Journal: Cell Death Discovery

Article Title: Combined anti-PD-L1 and anti-VEGFR2 therapy promotes the antitumor immune response in GBM by reprogramming tumor microenvironment

doi: 10.1038/s41420-025-02427-7

Figure Lengend Snippet: Analysis of the TCGA ( A ) and CGGA ( B ) databases showed that PD-L1 expression was highest in GBM, and Kaplan‒Meier analysis indicated that glioma patients with low PD-L1 expression had better survival than those with high PD-L1 expression ( P < 0.0001). The TCGA ( C ) and CGGA ( D ) databases showed that VEGFR2 was more highly expressed in high-grade gliomas than in low-grade gliomas, and Kaplan‒Meier analysis indicated that glioma patients with low VEGFR2 expression had better survival times than those with high VEGFR2 expression ( P < 0.0001). E , F The protein expression levels of PD-L1 and VEGFR2 in normal and glioma tissues were detected by immunohistochemistry staining. Scale bar = 50 μm. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, Student’s t -test.

Article Snippet: The mice in the fourth group were intraperitoneally injected with TMZ combined with anti-mouse VEGFR2 (10 mg/kg, once a week; Mabspace Biosciences, MSB0254m).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Cell Death Discovery

Article Title: Combined anti-PD-L1 and anti-VEGFR2 therapy promotes the antitumor immune response in GBM by reprogramming tumor microenvironment

doi: 10.1038/s41420-025-02427-7

Figure Lengend Snippet: A , B Flow cytometry showed that VEGFR2 knockdown induced cell cycle arrest in the G2-M phase on LN229, U251, and SHG141 cells. C , D Flow cytometry showed that VEGFR2 knockdown did not induce tumor cell apoptosis. E ELISA results showed that the concentrations of IL-7 and IL-12 were significantly greater in the VEGFR2 knockdown group than in the control group. F , G , K , L Flow cytometry analysis of GBM cells apoptosis in each treatment group. H – J , M – O The expression of cleaved PARP and cleaved caspase-3 in each treatment group was detected by western blotting. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, Student’s t -test.

Article Snippet: The mice in the fourth group were intraperitoneally injected with TMZ combined with anti-mouse VEGFR2 (10 mg/kg, once a week; Mabspace Biosciences, MSB0254m).

Techniques: Flow Cytometry, Knockdown, Enzyme-linked Immunosorbent Assay, Control, Expressing, Western Blot

Journal: Cell Death Discovery

Article Title: Combined anti-PD-L1 and anti-VEGFR2 therapy promotes the antitumor immune response in GBM by reprogramming tumor microenvironment

doi: 10.1038/s41420-025-02427-7

Figure Lengend Snippet: A Volcano plot of the RNA-Seq results. Red dots indicate upregulated genes, green dots indicate downregulated genes, and black dots indicate genes without differential expression. B Heatmap of genes expressed in the VEGFR2 knockdown and control groups. C, D Analysis of the TCGA ( C ) and CGGA ( D ) databases showed that PAK4 was most highly expressed in GBM among different glioma grades. Kaplan‒Meier analysis indicated that glioma patients with low PAK4 expression had better survival times than those with high PAK4 expression. PAK4 protein expression in normal and glioma tissues was detected by immunohistochemistry ( E ) and western blotting ( F , G ). Scale bar = 50 μm. H The correlations between PAK4 expression and CD8+ T cells immune score were analyzed with Spearman. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, Student’s t -test.

Article Snippet: The mice in the fourth group were intraperitoneally injected with TMZ combined with anti-mouse VEGFR2 (10 mg/kg, once a week; Mabspace Biosciences, MSB0254m).

Techniques: RNA Sequencing, Quantitative Proteomics, Knockdown, Control, Expressing, Immunohistochemistry, Western Blot

Journal: Cell Death Discovery

Article Title: Combined anti-PD-L1 and anti-VEGFR2 therapy promotes the antitumor immune response in GBM by reprogramming tumor microenvironment

doi: 10.1038/s41420-025-02427-7

Figure Lengend Snippet: A qRT‒PCR was used to investigate the expression of PAK4 after VEGFR2 knockdown. Western blotting ( B , C ) and immunofluorescence staining ( D ) showing the expression of VEGFR2, PAK4, STAT3, and p-STAT3 after VEGFR2 knockdown. Scale bar = 50 μm. E The TCGA database was used to determine the correlation between the expression profiles of PAK4 and STAT3. F Potential promoter binding sites and ChIP‒qPCR data showing the binding of p-STAT3 to the PAK4 gene. IgG was used as a reference. G Co-IP assays demonstrated that VEGFR2 can interact with STAT3. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, Student’s t -test.

Article Snippet: The mice in the fourth group were intraperitoneally injected with TMZ combined with anti-mouse VEGFR2 (10 mg/kg, once a week; Mabspace Biosciences, MSB0254m).

Techniques: Expressing, Knockdown, Western Blot, Immunofluorescence, Staining, Binding Assay, Co-Immunoprecipitation Assay

Journal: Cell Death Discovery

Article Title: Combined anti-PD-L1 and anti-VEGFR2 therapy promotes the antitumor immune response in GBM by reprogramming tumor microenvironment

doi: 10.1038/s41420-025-02427-7

Figure Lengend Snippet: A , B Flow cytometry assay showing the apoptosis of GBM cells in the control and experimental groups. C – E The expression of cleaved PARP and cleaved caspase-3 in the control and experimental groups was detected by western blotting. F – H Activated CD8+ T cells were cocultured with GBM cells at a 1:2 ratio, and were treated with anti-PD-L1/anti-VEGFR2 for 48 h. ELISA was used to evaluate the levels of IFN-γ, GZMB, and perforin secreted by CD8+ T cells in the control and experimental groups ( F ). Flow cytometry assay showed the expression of PD-1 on CD8+ T cells in the control and experimental groups ( G , H ). ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, Student’s t -test.

Article Snippet: The mice in the fourth group were intraperitoneally injected with TMZ combined with anti-mouse VEGFR2 (10 mg/kg, once a week; Mabspace Biosciences, MSB0254m).

Techniques: Flow Cytometry, Control, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Cell Death Discovery

Article Title: Combined anti-PD-L1 and anti-VEGFR2 therapy promotes the antitumor immune response in GBM by reprogramming tumor microenvironment

doi: 10.1038/s41420-025-02427-7

Figure Lengend Snippet: A Representative fluorescence images of mice in the control and experimental groups on days 7, 14, and 28. B Quantitative analysis of fluorescence images. C The overall survival of mice in the control and experimental groups. D Representative images of H&E-stained tumor sections from mice. Scale bar = 200 μm, enlarged scale bar = 50 μm. E Representative images of immunofluorescence staining of tumor sections from mice. Scale bar = 100 μm. F Representative images of immunohistochemistry staining of mouse tumor sections. Scale bar = 50 μm. ns P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, Student’s t -test. G Mechanistic diagram showing that anti-VEGFR2 therapy in GBM can downregulate PAK4, increase cytotoxic CD8+ T cells infiltration and activation, and enhance the therapeutic effect of anti-PD-L1 therapy.

Article Snippet: The mice in the fourth group were intraperitoneally injected with TMZ combined with anti-mouse VEGFR2 (10 mg/kg, once a week; Mabspace Biosciences, MSB0254m).

Techniques: Fluorescence, Control, Staining, Immunofluorescence, Immunohistochemistry, Activation Assay

Journal: EMBO Molecular Medicine

Article Title: Selective tubulin-binding drugs induce pericyte phenotype switching and anti-cancer immunity

doi: 10.1038/s44321-025-00222-6

Figure Lengend Snippet: ( A ) 8-week drug treatment scheme in RIP1-Tag5 mice including a 2-week priming and 6-week maintenance phase. ( B ) Representative fluorescent micrographs show vessel perfusion in untreated mice (U), or mice treated with CA-4 (C), eribulin (E), or anti-VEGFR2 antibodies (DC101, D). FITC-lectin (green) overlay (yellow, marked by arrows) with CD31 + (red) blood vessels is shown. Scale bar, 50 µm. ( C ) Representative fluorescent micrographs show staining of the contractile marker CNN1 (red) in NG2 + pericytes (green) and overlay (yellow, marked by arrows). Scale bar, 50 µm. ( D ) Quantification of lectin-perfusion in treatment groups: n = 4 mice for untreated, CA-4 and DC101, n = 11 mice for erbulin treatment groups, ** P = 0.0024, *** P = 0.0002, **** P <0.0001. CD31 + tumor blood vessels: n = 4 mice for untreated, CA-4 and DC101, n = 11 mice for erbulin treatment groups, ** P = 0.0081, DC101 compared to U. CNN1: n = 10 mice for untreated and CA-4, n = 5 mice for eribulin and n = 6 mice for DC101 treatment groups, * P = 0.0139, ** P = 0.0048, *** P = 0.006. NG2 + intratumoral pericytes: n = 10 mice for untreated and CA-4, n = 5 mice for eribulin and n = 6 mice for DC101 treatment groups, * P = 0.0365, DC101 compared to untreated. ( E ) Representative H&E micrographs showing tumor cellularity and collagen-rich tumor capsule (black dotted line) in treatment groups. Quantification of percentage of RIP1-Tag5 tumors displaying an intact collagen capsule (dotted line), n = 8 mice in untreated, n = 5 mice in all treatment groups, **** P <0.0001, DC101 compared to untreated. Scale bar 200 µm (upper images), scale bars for DC101 (lower images), 50 µm. All data were analyzed using one-way ANOVA. Data are expressed as mean ± SD. .

Article Snippet: Combretastatin (CA-4, S7783, 1.5–2.5 mg/kg in 5% DMSO, 10% PEG300 and 85% PBS, Selleck Chemical), paclitaxel (AG-CN2-0045, 0.05, 0.25, 0.5 or 2 mg/kg in 5% DMSO, 95% PBS, AdipoGen Life Sciences), vinorelbine (A10976, Navelbine, 0.25 mg/kg in 5% DMSO, 95% PBS, AdooQ Bioscience), eribulin (0.05 and 0.25 mg/kg in 5% DMSO, 95% PBS, Eisai Inc.) were injected i.v.; for VEGF blocking studies, anti-VEGFR2 antibodies (DC101, 15 mg/kg in PBS, BioXCell) was injected intraperitoneally (i.p.).

Techniques: Staining, Marker

Journal: Angiogenesis

Article Title: Clioquinol inhibits angiogenesis by promoting VEGFR2 degradation and synergizes with AKT inhibition to suppress triple-negative breast cancer vascularization

doi: 10.1007/s10456-024-09965-1

Figure Lengend Snippet: Clioquinol selectively down-regulates VEGFR2 in ECs. a Western blots showing VEGFR2, VEGFR1, Tie2, FGFR1, and β-actin expression in HUVECs after 4-hour treatment with 0, 2.5, 5, or 10 µM clioquinol. b - e Expression level (% of 0 µM) of VEGFR2 ( b ), VEGFR1 ( c ), Tie2 ( d ), and FGFR1 ( e ) normalized to β-actin in HUVECs treated as described in (a) ( n = 3 independent experiments). f Mean fluorescence intensity (MFI) of membrane VEGFR2 on HUVECs treated with or without 10 µM clioquinol for 0.5, 1, 2, 3, and 4 h, as assessed by flow cytometry ( n = 4). g Phase-contrast microscopic images of HUVEC spheroids after 24-hour treatment without or with clioquinol in the absence or presence of 25 ng/mL VEGF. Scale bar: 135 μm. h Sprouting (% of control) of HUVEC spheroids treated as described in (g) ( n = 13–15). i Western blots showing VEGFR2 and β-actin expression in HUVECs, HDMECs, hPC-PLs, NHDFs, MCF-7, MDA-MB-231, and 4T1-Luc2 cells. j Expression level (% of HUVEC) of VEGFR2 normalized to β-actin in different cell types as described in (i) ( n = 3 independent experiments). k Correlation between cell viability and VEGFR2 expression following exposure to 10 or 25 µM clioquinol. Means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant. (b-e, h, j: one-way ANOVA with Tukey’s multiple comparisons test; f: unpaired Student’s t-test; k: Pearson correlation coefficient)

Article Snippet: Microvascular VEGFR2 expression was analyzed by sequential staining with a monoclonal rat anti-mouse CD31 antibody (1:100; ab56299; Abcam), a monoclonal rabbit anti-mouse VEGFR2 antibody (1:100; Cat# 2479; RRID: AB_2212507; Cell Signaling Technology), a goat anti-rat Alexa Fluor488-labeled secondary antibody (1:150; Cat# A11006; RRID: AB_2534074; Thermo Fisher Scientific), a goat anti-rabbit Cy3-conjugated secondary antibody (1:100; Cat# A10520; RRID: AB_10563288; Thermo Fisher Scientific), and Hoechst 33342 (2 μg/mL; Sigma-Aldrich).

Techniques: Western Blot, Expressing, Fluorescence, Membrane, Flow Cytometry, Control

Journal: Angiogenesis

Article Title: Clioquinol inhibits angiogenesis by promoting VEGFR2 degradation and synergizes with AKT inhibition to suppress triple-negative breast cancer vascularization

doi: 10.1007/s10456-024-09965-1

Figure Lengend Snippet: Clioquinol binds to the ATP-binding pocket of VEGFR2 and causes its degradation. a Western blots showing VEGFR2 and β-actin expression in HUVECs treated with 0.1% DMSO (vehicle) or 10 µM clioquinol in the presence of 100 µM cycloheximide (CHX) for 0, 1, 2, 4, 6, and 8 h. b Expression level (% of 0 h) of VEGFR2 normalized to β-actin in HUVECs treated as described in ( a ) ( n = 3 independent experiments). c mRNA level of VEGFR2 (% of control) in HUVECs treated with 0.1% DMSO (control) or 10 µM clioquinol for 4 h, as assessed by real-time PCR ( n = 3). d Western blots showing VEGFR2 and β-actin expression in HUVECs that were pre-treated without or with 20 µM MG132 or 200 µM chloroquine (CQ) for 2 h and then exposed to 0.1% DMSO or 10 µM clioquinol for another 4 h. e Expression level (% of control) of VEGFR2 normalized to β-actin in HUVECs treated as described in (d) ( n = 3 independent experiments). f Western blots showing VEGFR2 and β-actin expression in HUVECs that were pre-treated with 4 µg/mL IgG, 4 µg/mL anti-VEGFR2 NAb, 0.1% DMSO (vehicle), 100 nM lenvatinib, 250 nM tivozanib, or 1 mM ATP for 2 h and then exposed to 0.1% DMSO or 10 µM clioquinol for another 4 h. g , h Expression level (% of IgG or control) of VEGFR2 normalized to β-actin in HUVECs treated as described in (f) ( n = 4 independent experiments). i Western blots showing p-VEGFR2, VEGFR2, and β-actin expression in HUVECs that were treated with 0.1% DMSO, 100 nM lenvatinib, 250 nM tivozanib or 10 µM clioquinol for 1 h and then stimulated with 25 ng/mL VEGF for 7 min. j Expression level (% of control) of p-VEGFR2 normalized to VEGFR2 in HUVECs treated as described in (i) ( n = 4 independent experiments). k VEGFR2 kinase activity (% of control) in the presence of serial dilutions of clioquinol at an ATP concentration of 10 µM, as assessed by VEGFR2 kinase assay ( n = 2). l VEGFR2 kinase activity (% of control) in the presence of lenvatinib (0.3, 1, and 3 nM) or clioquinol (10, 50, and 250 µM) at ATP concentrations of 10 or 500 µM, as assessed by VEGFR2 kinase assay ( n = 3). Means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant. (e, g, h, j: one-way ANOVA with Tukey’s multiple comparisons test; b, c, l: unpaired Student’s t-test)

Article Snippet: Microvascular VEGFR2 expression was analyzed by sequential staining with a monoclonal rat anti-mouse CD31 antibody (1:100; ab56299; Abcam), a monoclonal rabbit anti-mouse VEGFR2 antibody (1:100; Cat# 2479; RRID: AB_2212507; Cell Signaling Technology), a goat anti-rat Alexa Fluor488-labeled secondary antibody (1:150; Cat# A11006; RRID: AB_2534074; Thermo Fisher Scientific), a goat anti-rabbit Cy3-conjugated secondary antibody (1:100; Cat# A10520; RRID: AB_10563288; Thermo Fisher Scientific), and Hoechst 33342 (2 μg/mL; Sigma-Aldrich).

Techniques: Binding Assay, Western Blot, Expressing, Control, Real-time Polymerase Chain Reaction, Activity Assay, Concentration Assay, Kinase Assay

Journal: Angiogenesis

Article Title: Clioquinol inhibits angiogenesis by promoting VEGFR2 degradation and synergizes with AKT inhibition to suppress triple-negative breast cancer vascularization

doi: 10.1007/s10456-024-09965-1

Figure Lengend Snippet: Clioquinol and MK-2206 inhibit TNBC development, as assessed by histology and immunohistochemistry. a Light microscopic images of H&E-stained 4T1 tumors (bordered by dotted line) in control, clioquinol, MK-2206, and combination group. Scale bar: 160 μm. b Tumor size (mm 2 ) in control, clioquinol, MK-2206, and combination group, as assessed by histology ( n = 10). c Fluorescence microscopic images of tumor microvessels in control, clioquinol, MK-2206, and combination group. Tumor sections were stained with an anti-CD31 antibody (red) and Hoechst 33342 (blue) for the visualization of ECs and cell nuclei, respectively. Scale bar: 70 μm. d Microvessel density (mm − 2 ) of 4T1 tumors in control, clioquinol, MK-2206, and combination group, as assessed by immunohistochemistry ( n = 10). e , g Light microscopic images of Ki67- (e) and cleaved caspase-3-positive (g) tumor cells in control, clioquinol, MK-2206, and combination group. Scale bars: 55 μm. f , h Ki67- (f) and cleaved caspase-3-positive tumor cells (h) (% of total cell number) in control, clioquinol, MK-2206, and combination group, as assessed by immunohistochemistry ( n = 10). i Fluorescence microscopic images of tumor microvessels in control and clioquinol group. Tumor sections were stained with an anti-VEGFR2 antibody (red), an anti-CD31 antibody (green), and Hoechst 33342 (blue). Scale bar: 60 μm. j Area of VEGFR2 signal normalized to CD31 area (% of control) in tumors of control and clioquinol group. (k) VEGFR2 MFI (% of control) in tumors of control and clioquinol group. Means ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, not significant. (b, d, f, h: one-way ANOVA with Tukey’s multiple comparisons test; j, k: unpaired Student’s t-test)

Article Snippet: Microvascular VEGFR2 expression was analyzed by sequential staining with a monoclonal rat anti-mouse CD31 antibody (1:100; ab56299; Abcam), a monoclonal rabbit anti-mouse VEGFR2 antibody (1:100; Cat# 2479; RRID: AB_2212507; Cell Signaling Technology), a goat anti-rat Alexa Fluor488-labeled secondary antibody (1:150; Cat# A11006; RRID: AB_2534074; Thermo Fisher Scientific), a goat anti-rabbit Cy3-conjugated secondary antibody (1:100; Cat# A10520; RRID: AB_10563288; Thermo Fisher Scientific), and Hoechst 33342 (2 μg/mL; Sigma-Aldrich).

Techniques: Immunohistochemistry, Staining, Control, Fluorescence

Journal: Angiogenesis

Article Title: Clioquinol inhibits angiogenesis by promoting VEGFR2 degradation and synergizes with AKT inhibition to suppress triple-negative breast cancer vascularization

doi: 10.1007/s10456-024-09965-1

Figure Lengend Snippet: Scheme illustrating the molecular mechanisms underlying the potent inhibitory effects of clioquinol alone and its synergistic inhibitory effects with MK-2206 on angiogenesis. Clioquinol binds directly to the ATP-binding site of VEGFR2 on ECs, leading to a transient inhibition of VEGFR2 phosphorylation induced by VEGF and eventual promotion of VEGFR2 degradation via both the proteasome and lysosome systems. Consequently, the downstream ERK pathway is down-regulated. Furthermore, clioquinol increases AKT phosphorylation, while the inhibition of AKT by MK-2206 synergistically enhances the anti-angiogenic efficacy of clioquinol

Article Snippet: Microvascular VEGFR2 expression was analyzed by sequential staining with a monoclonal rat anti-mouse CD31 antibody (1:100; ab56299; Abcam), a monoclonal rabbit anti-mouse VEGFR2 antibody (1:100; Cat# 2479; RRID: AB_2212507; Cell Signaling Technology), a goat anti-rat Alexa Fluor488-labeled secondary antibody (1:150; Cat# A11006; RRID: AB_2534074; Thermo Fisher Scientific), a goat anti-rabbit Cy3-conjugated secondary antibody (1:100; Cat# A10520; RRID: AB_10563288; Thermo Fisher Scientific), and Hoechst 33342 (2 μg/mL; Sigma-Aldrich).

Techniques: Binding Assay, Inhibition, Phospho-proteomics