. RAP did not affect the growth of B16F10-Luc2 cells, however, C57BL/6 mice undergoing tail vein injection of MOC2-Luc2 cells at the time of maximal RAP experienced early lung tumor-attributable death. Lung tumor growth was normalized when RAP was reduced by treatment with resiniferatoxin (300 µg/kg, subcutaneously, once).

Discussion: This research points towards radiation-induced activation of capsaicin-responsive (TRPV1) neurons as the cause for accelerated growth of tumors at distant (unirradiated) sites.

Keywords: TRPV1; cancer; mouse; pain; radiotherapy; sensory nerves.

Figure 1

RAP is associated with rapid growth of 4T1-Luc2 cells following tail vein injection in the syngeneic host. In female mice (n=12-18/group), transthoracic BLI (A) and ex vivo manual count (B) were significantly higher in the irradiated mice. These findings were recapitulated in male mice (n=11/group), where transthoracic BLI (C) and number of surface pulmonary nodules (D) were significantly higher in tongue-irradiated mice. The experiment was then repeated in female mice, with addition of a second control group in which mice underwent total body irradiation (TBI) to assess the effects of off-target radiation to non-lingual tissues. In this study (n=12 mice/group) we found a significant increase in transthoracic BLI for irradiated versus control mice; there was no detectable statistical difference between the two control groups (E). Likewise, IR mice had more lung tumors as compared to SHAM and TBI mice. Tumor burden was markedly decreased in the TBI versus SHAM group (F). We also evaluated the effects of cage temperature (n=7-8/group). Results (G, H) were as expected for standard (STDt) housing conditions, recapitulating what was already described in A through (F) However, when housed in warm cages (TNt), there was no detectable difference in lung tumor burden for IR and SHAM mice, and the tumor burden was higher for both groups than it was for the STDt-SHAM mice. Data for BLI imaging are presented as mean ± standard error of mean (SEM), and data from post-mortem evaluations are presented as mean ± standard deviation (SD). *p<0.05, **p<0.01; ****p<0.0001; ns., not significant.

Figure 2

The effect that acute RAP has on distant tumor growth varies with tumor model. In the B16F10-Luc2model (n=19-20/group) there were no significant differences in lung tumor burden (transthoracic BLI) between IR and SHAM C57BL/6 mice at days 25, 27, and 29 post-injection (A) or by post-mortem counting (B). Similarly, there was no detectable difference in transthoracic BLI strength in C57BL/6 mice with MOC2-Luc2 tumors (n=13-16/group) (C). However, pulmonary metastasis-attributable deaths occurred earlier for IR mice versus SHAM (D). Data are presented as mean ± standard deviation (SD). ****p<0.0001; ns., not significant.

Figure 3

Systemic ablation of TRPV1-expressing neurons reduces the severity of RIM, and mitigates the tumor-promoting effects of RAP. In all mice, tongue irradiation caused glossitis (A) that was accompanied by weight loss (B). The severity of these changes was reduced with pre-irradiation resiniferatoxin treatment. Resiniferatoxin pre-treatment also reduced the tumor-promoting effect of high-dose tongue irradiation as measured in vivo using transthoracic BLI (C) and postmortem enumeration of nodules (D). Representative images of the India-ink treated lungs used for manual nodule enumeration are depicted in (E). Data are presented as mean ± standard deviation (SD); *p<0.05; ***p<0.001; ****p<0.0001; ns., not significant.