Fig 1: CXCL16/CXCR6 axis is involved in establishing a pro-tumor microenvironment. (A) Representative scheme of GL261 transplantation in wt and cxcr6ko mice; (B) bar histogram of the mean (± s.e.m.) of tumor volume in wt and cxcr6ko mice, and representative hematoxylin-eosin stained coronal brain sections of GL261 bearing mice; (C) Kaplan–Meier survival curves of wt and cxcr6ko GL261 bearing mice; (D) Immunofluorescence analysis of Iba1 and CD68 expression in tumor bearing brain slice 17 days after GL261 transplantation in wt and cxcr6ko mice: (central panels) representative immune-fluorescence images for Iba1+, CD68+ (red signals), nuclei are evidenced with Hoechst (blue signal), scale bar = 20 µm; (left and right panels): bar histograms representative of the immunofluorescence analysis of Iba1+, CD68+. Data are expressed as % of Iba+ or CD68+ staining per tumor area; (E,F) RT-qPCR for pro-inflammatory and anti-inflammatory genes in CD11b+ cells isolated from the ipsilateral and controlateral brain hemispheres of wt and cxcr6ko mice transplanted with GL261 cells. Data are expressed as mRNA fold increase in the ipsilateral hemisphere vs. the controlateral hemisphere, normalized for gapdh mRNA, and are represented as the mean (± s.e.m.); (G) Left, bar histogram representative of immunofluorescence analysis of GFAP+ positive cells in tumor bearing brain slice (data are expressed as % of GFAP+ area in brain slice), and representative images of GFAP+ staining (red signal); right, analysis of glioma cell invasion of surrounding brain tissue in cxcr6ko or wt mice injected with GL261.Representative coronal brain sections stained with hematoxylin/eosin. Black arrows indicate glioma cells invading the brain parenchyma beyond the main tumor border (dashed line) for more than 150 µm (scale bars, 20 µm) and bar histogram of the number of invading tumor cells 17 days after glioma cell transplantation. Statistical analysis: Data are expressed as the mean number (± s.e.m.) (B) n = 7–12, **p < 0.001, Student's t-test; (C) n = 6–10, **p < 0.001, long-rank test; (D) n = 4, *p < 0.05, Student's t-test; (E,F) n = 4–5, *p < 0.05, Student's t-test. (G) n = 3–4, **p < 0.001, *p < 0.05, Student's t-test.
Fig 2: Biological effects of CXCL16/CXCR6 axis on mouse glioma cells. (A) Left panel, chemotactic assay (24 h) of GL261 cells in the absence (C) or presence of different doses of CXCL16. Data are expressed as percentage of cell migration toward CXCL16 vs. control. Central panel, invasion assay of GL261 cells in the absence (C) or presence of different CXCL16 concentrations (1, 10 nM, 48 h). Data are expressed as percentage of cell invasion in CXCL16 stimulated cells vs. control. Right panel, expression analysis of mmp2, mmp9 metalloproteinases mRNAs by RT-qPCR in GL261 cells not stimulated (vehicle), or stimulated with CXCL16 (10 nM, 24 h). For each gene data are expressed as specific mRNA fold increase in CXCL16 treated cells normalized to specific mRNA expression in vehicle; (B) Left panel, growth curve (0, 24, 48 h) of GL261 cells unstimulaed (C) or stimulated with CXCL16 (10 nM); data are expressed as MTT conversion optical density; middle panel, BrdU incorporation assay on GL261 stimulated with CXCL16 (10 nM, 4 h), or not (C). Data are expressed as number of BrdU+ cells (black or gray bars) within total counted cells (white bar); Right panel, representative images (scale bar = 20 μm) of BrdU incorporation in GL261 cells unstimulated or stimulated with CXCL16 (BrdU, red signal; Hoechst, blue signal); (C) RT-PCR for cxcr6 and actin mRNAs expression in GL261 shCXCR6 cells induced or not with IPTG (+/–IPTG); (D) Chemotactic assay of GL261shCXCR6 induced or not with IPTG, not stimulated (C) and stimulated with CXCL16 (10 nM; 24 h); data are expressed as percentage of cell migration toward CXCL16 vs. control; (E) Growth curve (0, 24, 48, 72, 96 h) of GL261shCXCR6 cells induced or not with IPTG (+/–IPTG); data are expressed as MTT conversion optical density; (F) (left) representative scheme of GL261shCXCR6 cells transplantation in wt mice; (right) bar histogram of the mean (± s.e.m.) of tumor volume in shCXCR6-IPTG or shCXCR6+IPTG treated mice, and representative hematoxylin-eosin stained coronal brain sections of glioma bearing mice; (G) BrdU proliferation analysis in mice transplanted with GL261shCXCR6 cells induced or not with IPTG. (Top) representative images (scale bar = 20 μm) of proliferating BrdU+ cells (red) within tumor area; (bottom) bar histograms of immunofluorescence analysis of BrdU+ cells; data are expressed as % of BrdU+ staining per tumor area. (H) Analysis of glioma cells invasion of surrounding brain tissue in mice injected with GL261shCXCR6 treated or not with IPTG. (Top), Representative coronal brain sections stained with hematoxylin/eosin. Black arrows indicate glioma cells invading the brain parenchyma beyond the main tumor border (dashed line) for more than 150 μm. Bottom, bar histogram of the number of glioma invading cells. Statistical analysis: Data are expressed as the mean number (± s.e.m.) (A) left panel n = 3 in triplicate, *p < 0.05, one-way ANOVA followed by Holm–Sidak post-hoc test; right panel n = 3, *p < 0.05, one-way ANOVA followed by Holm–Sidak post-hoc test; (B) left panel n = 3 six-replicates, *p < 0.05, one-Way ANOVA followed by Student-Newman-Keuls Method; central panel n = 3, *p < 0.05, Student's t-test; (D) n = 4 in duplicate, *p < 0.05, two-way ANOVA followed by Holm–Sidak post-hoc test; (E) n = 4 in quadruplicates, *p < 0.05, one-Way ANOVA followed by Student-Newman-Keuls Method; (F) n = 5, *p < 0.05, Student's t-test); (G) n = 3, *p < 0.05, Student's t-test; (H) n = 3–4, *p < 0.05, Student's t-test.
Fig 3: Biological effects of CXCL16/CXCR6 axis on primary human glioblastoma cells.(A) cxcl16 and cxcr6 mRNAs expression in human GBM tissues (black and white circles), in human primary GBM cells derived from the same tissues and in human primary T cells and fibroblasts (black and white triangles) determined by RT-qPCR; (B) CXCR6 surface expression on primary GBM19 and GBM45 as evaluated by flow cytometry. Black and gray lines represent CXCR6 staining and isotype control, respectively; (C) Chemotaxis assay of human primary glioblastoma cells: GBM19 and GBM45, toward CXCL16 (10 nM, 4 h), CXCL12 (50 nM, 4 h) and vehicle (C). Data are expressed as percentage of cell migration vs. control; (D) Left panel, matrigel invasion assay on human GBM19, toward CXCL16 (10 nm, 24 h) and vehicle (C). Data are expressed as percentage of cell invasion vs. control (n = 3, p < 0.05, Student's t-test); right panel, proliferation assay of GBM19 upon stimulation with CXCL16 (10 nM) at different time points (0, 24, 48, and 72 h), data are expressed as MTT conversion optical density. Statistical analysis: Data are expressed as the mean (± s.e.m.) (C) n = 4, *p < 0.05, one-way ANOVA followed by Holm–Sidak post-hoc test; (D) left panel n = 3, *p < 0.05, Student's t-test; right panel n = 3 in five replicates, *p < 0.05, Student's t-test.
Fig 4: CXCL16 released by glioma cells modulates microglia phenotype. (A) Left panel, RT-qPCR for cxcl16 and cxcr6 (black and white circles) mRNA expression in human GBM tissues and in human control tissues (temporal or frontal cortex); right panel, qRT-PCR for cxcr6 mRNA expression in human CD11b+ cells (microglia/machrophages) isolated by GBM tissues. Mean values of double measurements from individual patients; ΔCT values = CT gene of interest—CT gapdh (housekeeping gene). A ΔCT of 3.33 corresponds to one magnitude lower gene expression compared to gapdh; (B) Left panel, representative PCR for cxcl16, cxcr6, and actin mRNAs expression in mouse GL261 cells and mouse GL261 derived stem cells (cd133+); right panel RT-qPCR for cxcl16 and cxcr6 (black and white circles) mRNAs expression in GL261, cd133+ cells, and in mouse T-cells and fibroblasts; CXCR6 surface expression on mouse GL261 cells as evaluated by flow cytometry. Black and gray lines represent CXCR6 staining and isotype control, respectively. (C) Expression analysis by RT-qPCR for mRNAs of pro-inflammatory related genes (nos2, il1b, cd86, tnfa) (left panel), or anti-inflammatory related genes (arg1, chil3, retnla, cd163) (right panel) in primary wt microglia treated with GCM in the presence of AbCXCL16 neutralizing antibody (GCM + AbCXCL16) or control IgG (GCM + IgG). For each gene data are expressed as specific mRNA fold change in GCM + AbCXCL16 treated cells normalized to specific mRNA expression in GCM + IgG treated cells and are the mean (± s.e.m.); (D) Phagocytosis of fluorescent beads in microglia cells stimulated with GCM or GCM + AbCXCL16. Data are expressed as number of cells containing 5 or more beads (black/gray bars) within total counted cells (white bars); (E) Western-blot analysis of ARG-1 protein expression in microglia cells incubated with GCM or GCM + AbCXCL16. Top representative image; bottom histogram bar of the quantification of ARG-1 expression (data are expressed as ARG-1 signal normalized to ACTIN signal). Statistical analysis: data are expressed as the mean (± s.e.m.) (A) **p < 0.001 cxcl16 expression, #p < 0.05 cxcr6 expression, Student's t-test; (C) n = 14, *p < 0.05, Student's t-test; for each gene, variability in its expression between control conditions in different experiments never exceeded 10%; (D) n = 4, **p < 0.001, Student's paired t-test; (E) n = 3, *p < 0.05, Student's t-test.
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