Fig. 1. Tumor-intrinsic NLRP3 promotes PMN-MDSC accumulation in distant lung tissues.

(A) Representative example of frequencies of live⁺CD45⁺CD11b⁺Ly6G⁺ Ly6C^loF4/80⁻ PMN-MDSCs in the lungs of tumor-bearing and non-tumor-bearing autochthonous BRAF^V600EPTEN^−/− mice. PerCP, peridinin-chlorophyll-protein; FITC, fluorescein isothiocyanate. (B) qrt-PCR analysis of Cxcl5 and Cxcl2 expression by CD45⁺EpCAM⁻ and CD45⁻EpCAM⁺ cell populations derived from the lung tissues of non–tumor-bearing and tumor-bearing BRAF^V600EPTEN^−/− mice (n = 3). Statistical analysis was performed by two-way ANOVA followed by Sidak’s multiple comparisons test. (C) Experimental schematic to investigate the role of tumor NLRP3 on lung PMN-MDSC accumulation. KD, knockdown; NTC, nontarget control. (D) Flow cytometry analysis of PMN-MDSCs in the lung tissues of non–tumor-bearing, BRAF^V600EPTEN^−/− tumor–bearing, and BRAF^V600EPTEN^−/−−NLRP3^KD tumor–bearing mice (n = 3). Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparisons test. (E) qrt-PCR analysis of Cxcl1, Cxcl2, Cxcl3, and Cxcl5 expression in FACS-purified CD45⁻EpCAM⁺ lung epithelial cells derived from BRAF^V600EPTEN^−/− tumor–bearing and BRAF^V600EPTEN^−/−−NLRP3^KD tumor–bearing mice (n = 3). (F) Experimental schematic to verify the role of tumor-intrinsic NLRP3 in metastatic progression. NLRP3i, NLRP3 inhibitor. (G) Flow cytometry analysis of PMN-MDSCs in lung tissues of BRAF^V600EPTEN^−/− tumor–bearing mice after treatment with either NLRP3i or vehicle control (Ctrl; n = 4). (H) Left: Low-magnification imaging and quantification of resected lung tissues after treatment with either NLRP3i or Ctrl. Images are shown at 4×. Scale bars, 2000 μm. Right: Survival curve analysis of BRAF^V600EPTEN^−/− tumor–bearing mice after treatment with either NLRP3i or Ctrl (n = 5). Statistical analysis for the right panel of (H) was performed by log-rank test. All two-group comparisons were analyzed using unpaired t tests. All data are representative of two to three independent experiments and expressed as mean values ± SEM. *P < 0.05, **P < 0.005, and ***P < 0.0005.