opn expression inhibitor 1 opni 1  (MedChemExpress)

Journal: Cancer Communications

Article Title: Hypoxia-Induced Osteopontin-Positive Glioma-Associated Macrophages Facilitate Glioma Mesenchymal Transition via NF-κB Pathway Activation

doi: 10.34133/cancomm.0007

Figure Lengend Snippet: OPN facilitates the mesenchymal transition, migration, and proliferation of glioma cells. (A and B) Chord diagrams showing the correlations among SPP1 expression, CD44 expression, hypoxia score, mesenchymal score, GAM score, and proneural score in glioma samples from TCGA (A) and CGGA (B) datasets (red, positive correlation; green, negative correlation; the scale for each item represents the sum of the absolute values of the correlation coefficient of that item with the other items). (C to F) Distribution of SPP1 expression (C), hypoxia score (D), GAM score (E), and mesenchymal score (F) in samples from the IVY Glioblastoma Atlas dataset. (G) Spatial coexpression rate of SPP1 with hypoxia score, GAM score, and mesenchymal score in ZH_881 1A bulk spatial transcriptomic dataset. (H to K) Violin plots illustrating hypoxia score (H), GAM score (I), mesenchymal score (J), and proneural score (K) generated from RNA-seq data of BNI_2-4 cells cultured with the indicated CM ( n = 3 per group; control, standard growth medium for macrophages; THP-1-hypoCM, CM obtained from THP-1 cells cultured under hypoxic conditions for 24 h; U937-hypoCM, CM obtained from U937 cells cultured under hypoxic conditions for 24 h). ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. (L and M) Western blotting showing the expression levels of representative mesenchymal phenotype-related proteins (YKL40, EFEMP1, and CD44) in BNI_2-4 cells after culture with CM derived from THP-1 (L) and U937 (M) cells under different treatment conditions (normoxia, CM obtained from THP-1 or U937 cells cultured under normoxic condition for 24 h; Hypoxia, CM obtained from THP-1 or U937 cells cultured under hypoxic condition for 24 h; Hypo + siCtrl, CM obtained from siCtrl transfected THP-1 or U937 cells cultured under hypoxic condition for 24 h; Hypo + siSPP1-1, CM obtained from siSPP1-1 transfected THP-1 or U937 cells cultured under hypoxic condition for 24 h; Hypo + DMSO, CM obtained from THP-1 or U937 cells cultured under hypoxic condition for 24 h in the presence of DMSO; Hypo + OPNi-1, CM obtained from THP-1 or U937 cells cultured under hypoxic condition for 24 h in the presence of OPNi-1). (N) Transwell assays showing the migration of BNI_2-4 cells cultured with the indicated CM from THP-1 and U937 cells (scale bar = 100 μm). (O and P) Quantification of migrated BNI_2-4 cells cultured with indicated CM from THP-1 (O) and U937 (P) cells ( n = 5 per group). ** P < 0.01, *** P < 0.001. (Q and R) CCK-8 assays showing the proliferation of BNI_2-4 cells cultured with indicated CM from THP-1 (Q) and U937 (R) cells ( n = 5 per group). *** P < 0.001. The data are presented as the means ± SD. TCGA, The Cancer Genome Atlas; CGGA, Chinese Glioma Genome Atlas; SPP1, secreted phosphoprotein 1; CT, cellular tumor; HBV, hyperplastic blood vessels; IT, infiltrating tumor; LE, leading edge; MVP, microvascular proliferation; PNZ, perinecrotic zone; PAN, pseudopalisading cells around necrosis; CM, conditioned media; YKL40, chitinase-3 like protein 1; EFEMP1, EGF-containing fibulin extracellular matrix protein 1; CD44, cluster of differentiation 44; DMSO, dimethyl sulfoxide; OPNi-1, OPN expression inhibitor 1; kDa, kilodaltons.

Article Snippet: All the cells were treated with OPN expression inhibitor 1 (OPNi-1) (50 μM, catalog no. HY-146064, MedChemExpress) for 24 h under hypoxic or normoxic conditions.

Techniques: Migration, Expressing, Generated, RNA Sequencing, Cell Culture, Control, Western Blot, Derivative Assay, Transfection, CCK-8 Assay

Journal: Cancer Communications

Article Title: Hypoxia-Induced Osteopontin-Positive Glioma-Associated Macrophages Facilitate Glioma Mesenchymal Transition via NF-κB Pathway Activation

doi: 10.34133/cancomm.0007

Figure Lengend Snippet: CD44 mediates the protumor effects of OPN on glioma cells. (A) Bubble plot showing ligand–receptor signaling from macrophage to glioma cells in GSE141383 dataset. (B) Representative images of IHC staining for CD68, CD163, OPN, and CD44 in the tissue microarray of glioma samples with different histological subtypes. (C) GAM (CD163 + CD68 + ) distribution in glioma tissue microarray samples. ns, not significant and *** P < 0.001. (D) Scatter plot showing the relationship between OPN and CD44 H-scores in a glioma tissue microarray. Each dot represents one sample (blue, oligodendroglioma; green, astrocytoma; red, GBM). (E) IF staining showing the colocalization of OPN (red) and CD44 (green) in BNI_2-4 cells cultured with the indicated CM derived from THP-1 cells (normoxia, CM obtained from THP-1 cells cultured under normoxic condition for 24 h; Hypoxia, CM obtained from THP-1 cells cultured under hypoxic condition for 24 h; Hypo + siCtrl, CM obtained from siCtrl transfected THP-1 cells cultured under hypoxic condition for 24 h; Hypo + siSPP1-1, CM obtained from siSPP1-1 transfected THP-1 cells cultured under hypoxic condition for 24 h; Hypo + DMSO, CM obtained from THP-1 cells cultured under hypoxic condition for 24 h in the presence of DMSO; Hypo + OPNi-1, CM obtained from THP-1 cells cultured under hypoxic condition for 24 h in the presence of OPNi-1). Nuclei were counterstained with DAPI (blue). The yellow line in the first column indicates the line of interest used for intensity analysis. Line intensity profiles on the right display the fluorescence signal distribution along the marked line. (F) Western blotting showing the expression levels of representative mesenchymal phenotype-related proteins (YKL40, EFEMP1, and CD44) in BNI_2-4 cells cultured under the indicated conditions (normo + shCtrl , glioma cells that were transinfected with negative control lentivirus cultured in normoxic CM; hypo + shCtrl , glioma cells that were transinfected with negative control lentivirus cultured in hypoxic CM; normo + shCD44-2, glioma cells that were transinfected with shCD44-2 lentivirus cultured in normoxic CM; hypo + shCD44-2, glioma cells that were transinfected with shCD44-2 lentivirus cultured in hypoxic CM). (G) Western blotting showing the expression levels of representative mesenchymal phenotype-related proteins (YKL40, EFEMP1, and CD44) in BNI_1-3 cells cultured under the indicated conditions. (H and I) Transwell assays showing the migration of BNI_2-4 cells cultured under the indicated conditions with CM of THP-1 (H) and U937 (I) cells (scale bar = 100 μm). (J and K) Quantification of migrated BNI_2-4 cells cultured under the indicated conditions with CM of THP-1 (J) and U937 (K) cells ( n = 5 per group). ns, not significant, *** P < 0.001. (L and M) CCK-8 assays showing the proliferation of BNI_2-4 cells cultured under the indicated conditions with CM of THP-1 (L) and U937 (M) cells. ns, not significant, *** P < 0.001. (N and O) CCK-8 assays showing the proliferation of BNI_1-3 cells cultured under the indicated conditions with CM of THP-1 (N) and U937 (O) cells. ns, not significant, *** P < 0.001. The data are presented as the means ± SD. OPN, osteopontin; IHC, immunohistochemistry; CD68, cluster of differentiation 68; CD163, cluster of differentiation 163; GAM, glioma-associated macrophage; OPN + GAM, osteopontin-positive glioma-associated macrophage; KD, knockdown; CM, conditioned media; EFEMP1, EGF-containing fibulin extracellular matrix protein 1; YKL40, chitinase-3 like protein 1; kDa, kilodaltons.

Article Snippet: All the cells were treated with OPN expression inhibitor 1 (OPNi-1) (50 μM, catalog no. HY-146064, MedChemExpress) for 24 h under hypoxic or normoxic conditions.

Techniques: Immunohistochemistry, Microarray, Staining, Cell Culture, Derivative Assay, Transfection, Fluorescence, Western Blot, Expressing, Negative Control, Migration, CCK-8 Assay, Knockdown

Journal: Cancer Communications

Article Title: Hypoxia-Induced Osteopontin-Positive Glioma-Associated Macrophages Facilitate Glioma Mesenchymal Transition via NF-κB Pathway Activation

doi: 10.34133/cancomm.0007

Figure Lengend Snippet: OPN promotes PD-L1 expression in glioma cells by activating the NF-κB signaling pathway. (A) Heatmap showing commonly up-regulated DEGs in BNI_1-3, BNI_2-4, and U87-MG cells treated with the hypoxic CM, as identified by transcriptome sequencing. Gene expression was normalized and clustered across samples. (B) Bubble plot showing the results of GO and KEGG enrichment analyses of commonly up-regulated DEGs in glioma cells cultured with hypoxic CM. (C) Western blotting showing the expression levels of PD-L1 and proteins in the NF-κB signaling pathway in BNI_2-4 cells cultured with the indicated CM (normoxia, CM obtained from THP-1 or U937 cells cultured under normoxic condition for 24 h; Hypoxia, CM obtained from THP-1 or U937 cells cultured under hypoxic condition for 24 h; Hypo + siCtrl, CM obtained from siCtrl transfected THP-1 or U937 cells cultured under hypoxic condition for 24 h; Hypo + siSPP1-1, CM obtained from siSPP1-1 transfected THP-1 or U937 cells cultured under hypoxic condition for 24 h; Hypo + DMSO, CM obtained from THP-1 or U937 cells cultured under hypoxic condition for 24 h in the presence of DMSO; Hypo + OPNi-1, CM obtained from THP-1 or U937 cells cultured under hypoxic condition for 24 h in the presence of OPNi-1). (D and E) Western blotting showing NF-κB (p65 subunit) expression in the cytoplasmic and nuclear fractions of BNI_2-4 (D) and BNI_1-3 (E) cells cultured with normoxic or hypoxic CM derived from THP-1 and U937 cells. β-Actin and H3 were used as cytoplasmic and nuclear loading controls, respectively (P, cytoplasm; N, nuclei). (F and G) IF staining showing the localization of the NF-κB (p65 subunit, green) in BNI_2-4 (F) and BNI_1-3 (G) cells cultured with CM derived from normoxic and hypoxic THP-1 and U937 cells. Nuclei were counterstained with DAPI (blue). The red line in the first row indicates the line of interest used for intensity analysis. The line intensity profiles below display the fluorescence signal distribution along the marked line. (H) qPCR analysis of NF-κB (p65 subunit) enrichment at CD274 promoter area in BNI_2-4 cells cultured with indicated CM derived from THP-1 cells ( n = 3 per group). (I) Agarose gel electrophoresis showing ChIP-PCR products (CD274 promoter sequence) from BNI_2-4 cells cultured with indicated CM derived from THP-1 cells. Chromatin was immunoprecipitated using anti-NF-κB (p65 subunit) and IgG control antibodies. (J) qPCR analysis of NF-κB (p65 subunit) enrichment at CD274 promoter area in BNI_2-4 cells cultured with indicated CM derived from U937 cells ( n = 3 per group). (K) Agarose gel electrophoresis showing ChIP-PCR products (CD274 promoter sequence) from BNI_2-4 cells cultured with indicated CM derived from U937 cells. Chromatin was immunoprecipitated using anti-NF-κB (p65 subunit) and IgG control antibodies. The data are presented as the means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; PD-L1, programmed cell death ligand 1; PI3K, phosphatidylinositol kinase 3; p-PI3K, phosphorylated phosphatidylinositol kinase 3; Akt, protein kinase B; p-Akt, phosphorylated protein kinase B; ERK1/2, extracellular signal-regulated protein kinases 1/2; p-ERK1/2, phosphorylated extracellular signal-regulated protein kinases 1/2; NF-κB, nuclear factor κB; p-p65, phosphorylated nuclear factor κB; CM, conditioned media; DMSO, dimethyl sulfoxide; OPNi-1, OPN expression inhibitor 1; kDa, kilodaltons.

Article Snippet: All the cells were treated with OPN expression inhibitor 1 (OPNi-1) (50 μM, catalog no. HY-146064, MedChemExpress) for 24 h under hypoxic or normoxic conditions.

Techniques: Expressing, Sequencing, Gene Expression, Cell Culture, Western Blot, Transfection, Derivative Assay, Staining, Fluorescence, Agarose Gel Electrophoresis, Immunoprecipitation, Control

Journal: Cancer Communications

Article Title: Hypoxia-Induced Osteopontin-Positive Glioma-Associated Macrophages Facilitate Glioma Mesenchymal Transition via NF-κB Pathway Activation

doi: 10.34133/cancomm.0007

Figure Lengend Snippet: Targeting OPN increases the therapeutic effectiveness of TMZ in a C57BL/6J in vivo glioma model. (A) Schematic illustration of the in vivo experimental design in C57BL/6J mice. The blue arrow represents intracranial implantation; the red arrows represent treatments (control, treatment with PBS; OPNi-1, treatment with OPNi-1 alone; TMZ, treatment with TMZ alone; TMZ + OPNi-1, combined treatment with TMZ and OPNi-1); the green arrows represent MRI scans. (B) Representative MR images showing the intracranial tumor burden in C57BL/6J mice from the indicated treatment groups. (C) Quantification of the tumor volume in C57BL/6J mice from the indicated groups on days 0, 7, and 14 since initial treatment ( n = 7 mice per group). (D) Kaplan–Meier survival curves of glioma-bearing mice receiving the indicated treatments ( n = 7 mice per group). (E) Representative mIHC images of glioma tissues from mouse brain sections. Eight markers were divided into 2 staining panels and applied to serial tumor sections to preserve spatial consistency. Panel 1 includes PD-L1, F4/80, CD163, and OPN, and panel 2 includes CD20, NK1.1, CD8a, and CD4. Images for both panels were acquired from matched anatomical regions on adjacent serial sections. (F) Quantification of PD-L1 positivity based on mIHC staining ( n = 3 per group). (G) Quantification of macrophage (F4/80 + cells) proportion based on mIHC staining ( n = 3 per group). (H) Quantification of GAM (CD163 + F4/80 + cells) proportion based on mIHC staining ( n = 3 per group). (I) Quantification of OPN + GAM (OPN + CD163 + F4/80 + cells) proportion based on mIHC staining ( n = 3 per group). (J) Quantification of B cell (CD20 + cells) proportion based on mIHC staining ( n = 3 per group). (K) Quantification of NK cell (NK1.1 + cells) proportion based on mIHC staining ( n = 3 per group). (L) Quantification of CD8 + T cell (CD8a + cells) proportion based on mIHC staining ( n = 3 per group). (M) Quantification of CD4 + T cell (CD4 + cells) proportion based on mIHC staining ( n = 3 per group). Data are presented as the mean ± SD; * P < 0.05, ** P < 0.01. PD-L1, programmed cell death ligand 1; F4/80, mouse EGF-like module-containing mucin-like hormone receptor-like 1; CD163, cluster of differentiation 163; OPN, osteopontin; GAM, glioma-associated macrophage; OPN + GAM, osteopontin positive glioma-associated macrophage; NK, natural killer; CD20, cluster of differentiation 20; SD, standard deviation. Hypoxia induces the emergence of OPN + GAMs through the epigenetic activation of the H3K4me3-WDR5 axis. The secreted OPN promotes the mesenchymal transition and PD-L1 expression in glioma cells via CD44-mediated activation of the NF-κB signaling pathway.

Article Snippet: All the cells were treated with OPN expression inhibitor 1 (OPNi-1) (50 μM, catalog no. HY-146064, MedChemExpress) for 24 h under hypoxic or normoxic conditions.

Techniques: In Vivo, Control, Staining, Standard Deviation, Activation Assay, Expressing

Journal: Cancer Communications

Article Title: Hypoxia-Induced Osteopontin-Positive Glioma-Associated Macrophages Facilitate Glioma Mesenchymal Transition via NF-κB Pathway Activation

doi: 10.34133/cancomm.0007

Figure Lengend Snippet: OPN facilitates the mesenchymal transition, migration, and proliferation of glioma cells. (A and B) Chord diagrams showing the correlations among SPP1 expression, CD44 expression, hypoxia score, mesenchymal score, GAM score, and proneural score in glioma samples from TCGA (A) and CGGA (B) datasets (red, positive correlation; green, negative correlation; the scale for each item represents the sum of the absolute values of the correlation coefficient of that item with the other items). (C to F) Distribution of SPP1 expression (C), hypoxia score (D), GAM score (E), and mesenchymal score (F) in samples from the IVY Glioblastoma Atlas dataset. (G) Spatial coexpression rate of SPP1 with hypoxia score, GAM score, and mesenchymal score in ZH_881 1A bulk spatial transcriptomic dataset. (H to K) Violin plots illustrating hypoxia score (H), GAM score (I), mesenchymal score (J), and proneural score (K) generated from RNA-seq data of BNI_2-4 cells cultured with the indicated CM ( n = 3 per group; control, standard growth medium for macrophages; THP-1-hypoCM, CM obtained from THP-1 cells cultured under hypoxic conditions for 24 h; U937-hypoCM, CM obtained from U937 cells cultured under hypoxic conditions for 24 h). ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. (L and M) Western blotting showing the expression levels of representative mesenchymal phenotype-related proteins (YKL40, EFEMP1, and CD44) in BNI_2-4 cells after culture with CM derived from THP-1 (L) and U937 (M) cells under different treatment conditions (normoxia, CM obtained from THP-1 or U937 cells cultured under normoxic condition for 24 h; Hypoxia, CM obtained from THP-1 or U937 cells cultured under hypoxic condition for 24 h; Hypo + siCtrl, CM obtained from siCtrl transfected THP-1 or U937 cells cultured under hypoxic condition for 24 h; Hypo + siSPP1-1, CM obtained from siSPP1-1 transfected THP-1 or U937 cells cultured under hypoxic condition for 24 h; Hypo + DMSO, CM obtained from THP-1 or U937 cells cultured under hypoxic condition for 24 h in the presence of DMSO; Hypo + OPNi-1, CM obtained from THP-1 or U937 cells cultured under hypoxic condition for 24 h in the presence of OPNi-1). (N) Transwell assays showing the migration of BNI_2-4 cells cultured with the indicated CM from THP-1 and U937 cells (scale bar = 100 μm). (O and P) Quantification of migrated BNI_2-4 cells cultured with indicated CM from THP-1 (O) and U937 (P) cells ( n = 5 per group). ** P < 0.01, *** P < 0.001. (Q and R) CCK-8 assays showing the proliferation of BNI_2-4 cells cultured with indicated CM from THP-1 (Q) and U937 (R) cells ( n = 5 per group). *** P < 0.001. The data are presented as the means ± SD. TCGA, The Cancer Genome Atlas; CGGA, Chinese Glioma Genome Atlas; SPP1, secreted phosphoprotein 1; CT, cellular tumor; HBV, hyperplastic blood vessels; IT, infiltrating tumor; LE, leading edge; MVP, microvascular proliferation; PNZ, perinecrotic zone; PAN, pseudopalisading cells around necrosis; CM, conditioned media; YKL40, chitinase-3 like protein 1; EFEMP1, EGF-containing fibulin extracellular matrix protein 1; CD44, cluster of differentiation 44; DMSO, dimethyl sulfoxide; OPNi-1, OPN expression inhibitor 1; kDa, kilodaltons.

Article Snippet: The mice were randomly assigned to the following 4 groups: the control group (treated with PBS), the OPNi-1 group (treated with 0.5 mg/kg OPNi-1, catalog no. HY-146064; MedChemExpress), the TMZ group (treated with 50 mg/kg TMZ, catalog no. HY-17364, MedChemExpress), and the TMZ + OPNi-1 group (treated with 50 mg/kg TMZ and 0.5 mg/kg OPNi-1).

Techniques: Migration, Expressing, Generated, RNA Sequencing, Cell Culture, Control, Western Blot, Derivative Assay, Transfection, CCK-8 Assay

Journal: Cancer Communications

Article Title: Hypoxia-Induced Osteopontin-Positive Glioma-Associated Macrophages Facilitate Glioma Mesenchymal Transition via NF-κB Pathway Activation

doi: 10.34133/cancomm.0007

Figure Lengend Snippet: CD44 mediates the protumor effects of OPN on glioma cells. (A) Bubble plot showing ligand–receptor signaling from macrophage to glioma cells in GSE141383 dataset. (B) Representative images of IHC staining for CD68, CD163, OPN, and CD44 in the tissue microarray of glioma samples with different histological subtypes. (C) GAM (CD163 + CD68 + ) distribution in glioma tissue microarray samples. ns, not significant and *** P < 0.001. (D) Scatter plot showing the relationship between OPN and CD44 H-scores in a glioma tissue microarray. Each dot represents one sample (blue, oligodendroglioma; green, astrocytoma; red, GBM). (E) IF staining showing the colocalization of OPN (red) and CD44 (green) in BNI_2-4 cells cultured with the indicated CM derived from THP-1 cells (normoxia, CM obtained from THP-1 cells cultured under normoxic condition for 24 h; Hypoxia, CM obtained from THP-1 cells cultured under hypoxic condition for 24 h; Hypo + siCtrl, CM obtained from siCtrl transfected THP-1 cells cultured under hypoxic condition for 24 h; Hypo + siSPP1-1, CM obtained from siSPP1-1 transfected THP-1 cells cultured under hypoxic condition for 24 h; Hypo + DMSO, CM obtained from THP-1 cells cultured under hypoxic condition for 24 h in the presence of DMSO; Hypo + OPNi-1, CM obtained from THP-1 cells cultured under hypoxic condition for 24 h in the presence of OPNi-1). Nuclei were counterstained with DAPI (blue). The yellow line in the first column indicates the line of interest used for intensity analysis. Line intensity profiles on the right display the fluorescence signal distribution along the marked line. (F) Western blotting showing the expression levels of representative mesenchymal phenotype-related proteins (YKL40, EFEMP1, and CD44) in BNI_2-4 cells cultured under the indicated conditions (normo + shCtrl , glioma cells that were transinfected with negative control lentivirus cultured in normoxic CM; hypo + shCtrl , glioma cells that were transinfected with negative control lentivirus cultured in hypoxic CM; normo + shCD44-2, glioma cells that were transinfected with shCD44-2 lentivirus cultured in normoxic CM; hypo + shCD44-2, glioma cells that were transinfected with shCD44-2 lentivirus cultured in hypoxic CM). (G) Western blotting showing the expression levels of representative mesenchymal phenotype-related proteins (YKL40, EFEMP1, and CD44) in BNI_1-3 cells cultured under the indicated conditions. (H and I) Transwell assays showing the migration of BNI_2-4 cells cultured under the indicated conditions with CM of THP-1 (H) and U937 (I) cells (scale bar = 100 μm). (J and K) Quantification of migrated BNI_2-4 cells cultured under the indicated conditions with CM of THP-1 (J) and U937 (K) cells ( n = 5 per group). ns, not significant, *** P < 0.001. (L and M) CCK-8 assays showing the proliferation of BNI_2-4 cells cultured under the indicated conditions with CM of THP-1 (L) and U937 (M) cells. ns, not significant, *** P < 0.001. (N and O) CCK-8 assays showing the proliferation of BNI_1-3 cells cultured under the indicated conditions with CM of THP-1 (N) and U937 (O) cells. ns, not significant, *** P < 0.001. The data are presented as the means ± SD. OPN, osteopontin; IHC, immunohistochemistry; CD68, cluster of differentiation 68; CD163, cluster of differentiation 163; GAM, glioma-associated macrophage; OPN + GAM, osteopontin-positive glioma-associated macrophage; KD, knockdown; CM, conditioned media; EFEMP1, EGF-containing fibulin extracellular matrix protein 1; YKL40, chitinase-3 like protein 1; kDa, kilodaltons.

Article Snippet: The mice were randomly assigned to the following 4 groups: the control group (treated with PBS), the OPNi-1 group (treated with 0.5 mg/kg OPNi-1, catalog no. HY-146064; MedChemExpress), the TMZ group (treated with 50 mg/kg TMZ, catalog no. HY-17364, MedChemExpress), and the TMZ + OPNi-1 group (treated with 50 mg/kg TMZ and 0.5 mg/kg OPNi-1).

Techniques: Immunohistochemistry, Microarray, Staining, Cell Culture, Derivative Assay, Transfection, Fluorescence, Western Blot, Expressing, Negative Control, Migration, CCK-8 Assay, Knockdown

Journal: Cancer Communications

Article Title: Hypoxia-Induced Osteopontin-Positive Glioma-Associated Macrophages Facilitate Glioma Mesenchymal Transition via NF-κB Pathway Activation

doi: 10.34133/cancomm.0007

Figure Lengend Snippet: OPN promotes PD-L1 expression in glioma cells by activating the NF-κB signaling pathway. (A) Heatmap showing commonly up-regulated DEGs in BNI_1-3, BNI_2-4, and U87-MG cells treated with the hypoxic CM, as identified by transcriptome sequencing. Gene expression was normalized and clustered across samples. (B) Bubble plot showing the results of GO and KEGG enrichment analyses of commonly up-regulated DEGs in glioma cells cultured with hypoxic CM. (C) Western blotting showing the expression levels of PD-L1 and proteins in the NF-κB signaling pathway in BNI_2-4 cells cultured with the indicated CM (normoxia, CM obtained from THP-1 or U937 cells cultured under normoxic condition for 24 h; Hypoxia, CM obtained from THP-1 or U937 cells cultured under hypoxic condition for 24 h; Hypo + siCtrl, CM obtained from siCtrl transfected THP-1 or U937 cells cultured under hypoxic condition for 24 h; Hypo + siSPP1-1, CM obtained from siSPP1-1 transfected THP-1 or U937 cells cultured under hypoxic condition for 24 h; Hypo + DMSO, CM obtained from THP-1 or U937 cells cultured under hypoxic condition for 24 h in the presence of DMSO; Hypo + OPNi-1, CM obtained from THP-1 or U937 cells cultured under hypoxic condition for 24 h in the presence of OPNi-1). (D and E) Western blotting showing NF-κB (p65 subunit) expression in the cytoplasmic and nuclear fractions of BNI_2-4 (D) and BNI_1-3 (E) cells cultured with normoxic or hypoxic CM derived from THP-1 and U937 cells. β-Actin and H3 were used as cytoplasmic and nuclear loading controls, respectively (P, cytoplasm; N, nuclei). (F and G) IF staining showing the localization of the NF-κB (p65 subunit, green) in BNI_2-4 (F) and BNI_1-3 (G) cells cultured with CM derived from normoxic and hypoxic THP-1 and U937 cells. Nuclei were counterstained with DAPI (blue). The red line in the first row indicates the line of interest used for intensity analysis. The line intensity profiles below display the fluorescence signal distribution along the marked line. (H) qPCR analysis of NF-κB (p65 subunit) enrichment at CD274 promoter area in BNI_2-4 cells cultured with indicated CM derived from THP-1 cells ( n = 3 per group). (I) Agarose gel electrophoresis showing ChIP-PCR products (CD274 promoter sequence) from BNI_2-4 cells cultured with indicated CM derived from THP-1 cells. Chromatin was immunoprecipitated using anti-NF-κB (p65 subunit) and IgG control antibodies. (J) qPCR analysis of NF-κB (p65 subunit) enrichment at CD274 promoter area in BNI_2-4 cells cultured with indicated CM derived from U937 cells ( n = 3 per group). (K) Agarose gel electrophoresis showing ChIP-PCR products (CD274 promoter sequence) from BNI_2-4 cells cultured with indicated CM derived from U937 cells. Chromatin was immunoprecipitated using anti-NF-κB (p65 subunit) and IgG control antibodies. The data are presented as the means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; PD-L1, programmed cell death ligand 1; PI3K, phosphatidylinositol kinase 3; p-PI3K, phosphorylated phosphatidylinositol kinase 3; Akt, protein kinase B; p-Akt, phosphorylated protein kinase B; ERK1/2, extracellular signal-regulated protein kinases 1/2; p-ERK1/2, phosphorylated extracellular signal-regulated protein kinases 1/2; NF-κB, nuclear factor κB; p-p65, phosphorylated nuclear factor κB; CM, conditioned media; DMSO, dimethyl sulfoxide; OPNi-1, OPN expression inhibitor 1; kDa, kilodaltons.

Article Snippet: The mice were randomly assigned to the following 4 groups: the control group (treated with PBS), the OPNi-1 group (treated with 0.5 mg/kg OPNi-1, catalog no. HY-146064; MedChemExpress), the TMZ group (treated with 50 mg/kg TMZ, catalog no. HY-17364, MedChemExpress), and the TMZ + OPNi-1 group (treated with 50 mg/kg TMZ and 0.5 mg/kg OPNi-1).

Techniques: Expressing, Sequencing, Gene Expression, Cell Culture, Western Blot, Transfection, Derivative Assay, Staining, Fluorescence, Agarose Gel Electrophoresis, Immunoprecipitation, Control

Journal: Cancer Communications

Article Title: Hypoxia-Induced Osteopontin-Positive Glioma-Associated Macrophages Facilitate Glioma Mesenchymal Transition via NF-κB Pathway Activation

doi: 10.34133/cancomm.0007

Figure Lengend Snippet: Targeting OPN increases the therapeutic effectiveness of TMZ in a C57BL/6J in vivo glioma model. (A) Schematic illustration of the in vivo experimental design in C57BL/6J mice. The blue arrow represents intracranial implantation; the red arrows represent treatments (control, treatment with PBS; OPNi-1, treatment with OPNi-1 alone; TMZ, treatment with TMZ alone; TMZ + OPNi-1, combined treatment with TMZ and OPNi-1); the green arrows represent MRI scans. (B) Representative MR images showing the intracranial tumor burden in C57BL/6J mice from the indicated treatment groups. (C) Quantification of the tumor volume in C57BL/6J mice from the indicated groups on days 0, 7, and 14 since initial treatment ( n = 7 mice per group). (D) Kaplan–Meier survival curves of glioma-bearing mice receiving the indicated treatments ( n = 7 mice per group). (E) Representative mIHC images of glioma tissues from mouse brain sections. Eight markers were divided into 2 staining panels and applied to serial tumor sections to preserve spatial consistency. Panel 1 includes PD-L1, F4/80, CD163, and OPN, and panel 2 includes CD20, NK1.1, CD8a, and CD4. Images for both panels were acquired from matched anatomical regions on adjacent serial sections. (F) Quantification of PD-L1 positivity based on mIHC staining ( n = 3 per group). (G) Quantification of macrophage (F4/80 + cells) proportion based on mIHC staining ( n = 3 per group). (H) Quantification of GAM (CD163 + F4/80 + cells) proportion based on mIHC staining ( n = 3 per group). (I) Quantification of OPN + GAM (OPN + CD163 + F4/80 + cells) proportion based on mIHC staining ( n = 3 per group). (J) Quantification of B cell (CD20 + cells) proportion based on mIHC staining ( n = 3 per group). (K) Quantification of NK cell (NK1.1 + cells) proportion based on mIHC staining ( n = 3 per group). (L) Quantification of CD8 + T cell (CD8a + cells) proportion based on mIHC staining ( n = 3 per group). (M) Quantification of CD4 + T cell (CD4 + cells) proportion based on mIHC staining ( n = 3 per group). Data are presented as the mean ± SD; * P < 0.05, ** P < 0.01. PD-L1, programmed cell death ligand 1; F4/80, mouse EGF-like module-containing mucin-like hormone receptor-like 1; CD163, cluster of differentiation 163; OPN, osteopontin; GAM, glioma-associated macrophage; OPN + GAM, osteopontin positive glioma-associated macrophage; NK, natural killer; CD20, cluster of differentiation 20; SD, standard deviation. Hypoxia induces the emergence of OPN + GAMs through the epigenetic activation of the H3K4me3-WDR5 axis. The secreted OPN promotes the mesenchymal transition and PD-L1 expression in glioma cells via CD44-mediated activation of the NF-κB signaling pathway.

Article Snippet: The mice were randomly assigned to the following 4 groups: the control group (treated with PBS), the OPNi-1 group (treated with 0.5 mg/kg OPNi-1, catalog no. HY-146064; MedChemExpress), the TMZ group (treated with 50 mg/kg TMZ, catalog no. HY-17364, MedChemExpress), and the TMZ + OPNi-1 group (treated with 50 mg/kg TMZ and 0.5 mg/kg OPNi-1).

Techniques: In Vivo, Control, Staining, Standard Deviation, Activation Assay, Expressing