Journal: Advanced Science

Article Title: Micro‐Organ Chip Deciphers Tumor‐Derived G‐CSF as Remote Commander of Lung Pre‐Metastatic Niche via VEGFA‐KDR Cascade

doi: 10.1002/advs.202518584

Figure Lengend Snippet: The VEGFA‐KDR Signaling Axis Drives Pulmonary PMN Formation. A) Left : Schematic of the micro‐organ chip‐based experimental workflow for validating VEGFA‐induced PMN formation . Right : Representative fluorescence images of 4T1‐GFP+ cell colonization in PBS‐ versus VEGFA‐treated lung tissue clusters and normalized colonization density (cells per cluster). (Scale bars: 500 µm; n=3; * p < 0.05, ** p < 0.01, *** p < 0.001 by two‐tailed t‐test). B) Immunofluorescence analysis of lung tissues after PBS/VEGFA treatment: Nuclei (DAPI, blue), Angiogenesis (CD31, yellow), Microvasculature (EMCN, red), KDR expression (green), ECM remodeling (Fibronectin, green; Vimentin, yellow). (Scale bars: 30 µm), and quantification of CD31+, CD31+EMCN+ (CAP cells), KDR+ fractions in CAP cells, Fibronectin and Vimentin deposition. (n=3; *p < 0.05, **p < 0.01, ***p < 0.001 by two‐tailed t‐test). C) IF and IHC of in vivo lung sections from VEGFA‐infused mice showing CD31, KDR, and VEGFA expression, and marker‐positive area quantification. (Scale bars: 60 µm (IF), 40 µm (IHC); n=3 mice/group; *p < 0.05 , **p < 0.01 , ***p < 0.01 by t‐test). D) Schematic of the micro‐organ chip based experimental workflow for validating receptor‐dependent KDR activation, and signaling specificity of the VEGFA‐KDR axis. Top : 4T1‐GFP+ colonization in VEGFA protein and vehicle versus VEGFA protein and cabozantinib groups. Bottom : 4T1‐GFP+ colonization in tumor co‐cultured lung tissue clusters with isotype control versus αVEGFA antibody groups. E) Left : Normalized colonization density (cells per cluster) of 4T1‐GFP+ cell colonization in VEGFA protein and vehicle versus VEGFA protein and cabozantinib groups. Right : Normalized colonization density (cells per cluster) of 4T1‐GFP+ cell colonization in tumor‐lung co‐cultures with isotype control versus anti‐VEGFA. (Scale bars: 500 µm; n=3; *p < 0.05, **p < 0.01, ***p < 0.001 by two‐tailed t‐test). F) Quantification of CD31+EMCN+ (CAP cells), KDR+ fractions in CAP cells, Fibronectin deposition in VGEFA protein combine with cabozantinib or vehicle. (n=3; *p < 0.05, **p < 0.01, ***p < 0.001 by two‐tailed t‐test). G) IF and IHC analysis of lung tissues from orthotopic tumor‐bearing and VEGFA antibody or isotype control‐treated mice. Representative images of CD31 (angiogenesis), KDR (target engagement), and VEGFA (pro‐angiogenic signaling) in lung sections and quantification of marker‐positive areas. (Scale bars: 60 µm (IF), 100 and 40 µm (IHC); n = 3 mice/group; *p < 0.05, **p < 0.01, ***p < 0.001 by two‐tailed t‐test).

Article Snippet: For pharmacological interventions, the system was treated with cabozantinib malate (XL184, 1.5 μ m ; Selleck) or vehicle (DMSO), recombinant VEGF164 (5 ng μL −1 ; MedChemExpress), anti‐mouse VEGF‐A neutralizing antibody (2G11‐2A05, 1 ng μL −1 ; Bio X Cell), recombinant G‐CSF (10 ng μL −1 ; Servicebio), or anti‐mouse G‐CSF antibody (MAB414, 1 ng μL −1 ; R&D Systems).

Techniques: Fluorescence, Two Tailed Test, Immunofluorescence, Expressing, In Vivo, Marker, Activation Assay, Cell Culture, Control, Drug discovery

Journal: Advanced Science

Article Title: Micro‐Organ Chip Deciphers Tumor‐Derived G‐CSF as Remote Commander of Lung Pre‐Metastatic Niche via VEGFA‐KDR Cascade

doi: 10.1002/advs.202518584

Figure Lengend Snippet: Tumor‐derived G‐CSF promotes the formation of pulmonary PMN. A) Cytokine/chemokine array profiling of normal mammary tissue versus three breast cancer subtypes (4T1, EMT6, JC), red boxes indicate the positions of G‐CSF, with each antibody array containing duplicate spots for reproducibility. B) Schematic of the micro‐organ chip experimental design for functional validation of G‐CSF. Lung tissue clusters were co‐cultured with PBS (negative control), 4T1 tumor tissue clusters (positive control), or recombinant G‐CSF. C) Left : Representative fluorescence images showing 4T1‐GFP+ cell colonization in lung tissues treated with PBS, 4T1 tumor clusters, or G‐CSF. Right : Quantification of normalized colonization density (cells per cluster). (Scale bar: 500 µm; n=3; one‐way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001). D) IF and IHC of lung sections from G‐CSF‐infused mice showing CD31, KDR, and VEGFA expression. Right: Marker‐positive area quantification. (Scale bars: 60 µm (IF), 100 and 20 µm (IHC); n=3 mice/group; *p < 0.05, **p < 0.01, ***p < 0.01 by two‐tailed t‐test). E) Left : Schematic of tumor co‐cultured lung tissue clusters were administrated with isotype control or αG‐CSF antibody. Right : Quantification of normalized colonization density (cells per cluster) about 4T1‐GFP+ cell colonization in 4T1 co‐cultured lung tissues treated with G‐CSF antibody or isotype control. (n=3; two‐tailed t‐test, *p < 0.05, **p < 0.01, ***p < 0.001). F) Quantitative assessment of CD31+EMCN+, CD31+EMCN+ KDR+, Fibronectin+ area fractions of 4T1‐preconditioned lung tissues treated with αG‐CSF antibody or isotype control. (mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 by two‐tailed t‐test). G) IF and IHC analysis of lung tissues from orthotopic tumor‐bearing and G‐CSF antibody or isotype control treated mice. Representative images of CD31 (angiogenesis), KDR (target engagement), and VEGFA (pro‐angiogenic signaling) in lung sections and quantification of marker‐positive areas. (Scale bars: 60 µm (IF), 100 and 20 µm (IHC); n = 3 mice/group; *p < 0.05, **p < 0.01, ***p <0.001 by two‐tailed t‐test).

Article Snippet: For pharmacological interventions, the system was treated with cabozantinib malate (XL184, 1.5 μ m ; Selleck) or vehicle (DMSO), recombinant VEGF164 (5 ng μL −1 ; MedChemExpress), anti‐mouse VEGF‐A neutralizing antibody (2G11‐2A05, 1 ng μL −1 ; Bio X Cell), recombinant G‐CSF (10 ng μL −1 ; Servicebio), or anti‐mouse G‐CSF antibody (MAB414, 1 ng μL −1 ; R&D Systems).

Techniques: Derivative Assay, Ab Array, Functional Assay, Biomarker Discovery, Cell Culture, Negative Control, Positive Control, Recombinant, Fluorescence, Expressing, Marker, Two Tailed Test, Control, Drug discovery

Journal: Advanced Science

Article Title: Micro‐Organ Chip Deciphers Tumor‐Derived G‐CSF as Remote Commander of Lung Pre‐Metastatic Niche via VEGFA‐KDR Cascade

doi: 10.1002/advs.202518584

Figure Lengend Snippet: Tumor‐derived G‐CSF regulates VEGFA‐KDR axis. A) ELISA quantification of VEGFA levels in conditioned media from differentially treated lung tissues. Tumor co‐culture and recombinant G‐CSF significantly increased VEGFA secretion compared to RPMI 1640 controls (n=3; *p < 0.05, **p < 0.01, ***p < 0.001 by one‐way ANOVA).B) Schematic of the experimental design for functional validation of G‐CSF‐mediated VEGFA‐KDR axis regulation in the micro‐organ chip system. Key interventions included: VEGFA neutralization (VEGFA antibody) and KDR inhibition (cabozantinib). C) Representative fluorescence images and quantitative analysis of 4T1‐GFP+ cell colonization in lung tissues treated with G‐CSF plus VEGFA antibody and parallel experiments with G‐CSF plus cabozantinib. Normalized colonization density (cells per cluster) demonstrates a significant reduction in tumor cell adhesion upon pathway disruption (Scale bar: 500 µm; n=3; two‐tailed t‐test, *p < 0.05, **p < 0.01, ***p < 0.01). D) Immunofluorescence analysis of lung tissues post‐treatment with G‐CSF combine with VEGFA antibody. Nuclei (DAPI, blue), angiogenesis (CD31, yellow), microvasculature (EMCN, red), and KDR expression (green). (Scale bars: 30 µm), and quantification of CD31+EMCN+ (CAP cells), KDR+ fractions in CAP cells. E) Immunofluorescence analysis of lung tissues post‐treatment with G‐CSF combined with VEGFA antibody. Nuclei (DAPI, blue), ECM remodeling (Fibronectin, green; Vimentin, yellow). (Scale bars: 30 µm), and quantification of Fibronectin deposition. F) Quantitative analysis of CD31+, CD31+EMCN+ (microvascular/CAP cells), KDR+ CAP cells, Fibronectin, and Vimentin deposition in G‐CSF‐cabozantinib. (n=3; two‐tailed t‐test, *p < 0.05, **p < 0.01, ***p < 0.001). G) Schematic illustration of how tumor‐secreted G‐CSF promotes pre‐metastatic niche formation in the lung by modulating the VEGFA‐KDR signaling axis.

Article Snippet: For pharmacological interventions, the system was treated with cabozantinib malate (XL184, 1.5 μ m ; Selleck) or vehicle (DMSO), recombinant VEGF164 (5 ng μL −1 ; MedChemExpress), anti‐mouse VEGF‐A neutralizing antibody (2G11‐2A05, 1 ng μL −1 ; Bio X Cell), recombinant G‐CSF (10 ng μL −1 ; Servicebio), or anti‐mouse G‐CSF antibody (MAB414, 1 ng μL −1 ; R&D Systems).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Recombinant, Functional Assay, Biomarker Discovery, Neutralization, Inhibition, Fluorescence, Disruption, Two Tailed Test, Immunofluorescence, Expressing

Journal: Frontiers in pharmacology

Article Title: Curcumin Derivative Cur20 Attenuated Cerebral Ischemic Injury by Antioxidant Effect and HIF-1α/VEGF/TFEB-Activated Angiogenesis.

doi: 10.3389/fphar.2021.648107

Figure Lengend Snippet: FIGURE 6 | Expression of HIF-1 a (A), VEGF (B), TFEB (C), and CD34 (D) in cerebral cortex analyzed by immunohistochemistry. *p < 0.05 vs. sham group, **p < 0.01 vs. sham group; #p < 0.05 vs. Model group, ##p < 0.01 vs. Model group.

Article Snippet: Brain slices were incubated with antibodies (BosterBioengineering, Wuhan, China) against HIF-1α, CD34, NF-κB, VEGF, TFEB (rabbit polyclonal IgGantibody; 1:100) at 4°C and reacted with avidin-biotin-peroxidase complex and DAB (Boster Bio-engineering, Wuhan, China).

Techniques: Expressing, Immunohistochemistry

Journal: Frontiers in pharmacology

Article Title: Curcumin Derivative Cur20 Attenuated Cerebral Ischemic Injury by Antioxidant Effect and HIF-1α/VEGF/TFEB-Activated Angiogenesis.

doi: 10.3389/fphar.2021.648107

Figure Lengend Snippet: FIGURE 9 | Effect of Cur20 on the HIF-1α/VEGF/TFEB pathway. A–E, The protein levels of HIF-1α, NF-κB, VEGF, and TFEB analyzed by western blot. F, The content and translocation of TFEB in nuclei observed by confocal microscopy with immunofluorescence. rBMECs were pretreated with Cur20 for 2 h then treated with OGD for 4 h. L, M, or H represents the concentration of Cur20 as 0.1, 1, 10 μM, respectively. In the immunofluorescence experiment, the concentration of Cur20 was 10 μM. The experimental data was expressed by mean ± SD, n 3.*p < 0.05 vs. control group, #p < 0.05 vs. Model group.

Article Snippet: Brain slices were incubated with antibodies (BosterBioengineering, Wuhan, China) against HIF-1α, CD34, NF-κB, VEGF, TFEB (rabbit polyclonal IgGantibody; 1:100) at 4°C and reacted with avidin-biotin-peroxidase complex and DAB (Boster Bio-engineering, Wuhan, China).

Techniques: Western Blot, Translocation Assay, Confocal Microscopy, Concentration Assay, Control

Journal: Cell death & disease

Article Title: B7-H3 promotes colorectal cancer angiogenesis through activating the NF-κB pathway to induce VEGFA expression.

doi: 10.1038/s41419-020-2252-3

Figure Lengend Snippet: Fig. 3 B7-H3 promoted the expression of VEGFA in CRC. a The expression of angiogenesis-related genes was detected by RT-qPCR in shB7-H3 HCT116 and RKO cells. b Western blot analysis of B7-H3 and VEGFA in the sh-NC and shB7-H3 CRC cell lines. β-actin served as a loading control. c Representative images of IHC for VEGFA in CRC tissues and matched normal tissues from the 125 clinical CRC patients. Scale bar, 100 μm. d VEGFA protein expression based on the staining index of CRC specimens and matched normal tissues. e VEGFA protein expression is shown for patients stratified into B7-H3 low (median value) groups. f Correlation analysis of the staining index of the protein expression levels of B7-H3 and CD31 in human CRC specimens (n = 125). The correlation coefficient (r) is shown. The data represent the means ± SEM. NS no significant difference; *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: After antigen retrieval with 10mM sodium citrate buffer (pH 6.0), the sections were incubated with goat anti-human 4IgB7-H3 antibody (R&D Systems, MN, USA, #AF1027, 1:100), mouse anti-human CD31 antibody (Abcam, Cambridge, MA, USA, #ab32457, 1:1500), or rabbit anti-human VEGFA antibody (Proteintech, Wuhan, China, #19003–1-AP, 1:500).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Staining

Journal: Cell death & disease

Article Title: B7-H3 promotes colorectal cancer angiogenesis through activating the NF-κB pathway to induce VEGFA expression.

doi: 10.1038/s41419-020-2252-3

Figure Lengend Snippet: Fig. 6 B7-H3 promoted angiogenesis via NF-κB/VEGFA pathway in Matrigel plugs in vivo. a, b CD31 (a) and VEGFA (b) protein expression based on their IHC staining index results in subcutaneous tumors formed by sh-NC-HCT116 and shB7-H3-HCT116 cells. N = 5. c, d CD31 (c) and VEGFA (d) protein expression based on their IHC staining index results in subcutaneous tumors formed by EV-HCT116 and B7-H3-HCT116 cells. N = 5. e, f CD31 (e) and VEGFA (f) protein expression based on their IHC staining index results in subcutaneous B7-H3-HCT116 tumors and B7-H3-HCT116 tumors treated with BAY11–7082 (6 mg/kg). g, h CD31 (g) and VEGFA (h) protein expression based on their IHC staining index results in subcutaneous B7-H3-HCT116 tumors and B7-H3-HCT116 tumors treated with bevacizumab (1 mg/kg). i, j CD31 (i) and VEGFA (j) protein expression based on their IHC staining index results in subcutaneous B7-H3-HCT116 tumors and B7-H3-HCT116 tumors co-treated with 3E8 (5 mg/kg) and BAY11–7082 (6 mg/kg) or bevacizumab (1 mg/kg). N = 5. The data represent the means ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: After antigen retrieval with 10mM sodium citrate buffer (pH 6.0), the sections were incubated with goat anti-human 4IgB7-H3 antibody (R&D Systems, MN, USA, #AF1027, 1:100), mouse anti-human CD31 antibody (Abcam, Cambridge, MA, USA, #ab32457, 1:1500), or rabbit anti-human VEGFA antibody (Proteintech, Wuhan, China, #19003–1-AP, 1:500).

Techniques: In Vivo, Expressing, Immunohistochemistry

Journal: Journal of Endocrinology

Article Title: The effects of recombinant human GH on promoting tumor growth depend on the expression of GH receptor in vivo

doi: 10.1530/joe-11-0100

Figure Lengend Snippet: Figure 3 The VEGF expression in tumor tissues. (A–C) VEGF staining in SGC-7901 observed with high microscope (200!original magnifications). (D–F) VEGF staining in MKN-45 observed with high microscope (200! original magnifications). A and D, control groups; B and E, low-dose rhGH groups; and C and F, high-dose rhGH groups. Three animals per group were used for immunohistochemistry assay. Full colour version of this figure available via http://dx.doi.org/10.1530/ JOE-11-0100.

Article Snippet: BALB-c/nunu male nude mice (4–5 weeks old, 14–16 g) were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences. rhGH was obtained from Serono Pharm Co., (Geneva, Switzerland); RPMI medium 1640 was purchased from Gibco Co.; rabbit anti-human GHR polyclonal antibody, rabbit anti-human VEGF monoclonal antibody, and HRPlabeled goat anti-rabbit secondary antibody were purchased from Boster Biological Company of China (Wuhan, China); signal transducers and activators of transcription 3 (STAT3), phosphorylated STAT3 (pSTAT3), hypoxia-inducible factor 1, alpha subunit (HIF-a), matrix metalloproteinase 2 (MMP-2), and other antibodies were purchased from Santa Cruz Co., (Santa Cruz, CA, USA).

Techniques: Expressing, Staining, Microscopy, Control, Immunohistochemistry

Journal: Journal of Endocrinology

Article Title: The effects of recombinant human GH on promoting tumor growth depend on the expression of GH receptor in vivo

doi: 10.1530/joe-11-0100

Figure Lengend Snippet: Figure 4 The mRNA expressions of angiogenesis-related factors in tumor tissues. (A) RT-PCR analysis of Ghr, Jak-2, Stat3, Vegf, Hif-1a, Fgf, and Mmp-2. Sc, control group of SGC-7901; Sl, low-rhGH treatment group of SGC-7901; Sh, high-rhGH treatment group of SGC-7901; Mc, control group of MKN-45; Ml, low-rhGH treatment group of MKN-45; Mh, high-rhGH treatment group of MKN-45. (B) Ratio of gray scale in SGC-7901 groups (*P!0.05 vs control and #P!0.05 vs low-dose rhGH group). (C) Ratio of gray scale in MKN-45 groups. Four animals per group were used for RT-PCR.

Article Snippet: BALB-c/nunu male nude mice (4–5 weeks old, 14–16 g) were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences. rhGH was obtained from Serono Pharm Co., (Geneva, Switzerland); RPMI medium 1640 was purchased from Gibco Co.; rabbit anti-human GHR polyclonal antibody, rabbit anti-human VEGF monoclonal antibody, and HRPlabeled goat anti-rabbit secondary antibody were purchased from Boster Biological Company of China (Wuhan, China); signal transducers and activators of transcription 3 (STAT3), phosphorylated STAT3 (pSTAT3), hypoxia-inducible factor 1, alpha subunit (HIF-a), matrix metalloproteinase 2 (MMP-2), and other antibodies were purchased from Santa Cruz Co., (Santa Cruz, CA, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Control

Journal: Journal of Endocrinology

Article Title: The effects of recombinant human GH on promoting tumor growth depend on the expression of GH receptor in vivo

doi: 10.1530/joe-11-0100

Figure Lengend Snippet: Figure 5 The protein expressions of angiogenesis-related factors in tumor tissues. (A) Western blot analysis of STAT3, pSTAT3, VEGF, HIF-1a, and MMP-2. Sc, control group of SGC-7901; Sl, low-rhGH treatment group of SGC-7901; Sh, high-rhGH treatment group of SGC-7901; Mc, control group of MKN-45; Ml, low-rhGH treatment group of MKN-45; Mh, high-rhGH treatment group of MKN-45. (B) Ratio of gray scale in SGC-7901 groups (*P!0.05 vs control and #P!0.05 vs low-dose rhGH group). (C) Ratio of gray scale in MKN-45 groups. Four animals per group were used for western blotting.

Article Snippet: BALB-c/nunu male nude mice (4–5 weeks old, 14–16 g) were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences. rhGH was obtained from Serono Pharm Co., (Geneva, Switzerland); RPMI medium 1640 was purchased from Gibco Co.; rabbit anti-human GHR polyclonal antibody, rabbit anti-human VEGF monoclonal antibody, and HRPlabeled goat anti-rabbit secondary antibody were purchased from Boster Biological Company of China (Wuhan, China); signal transducers and activators of transcription 3 (STAT3), phosphorylated STAT3 (pSTAT3), hypoxia-inducible factor 1, alpha subunit (HIF-a), matrix metalloproteinase 2 (MMP-2), and other antibodies were purchased from Santa Cruz Co., (Santa Cruz, CA, USA).

Techniques: Western Blot, Control