Pain signaling in dogs undergoing radiotherapy for head and neck cancer

A frequent side effect of radiotherapy (RT) for head and neck cancer is intense pain. While the biological basis of acute orofacial radiation-associated pain (RAP) remains unclear, it may involve pathways related to cold sensation and signaling. One important cold-sensing receptor is TRPM8 (Transient Receptor Potential Melastatin 8), which may be activated by the neurotrophic factor artemin (ARTN) through its main receptor, GFRalpha3. To investigate whether radiation induces the release of ARTN, resulting in TRPM8 signaling pathway modulation and subsequent RAP, a prospective clinical trial enrolled 24 pet dogs undergoing RT (32 or 36 Gy total, given in four-weekly fractions) for either oral melanoma or intranasal carcinoma. Radiotoxicity was scored at each visit, per Veterinary Radiation Therapy Oncology Group criteria. Pain was assessed using a previously validated composite oral and maxillofacial pain scale. Oral mucosal biopsies were collected from the RT field immediately before the first fraction, after the fourth/final fraction, and two weeks post-RT, to assess gene expression using bulk RNA-sequencing and protein expression of ARTN and GFRalpha3 via immunohistochemistry and immunofluorescence. Blood was collected at these time points and before the second fraction to quantify ARTN in serum using ELISA. Data were analyzed using two-way ANOVA. At the end of RT, 79% of all dogs had grade 1-2 oral mucositis; by the two-week recheck, only 58% had persistent mucositis. Pain severity scores were variable. In oral mucosa collected at the end of RT, there were 1,406 significantly differentially expressed genes at 32 Gy and 1,898 at 36 Gy. At the end of RT, ARTN increased 1.11-fold (P < 0.01) in 32 Gy-treated dogs and 1.35-fold in those received 36 Gy (P < 0.01) . GFRalpha3 expression decreased by 1.02-fold (P = 0.02) in 36 Gy-treated dogs at the end of RT and by 1.23-fold after RT (P = 0.01). There were no significant changes in TRPM8 gene expression. Protein expression of ARTN and GFRalpha3 was unchanged in oral mucosa and neither mucositis nor pain severity correlated with ARTN or GFRalpha3 levels. However, there was a significant increase in serum concentrations of ARTN levels (P= 0.01) in dogs whose pain worsened after RT. Elevated circulating ARTN mirrors prior observations in dogs with mild radiodermatitis. Increased ARTN mRNA expression in the oral mucosa after irradiation suggests that the irradiated tissue itself is a key source of the circulating ligand. Interestingly, this occurred alongside downregulation of GFRalpha3 mRNA and without a clear relationship between local protein expression and the severity of radiation-associated pathology. The unexpected ligand-receptor imbalance suggests that after irradiation, increased ARTN reflects nerve regeneration and tissue repair, while reduced GFRalpha3 may result from nerve damage or silencing. Thus, the apparent inverse correlation may reflect tissue reorganization and adaptive signaling.