Figure 6.

Opioid expression in cancer-stimulated immune cell subpopulations. The relationship between the number of Ly6G⁺ neutrophils in the tongue oSCC microenvironment and measured nociceptive behavior expressed as a percent change from baseline in (A) female (N = 10, P > 0.05) and (B) male (N = 15, P < 0.05) mice was evaluated with Pearson correlation. Note nociceptive behavior data in (A,B) are plotted on different scales due a greater spread in females. Following three consecutive injections of HSC-3 supernatant, mouse tongue tissue was dissociated and immune cell subpopulations were separated and collected for analysis by quantitative real-time polymerase chain reaction (qRT-PCR). (C) Representative gating strategy used to isolate cancer-activated tongue immune cells by fluorescence-activated cell sorting (FACS). (D) Quantification of mean Pomc and Penk expression in CD11b⁺Ly6G⁺ and CD11b⁺Ly6G⁻ immune cell subpopulations from HSC-3 supernatant-treated male (blue, N = 4) and female mice (red, N = 4) relative to housekeeping gene Actb. Data were analyzed using the Pfaffl method and normalized to the average expression ratios from female mice. *P < 0.05, **P < 0.01 by Unpaired Student’s t-test.