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. Author manuscript; available in PMC: 2018 Dec 1.
Published in final edited form as: Pain. 2017 Dec;158(12):2396–2409. doi: 10.1097/j.pain.0000000000001044

Figure 2. Cancer-secreted mediators recruited immune cells to the tongue microenvironment.

Figure 2

Tongue tissue from naïve mice (n = 9) and mice after 3 consecutive injections of cell line supernatant from HaCaT (n = 10), DOK (n = 6), HSC3 (n = 10), SCC9 (n = 10), and SkMel28 (n = 6) were analyzed using flow cytometry. A) Representative gating strategy used to isolate immune cell subpopulations from a mouse tongue treated with HSC3 supernatant. CD45+ immune cells were isolated first from the population of live cells as determined by vitality marker, propidium iodide (PI), and then sorted into macrophages/monocytes/neutrophils (CD11b+) and dendritic cells (CD11c+) (not shown) or sorted into T cells (CD3+). The CD11b+ cells were further sorted into macrophages/monocytes (CD11b+Ly6G−) and neutrophils (CD11b+Ly6G+). The CD3− cells were further sorted into natural killer (NK) cells (NK1.1+) or B cells (B220+). All pooled data are expressed as a percent of total live cells. One-way ANOVA analysis of pooled CD45+ cells (B) indicated a significant difference between oral cancer cell line supernatant (HSC3, SCC9) treatment and naïve tongues (* p < 0.05, ** p < 0.01). C) Further gating of CD45+ cells into immune cell subpopulations identified significantly increased neutrophils (Ly6G+) and macrophages/monocytes (CD11b+) in the tongue tissue in response to HSC3 and SCC9 (One-way ANOVA, * p < 0.05, ** p < 0.01). There was no change in T cells (CD3+), B cells (B220+) or NK cells (NK1.1) subpopulations in response to any cell line supernatant compared to naïve tongue tissue (One-way ANOVA, p > 0.05).