non biotinylated human vegf165  (R&D Systems)

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