Fig 1: (A) CIBERSORT evaluated tumor-infiltrating immune cell (TIIC) differences under high (top 50%)/low (bottom 50%) PANX1 expression in basal-like subtype breast cancer (The key difference immune cells are shown by star (* p < 0.05); TCGA-BRCA basal-like subtype (n = 186) and GSE103091(n = 238) data); (B) GO analysis of PANX1 and its coexpression immune-related genes; (C) TIMER analysis of PANX1 expression and TIIC correlation (TCGA-BRCA data; n = 1083; Spearman’s rank correlation); (D) neutrophil abundance was positively correlated with PANX1 expression in METABRIC basal-like subtype data (R2 = 0.32; p = 0.003; Pearson’s correlation) (CIBERSORT algorithms; n = 25, outliers have been filtered); (E) MPO (neutrophil marker) expression was positively correlated with PANX1 expression in TCGA-BRCA basal-like subtype data (n = 186; R2 = 0.19; p < 0.001; Pearson’s correlation); (F) immunofluorescence detection of PANX1/MPO coexpression in basal-like breast cancer paraffin-embedded pathological specimens (DAPI, 4′,6-diamidino-phenylindole; MPO, myeloperoxidase).
Fig 2: (A) Levels of exATP and exADO in MDA-MB-231, HCC-1937 and MCF-7 cell culture media; PANX1 knock down and probenecid (PRB) treatment reduced the levels of exATP and exADO in the supernatant of MDA-MB-231 and HCC-1937 cells (n = 19 for each group; p < 0.05; Student’s t test); (B) levels of exATP and exADO in the supernatant of digested tissue from triple-negative and Luminal A breast cancer surgical specimens (p < 0.05; n = 9 for each group; Student’s t test); (C) the correlation between PANX1 expression and infiltration levels of neutrophils, Tregs, M2-like macrophages, MDSC cells, CD8+ T cells, and NK cells in the tumor microenvironment for Luminal B, HER2 enriched and basal-like breast cancer by TIMER (TCGA-BRCA data; Spearman’s rank correlation). (* p < 0.05; ** p < 0.01).
Fig 3: Panx1 mimetic peptide 10Panx enhanced LPS-induced macrophage hemichannel activation. (a,b) 10Panx elevated the LPS-induced Lucifer Yellow dye uptake. RAW264.7 cells were stimulated with LPS (1.0 μg/ml) in the absence or presence of 10Panx at indicated concentrations for 16 h, and subsequently incubated with Lucifer Yellow (LY, 1.0 mg/ml) for 15 min. Following fixation and three extensive washes, the number of cells with diffused fluorescent signals was counted under a fluorescence microscope, and expressed as a percentage of total cell numbers (DAPI-stained nuclei) in six fields. (c) 10Panx enhanced the LPS-induced ATP release. RAW264.7 cells were cultured in serum-free DMEM medium, and stimulated with LPS (1.0 μg/ml) in the absence or presence of Panx1 mimetic peptide at indicated concentrations, and the cell-conditioned culture medium was collected and subjected to ATP measurement. *P < 0.05 versus “-LPS” Control; #P < 0.05 versus “+LPS” control.
Fig 4: Crude LPS and purified SAA induced Panx1 release in macrophage and monocyte cultures. (a,b) Extracellular release of Panx1 by murine macrophages. Murine macrophage-like RAW264.7 cells were stimulated with LPS or SAA at indicated concentrations for 16 h. The macrophage-conditioned culture medium was subjected to differential centrifugations, and each fraction was assayed for Panx1 by Western blotting analysis. Note Panx1 was found in the LPS- or SAA-stimulated macrophage-conditioned medium (“Cell Medium”), and the supernatant of the 20,000 × g centrifugation. (c,d) Extracellular release of Panx1 by primary human monocytes. Human PBMCs were stimulated with crude LPS at indicated concentrations for different time periods, and extracellular Panx1 levels were determined by Western blotting analysis. The relative optical intensity of the 48-kDa (Panx1) and 12-kDa (Panx1Δ) band was measured, and expressed as an arbitrary unit (AU).
Fig 5: (A) The proportion of infiltrating TANs in Luminal (n = 3) and basal-like subtype (High PANX1 expression: n = 6; low PANX1 expression: n = 6) surgical specimens; (B,C) basal-like breast cancer with high PANX1 expression had more infiltrating TANs than basal-like breast cancer with low PANX1 expression and the Luminal subtype (p < 0.05 for TIMER; p = 0.11 for quanTIseq; Student’s t test); (D) the correlation between ENTPD1/NT5E expression and TAN infiltration in basal-like breast cancer (n = 25; p < 0.05; R2 = 0.28 (ENTPD1) and 0.23 (NT5E); Pearson’s correlation; TCGA-BRCA data; CIBERSORT-LM22 algorithms; outliers have been filtered); (E) TIMER analysis suggested a positive correlation between ENTPD1/NT5E expression and TAN infiltration in the basal-like subtype (n = 186; p < 0.01; Rho = 0.35 (ENTPD1) and 0.26 (NT5E); Spearman’s rank correlation; TCGA-BRCA data); (F) heatmap of the transcriptome analysis of TANs (n = 6) and PBNs (n = 6) in basal-like breast cancer (TANs, tumor-associated neutrophils; PBNs, peripheral blood neutrophils) (* p < 0.05).
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