Journal: International Journal of Biological Sciences

Article Title: Apoptotic Cancer Cell-Primed Cancer-Associated Fibroblasts Suppress Immunosuppressive Macrophages via WISP-1-Integrin α5β3-STAT1 Signaling in Lung Cancer

doi: 10.7150/ijbs.124282

Figure Lengend Snippet: CM from CAFs exposed to apoptotic cancer cells reduces M2 macrophage survival, induces apoptosis, and promotes reprogramming toward an M1 phenotype in vitro . ( a, b ) Cell viability assay of M1 (M1) or M2 macrophages (M2) derived from THP-1 cells and BMDMs. ( c, d ) Apoptotic M1 or M2 macrophages were quantified as the sum of the percentages of early and late stages of apoptosis. Flow cytometry analysis after Annexin V-FICT/PI dual staining was employed to evaluate apoptosis. ( e ) Immunoblot analysis of Bax, Mcl-1, Bcl-xL, cleaved caspase-3, cleaved PARP, and β-actin in THP-1-derived M2 macrophages. ( a-e ) CAFs were exposed to apoptotic 344SQ cells (ApoSQ) or necrotic cancer cells (NecSQ) for 20 h. Conditioned medium from CAFs only (CAF CM), exposed to ApoSQ (ApoSQ-CAF CM) or NecSQ (NecSQ-CAF CM) was treated to THP-1- or BMDM-derived M1 and M2 macrophages for the indicated days ( a , b ), or 3 days ( c-e ). ( f ) Heatmap showing differentially expressed macrophage polarization-related genes in THP-1-derived M2 macrophages treated with CM for 3 days ( left ). Red: high expression; blue: low expression. Relative expression of selected genes from PCR array profiling of macrophage polarization markers ( right ). Log2 fold-change values (ApoSQ-CAF CM vs. CAF CM). ( g ) qRT-PCR analysis of relative mRNA levels of M1 ( Nos2 , MhcII , and Il12p40 ) and M2 ( Tgfβ1 , Il10 , and Il4 ) markers in THP-1-derived M2 macrophages treated with CM for 3 days. ( h ) ELISA of TNF-α, IL-1β, IL-4, and IL-13 in the culture supernatant of M2 macrophages treated with CM for 3 days. ( i ) Flow cytometry analysis of the population of CD80 + and CD206 + cells among M2 macrophages derived from THP-1 cells for 2 or 3 days. Mean fluorescence intensity (MFI) values ( right ). NS, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, two-tailed Student's t -test. Data are from one experiment representative of three independent experiments with similar results ( c , f and i left ; e ) or from three independent experiments (mean ± standard error: a , b , d , g , h ; c , f and i right ).

Article Snippet: To establish M1 macrophages, the unpolarized macrophages were stimulated with 20 ng/ml of IFN-γ (R&D Systems) and 100 ng/ml of lipopolysaccharide (Sigma-Aldrich) for an additional 48 h. To establish M2 macrophages, the unpolarized macrophages were stimulated with 20 ng/ml IL-4 and 20 ng/ml IL-13 (R&D Systems) for an additional 48 h. Following polarization, cells were harvested for immunoblot analysis or fixed for immunofluorescent staining.

Techniques: In Vitro, Viability Assay, Derivative Assay, Flow Cytometry, Staining, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Fluorescence, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: Apoptotic Cancer Cell-Primed Cancer-Associated Fibroblasts Suppress Immunosuppressive Macrophages via WISP-1-Integrin α5β3-STAT1 Signaling in Lung Cancer

doi: 10.7150/ijbs.124282

Figure Lengend Snippet: ApoSQ-CAF CM activates STAT1 in M2 macrophages. ( a, b ) Immunoblot analysis of the indicated proteins in THP-1-derived M1 (M1) and M2 macrophages (M2) treated with CAF CM or ApoSQ-CAF CM for the indicated time. ( c ) Immunofluorescence staining for phosphorylated STAT1 and p21 ( Left ) and quantitation ( Right ) in M2 macrophages for 1 h after treatment with CAF CM or ApoSQ-CAF CM. The imaging medium was VECTASHIELD fluorescence mounting medium containing DAPI. Original magnification: ×400. Scale bars = 20 μm. ( d ) Immunoblot analysis of STAT1 in M2 macrophages transfected with control or STAT1 siRNA ( upper ). Densitometric analysis of the relative STAT1 abundance ( lower ). ( e ) Cell viability assay of M2 macrophages. ( f ) Left: Flow cytometry analysis after Annexin V-FICT/PI dual staining was employed to evaluate the cell apoptosis of M2 macrophages. Right : Apoptotic cells were quantified as the sum of the percentages of early and late stages of apoptosis. ( g ) Immunoblot analysis of the indicated proteins in M2 macrophage lysates. ( h ) qRT-PCR analysis of relative mRNA levels of M1 ( Nos2 , MhcII , and Il12p40 ) and M2 ( Tgfβ1 , Il10 , and Il4 ) markers in M2 macrophages (M2). ( i ) ELISA of TNF-α, IL-1β, IL-4, and IL-13 in the culture supernatant of M2 macrophages. ( j ) Flow cytometry analysis of the population of CD80 + and CD206 + cells among M2 macrophages. Mean fluorescence intensity (MFI) values ( right ). ( e - j ) THP-1-derived M2 macrophages were transfected with control or STAT1 siRNA for 24 h before treatment with CM for 2 or 3 days. NS: not significant; ** P < 0.01, *** P < 0.001, two-tailed Student's t -test. The data are from one experiment representative of three independent experiments with similar results ( a , b, g ; c , f , and j left ; d upper ) or from three independent experiments (mean ± standard error in c , f , and j right ; d lower ; e , h , i ).

Article Snippet: To establish M1 macrophages, the unpolarized macrophages were stimulated with 20 ng/ml of IFN-γ (R&D Systems) and 100 ng/ml of lipopolysaccharide (Sigma-Aldrich) for an additional 48 h. To establish M2 macrophages, the unpolarized macrophages were stimulated with 20 ng/ml IL-4 and 20 ng/ml IL-13 (R&D Systems) for an additional 48 h. Following polarization, cells were harvested for immunoblot analysis or fixed for immunofluorescent staining.

Techniques: Western Blot, Derivative Assay, Immunofluorescence, Staining, Quantitation Assay, Imaging, Fluorescence, Transfection, Control, Viability Assay, Flow Cytometry, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: Apoptotic Cancer Cell-Primed Cancer-Associated Fibroblasts Suppress Immunosuppressive Macrophages via WISP-1-Integrin α5β3-STAT1 Signaling in Lung Cancer

doi: 10.7150/ijbs.124282

Figure Lengend Snippet: Recombinant WISP-1 reduces M2 macrophage survival, induces apoptosis, and promotes reprogramming toward an M1-like phenotype. ( a ) Cell viability assay of THP-1-derived M1 (M1) and M2 macrophages (M2) treated with 20-100 ng/ml mouse (rWISP-1) or human WISP-1 ( h rWISP-1) for 3 days. ( b ) Left: Flow cytometry analysis after Annexin V-FICT/PI dual staining was employed to evaluate the apoptosis of THP-1- derived M1 and M2 macrophages after treatment with h rWISP- (20-100 ng/ml) for 3 days. Right : Apoptotic cells were quantified as the sum of the percentages of early and late stages of apoptosis. ( c ) qRT-PCR analysis of relative mRNA levels of M1 ( Nos2 , MhcII , and Il12p40 ) and M2 ( Tgfβ1 , Il10 , and Il4 ) markers in M2 macrophages treated with 20-100 ng/ml h rWISP-1 for 3 days. ( d ) ELISA of TNF-α, IL-1β, IL-4, and IL-13 in the culture supernatant of M2 macrophages treated with 20-100 ng/ml h rWISP-1 for 3 days. ( e ) Flow cytometry analysis of the population of CD80 + and CD206 + cells among M2 macrophages after treatment with h rWISP-1 (20-100 ng/ml) for 2 or 3 days. Mean fluorescence intensity (MFI) values ( right ). ( f ) Heatmap showing differentially expressed macrophage polarization-related genes in THP-1-derived M2 macrophages ( left ). Red: high expression; blue: low expression. Relative expression of selected genes from PCR array profiling of macrophage polarization markers ( right ). Log2 fold-change values ( h rWISP-1 vs. Vehicle). THP-1-derived M2 macrophages were treated with h rWISP-1 (50 ng/ml) for 3 days. NS: not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, two-tailed Student's t -test. The data are from one experiment representative of three independent experiments with similar results ( b , e , and f left ) or from three independent experiments (mean ± standard error: a , c , d ; b , e and f right ).

Article Snippet: To establish M1 macrophages, the unpolarized macrophages were stimulated with 20 ng/ml of IFN-γ (R&D Systems) and 100 ng/ml of lipopolysaccharide (Sigma-Aldrich) for an additional 48 h. To establish M2 macrophages, the unpolarized macrophages were stimulated with 20 ng/ml IL-4 and 20 ng/ml IL-13 (R&D Systems) for an additional 48 h. Following polarization, cells were harvested for immunoblot analysis or fixed for immunofluorescent staining.

Techniques: Recombinant, Viability Assay, Derivative Assay, Flow Cytometry, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Fluorescence, Expressing, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: Apoptotic Cancer Cell-Primed Cancer-Associated Fibroblasts Suppress Immunosuppressive Macrophages via WISP-1-Integrin α5β3-STAT1 Signaling in Lung Cancer

doi: 10.7150/ijbs.124282

Figure Lengend Snippet: rWISP-1 acts through integrin α5β3 to activate STAT1 in M2 macrophages. ( a , i ) Cell viability assay of M2 macrophages treated with 50 ng/ml human rWISP-1 ( h rWISP-1) for 3 days. ( b , j ) Apoptotic cells were quantified as the sum of the percentages of early and late stages of apoptosis. Flow cytometry analysis after Annexin V-FICT/PI dual staining was employed to evaluate the cell apoptosis of M2 macrophages treated with h rWISP-1 for 3 days. ( c , l ) qRT-PCR analysis of relative mRNA levels of M1 ( Nos2 , MhcII , and Il12p40 ) and M2 ( Tgfβ1 , Il10 , and Il4 ) markers in M2 macrophages treated with h rWISP-1 for 3 days. ( d , m ) ELISA of the cytokines (TNFα, IL-1β, IL-4, and IL-13) in the culture supernatants of M2 macrophages treated with h rWISP-1 for 3 days. ( e , n ) Flow cytometry analysis of the population of CD80 + and CD206 + cells among M2 macrophages treated with h rWISP-1 for 2 or 3 days. Mean fluorescence intensity (MFI) values ( right ). ( f ) CoIP assays of protein interaction in M2 macrophages. Cell lysates were immunoprecipitated (IP) with anti-WISP-1 and then immunoblotted with anti-integrin α5 and anti-integrin β3 antibodies. ( g , h ) Immunoblot analysis of phosphorylated and total STAT1 in THP-1-derived M2 macrophages treated with ApoSQ-CAF CM or h rWISP-1 for the indicated time ( g ) or 30 min ( h ). ( k ) Immunoblot analysis of the indicated proteins in M2 macrophages treated with h rWISP-1 for 3 days. ( a-e ) THP-1-derived M2 macrophages were pretreated with an anti-integrin blocking antibody (3 μg/ml; anti-integrin αν, α5, β3 or β5) or corresponding IgG isotype control for 30 min before treatment with rWISP-1 (50 ng/ml). ( i-n ) THP-1-derived M2 macrophages were transfected with control or STAT1 siRNA before treatment with h rWISP-1 (50 ng/ml). NS: not significant; ** P < 0.01, *** P < 0.001, two-tailed Student's t -test. The data are from one experiment representative of three independent experiments with similar results ( e , j , and n left ; f , g , h , k ) or from three independent experiments (mean ± standard error: a-d , i , l , m ; e , j , and n right ).

Article Snippet: To establish M1 macrophages, the unpolarized macrophages were stimulated with 20 ng/ml of IFN-γ (R&D Systems) and 100 ng/ml of lipopolysaccharide (Sigma-Aldrich) for an additional 48 h. To establish M2 macrophages, the unpolarized macrophages were stimulated with 20 ng/ml IL-4 and 20 ng/ml IL-13 (R&D Systems) for an additional 48 h. Following polarization, cells were harvested for immunoblot analysis or fixed for immunofluorescent staining.

Techniques: Viability Assay, Flow Cytometry, Staining, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Fluorescence, Immunoprecipitation, Western Blot, Derivative Assay, Blocking Assay, Control, Transfection, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: Apoptotic Cancer Cell-Primed Cancer-Associated Fibroblasts Suppress Immunosuppressive Macrophages via WISP-1-Integrin α5β3-STAT1 Signaling in Lung Cancer

doi: 10.7150/ijbs.124282

Figure Lengend Snippet: ApoSQ-CAF CM promotes M2-to-M1 TAM reprogramming and activates STAT1 in M2 TAMs via WISP-1. The experimental design was described in Fig. a. ( a ) Heatmap showing differentially expressed genes encoding M1 and M2 marker-related molecules in isolated CD11b + TAMs from primary tumors (left). Red: high expression; blue: low expression. Relative expression of selected genes from PCR array profiling of macrophage polarization markers (right). Log2 fold-change values (ApoSQ-CAF CM vs. CAF CM). ( b ) qRT-PCR analysis of relative mRNA levels of M2 markers ( Arg1 , Cd206 , Cd163 , Il4 , Il10 , Tgfβ1 ), and M1 markers ( Tnfα , Cd80 , MhcII , Nos2 , Ifng , and Il12p40 ) in isolated CD11b + TAMs from primary tumors. NS: not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, Analysis of variance with Tukey's post hoc test. ( c ) Immunoblot analysis of Arg1, CD206, iNOS, and CD16/32 in isolated CD11b + TAMs from primary tumors. ( d , e ) Flow cytometry analysis of the population of M1 TAMs (MHCII + and CD80 + ) and M2 TAMs (CD163 + and CD206 + ) in CD11b + TAMs from primary tumors. Mean fluorescence intensity (MFI) values ( right ). ( f ) Upper: Representative flow cytometry plots in CD11b + TAMs. Lower : TAM ratio (CD163 + /MHCII + TAMs). ( g-k ) Flow cytometry analysis of the population of M2 macrophages ( g ), Tregs ( h ), M1 macrophages ( i ), CD8 + T cells ( j ), and CD4 + T cells ( k ). Tumor-infiltrating immune cells were stained with antibodies against CD45, CD11b, CD3, CD4, CD8, FoxP3, MHCII, and Ly6C. Absolute number of each cell type was counted using flow cytometry. ( a , d-k ) NS, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001, two-tailed Student's t -test. ( a - k ) The data are from three replicates per condition, with cells pooled from three mice per replicate. ( l, n ) Representative confocal images of primary tumor sections stained with an anti-phosphorylated STAT1 (red), anti-CD206 antibody (green), anti-iNOS antibody (green), and DAPI (blue). Original magnification: ×40. Scale bars = 100 μm. ( m , o ) Quantification of phosphorylated STAT1 + cells among CD206 + cells and iNOS + cells. NS, not significant; *** P < 0.001, Analysis of variance with Tukey's post hoc test. The data are from one experiment representative of three independent experiments with similar results ( a , d and e left ; c , l , n ; f upper ) or from three independent experiments (mean ± standard error: a , d and e right ; b, g-k , m , o ; f lower ).

Article Snippet: To establish M1 macrophages, the unpolarized macrophages were stimulated with 20 ng/ml of IFN-γ (R&D Systems) and 100 ng/ml of lipopolysaccharide (Sigma-Aldrich) for an additional 48 h. To establish M2 macrophages, the unpolarized macrophages were stimulated with 20 ng/ml IL-4 and 20 ng/ml IL-13 (R&D Systems) for an additional 48 h. Following polarization, cells were harvested for immunoblot analysis or fixed for immunofluorescent staining.

Techniques: Marker, Isolation, Expressing, Quantitative RT-PCR, Western Blot, Flow Cytometry, Fluorescence, Staining, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: Apoptotic Cancer Cell-Primed Cancer-Associated Fibroblasts Suppress Immunosuppressive Macrophages via WISP-1-Integrin α5β3-STAT1 Signaling in Lung Cancer

doi: 10.7150/ijbs.124282

Figure Lengend Snippet: Administration of rWISP-1 reduces TAM density, decrease the M2 fraction and marker expression, and activates STAT1 in M2 TAMs. The experimental design was described in Supplementary a. Where indicated, rWISP-1 (12.5 and 25 μg/kg) was administered intratumorally three times a week for 6 weeks starting 2 days after subcutaneous implantation of 344SQ cells into syngeneic (129/Sν) mice (n = 6 mice per group). Mice were necropsied 6 weeks later. ( a , b ) Upper : Immunofluorescent staining of primary tumor sections showing M2 TAM Markers Arg1 (green) and CD206 (green), along with the pan-macrophage marker CD11b (red). Original magnification: ×40. Scale bars = 100 μm. Lower : Quantitation of Arg1 + and CD206 + TAM (M2) density ( left ) and the fraction of M2 TAMs ( right ) in primary tumors. The fraction of M2 TAMs were determined by the percentage of M2 TAMs within CD11b + TAMs. ( c ) qRT-PCR analysis of relative mRNA levels of M2 markers ( Arg1 , CD206 , CD163 , IL-4 , IL-10 , TGF-β1 ), and M1 markers ( TNFα , CD80 , MhcII , NOS2, Ifng, and IL-12 p40 ) in isolated CD11b + TAMs from primary tumors. ( d ) Immunoblot analysis of Arg1, CD206, iNOS, and CD16/32 in isolated CD11b + TAMs from primary tumors. ( e, g ) Representative confocal images of primary tumor sections stained with an anti-phosphorylated STAT1 (red), anti-CD206 antibody (green), anti-CD86 antibody (green), and DAPI (blue). Original magnification: ×40. Scale bars = 100 μm. ( f , h ) Quantification of phosphorylated STAT1 + cells among CD206 + cells and CD86 + cells. NS, not significant; * P < 0.05, ** P < 0.01, *** P < 0.001 compared to Vehicle or as indicated; ### P < 0.001 compared to Vehicle, Analysis of variance with Tukey's post hoc test. The data are from one experiment representative of three independent experiments with similar results ( a and b upper ; d , e , g ). The data are represented as the means ± standard errors from three mice per group ( a and b lower ; c , f , h ).

Article Snippet: To establish M1 macrophages, the unpolarized macrophages were stimulated with 20 ng/ml of IFN-γ (R&D Systems) and 100 ng/ml of lipopolysaccharide (Sigma-Aldrich) for an additional 48 h. To establish M2 macrophages, the unpolarized macrophages were stimulated with 20 ng/ml IL-4 and 20 ng/ml IL-13 (R&D Systems) for an additional 48 h. Following polarization, cells were harvested for immunoblot analysis or fixed for immunofluorescent staining.

Techniques: Marker, Expressing, Staining, Quantitation Assay, Quantitative RT-PCR, Isolation, Western Blot

Journal: Journal of Biological Chemistry

Article Title: Tumor Microenvironment Macrophage Inhibitory Factor Directs the Accumulation of Interleukin-17-producing Tumor-infiltrating Lymphocytes and Predicts Favorable Survival in Nasopharyngeal Carcinoma Patients

doi: 10.1074/jbc.m112.367532

Figure Lengend Snippet: FIGURE 3. Characterization and cytokine expression profiles of the Th17 cell subset among PBMCs and TILs from NPC patients. A, T cell surface markers were detected in Th17 cells from NPC patients. T cells were stimulated with phorbol 12-myristate 13-acetate/ionomycin for 4 h, then stained with fluorescence- conjugated antibodies against the markers shown, and analyzed by flow cytometry. B, graph of the percentage of cytokine-secreting Th17 cells among PBMCs and TILs from NPC patients (n 5) and PBMCs from healthy donors (n 5). Error bars represent the S.E. Significance was determined by the 2 test or paired t test. (*, p 0.001).

Article Snippet: Cytokines in the culture medium were measured using the Human Cytokine Array Panel A (Proteome Profiler; R&D Systems).

Techniques: Expressing, Staining, Fluorescence, Flow Cytometry

Journal: Journal of Biological Chemistry

Article Title: Tumor Microenvironment Macrophage Inhibitory Factor Directs the Accumulation of Interleukin-17-producing Tumor-infiltrating Lymphocytes and Predicts Favorable Survival in Nasopharyngeal Carcinoma Patients

doi: 10.1074/jbc.m112.367532

Figure Lengend Snippet: FIGURE 4. Generation and migration of Th17 cells mediated by NPC tumor cell lines. A, NPC tumor cell lines induce the differentiation of naive T cells into Th17cellsinvitro.PurifiedCD4naiveTcellsfromhealthydonorswerestimulatedwithOKT3andthenco-culturedwiththeirradiatedNPCcelllineC666(EBV) or CNE2 (EBV) in IL-2-containing medium for 7 days. The NP69 and LCL lines and the cytokines IL-1 and TGF were used as controls. All cultured cells were stained for Foxp3 and IL-17 for FACS analysis after stimulation of phorbol 12-myristate 13-acetate/ionomycin. The data represent one of three independent experiments. B, mean percentage of cytokine-secreting Th17 cells induced from CD4 naive T cells from three experiments. C, migration of Th17 cells was increased in response to cultured supernatants from the NPC cell lines C666 and CNE2, relative to that from the normal NP cell line NP69 or with medium alone. The data represent one of three independent experiments. D, mean percentage of cytokine-secreting Th17 cells in the inner well and outer well after migration induced by supernatants from NP69, C666, or CNE2 cells from three experiments. Error bars represent the S.E. Significance was determined by the 2 test or paired t test. (*, p 0.05).

Article Snippet: Cytokines in the culture medium were measured using the Human Cytokine Array Panel A (Proteome Profiler; R&D Systems).

Techniques: Migration, Cell Culture, Staining