ATP in the HNSCC microenvironment activates neurons and causes pain through P2X2/3 heterotrimers. a. ATP concentration increases with HNSCC cell number (One-way ANOVA). b. ATP concentration in microdialysate of mouse tongue SCC was higher than normal mouse tongues (n = 6 in each group, Student’s t-test). c. Representative traces of calcium response (A340/A380) to culture media (DMEM), HNSCC supernatant, and 50 mM KCl (positive control). Each line represents one cell. d. Preincubation with A-317491 or AF-353 reduced the percentage of TG neurons responding to HNSCC supernatant based on calcium imaging (χ2-test). e. Representative whole cell recordings of TG neurons in response to HNSCC supernatant in presence and absence of A-317491 and AF-353. f. A-317491 and AF-353 reduced current ratio compared to vehicle controls in ATP-responding TG neurons (One-way ANOVA). g. HNSCC supernatant injection into the mouse hind paw caused mechanical nociception compared to DMEM injection. AF-353 reversed HNSCC supernatant-induced mechanical nociception measured at 30–60 minutes post injection. After 180–210 minutes, both antagonists reversed HNSCC supernatant-induced mechanical nociception (n = 7-9 in each group, One-way ANOVA compared to HNSCC supernatant group). h. Oral function was significantly impaired in tongue SCC mice compared to naïve mice. ATP hydrolyzing enzyme apyrase, AF-353 and A-317491 significantly improved oral function in HNSCC mice (n = 6-10 in each group; One-way ANOVA compared to HNSCC mice with vehicle treatment). i. In mice with paw SCC, twice daily (BID) treatment of AF-353 and A-317491 significantly reduced mechanical nociception compared to vehicle (n = 8 for each group; Two-way ANOVA).