Journal: The Journal of Experimental Medicine

Article Title: Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects

doi: 10.1084/jem.20020587

Figure Lengend Snippet: Viral gene expression in vitro and in vivo. (A) Comparison of Ad-VEGF-C156S and AdVEGF-C recombinant protein production in vitro. Top panels: the infected cells were metabolically labeled and cell culture media were subjected to precipitation with VEGFR-3-Ig or VEGFR-2-Ig fusion proteins. Medium from AdLacZ-infected cells was used as a negative control. Bottom panel: culture media of Ad-VEGF-C, AdVEGF-C156S, and AdLacZ infected cells were separated in 15% PAGE gel followed by Western blotting using antibodies against VEGF-C. (B) Viral transgene expression in vivo. Northern blot analysis of total RNA from mouse ears infected with recombinant adenoviruses or AAVs. The infecting virus and the duration of infection are indicated. (C) β-galactosidase staining of the ear three weeks after infection with AdLacZ. (D and E) EGFP expression in the ear 6 wk and 8 mo after AAV-EGFP infection.

Article Snippet: After 24–72 h, the cells were metabolically labeled for 8 h and subjected to immunoprecipitation with VEGF-C–specific antibodies or to a binding assay using soluble VEGFR-2-Ig (R&D Systems) and VEGFR-3-Ig ( ) fusion proteins.

Techniques: Gene Expression, In Vitro, In Vivo, Comparison, Recombinant, Infection, Metabolic Labelling, Labeling, Cell Culture, Negative Control, Western Blot, Expressing, Northern Blot, Virus, Staining

Journal: The Journal of Experimental Medicine

Article Title: Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects

doi: 10.1084/jem.20020587

Figure Lengend Snippet: Lymphangiogenesis in the skin of K14-VEGF-C156S and K14-VEGF-C transgenic embryos. (A–C) Superficial lymphatic vessels of VEGFR-3+/LacZ heterozygous E14.5 embryos visualized by β-galactosidase staining. Note the increase in the number of lymphatic capillaries in B and lymphatic capillary hyperplasia in A (arrows). (D–F) Dorsal views of the same embryos. (G–I) Cross sections of the superficial lymphatic vessels of E14.5 K14-VEGF-C156S and K14-VEGF-C transgenic embryos stained with antibodies against VEGFR-3. (J–L) Cutaneous lymphatic vessels of adult (age 8 wk) VEGFR-3+/LacZ mice. The lymphatic phenotype established during embryonic development is retained. Scale bars: A–C, 650 μm; D–F, 250 μm; G–I, 35 μm; J–L, 150 μm.

Article Snippet: After 24–72 h, the cells were metabolically labeled for 8 h and subjected to immunoprecipitation with VEGF-C–specific antibodies or to a binding assay using soluble VEGFR-2-Ig (R&D Systems) and VEGFR-3-Ig ( ) fusion proteins.

Techniques: Transgenic Assay, Staining

Journal: The Journal of Experimental Medicine

Article Title: Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects

doi: 10.1084/jem.20020587

Figure Lengend Snippet: Lymphangiogen-esis in AdVEGF-C156S and AdVEGF-C infected adult skin. Whole mount VEGFR-3 staining of the lymphatic vessels 1 wk (A–C) and 2 wk (D–F) after infection. Note enlarged lymphatic vessels (arrows) and sprouting or splitting lymphatic vessels (arrowheads) in AdVEGF-C156S (A) and AdVEGF-C (B) infected ears. 2 wk after infection, AdVEGF-C156S infected ear (D) contains large lymphatic vessels apparently still undergoing sprouting and splitting, whereas AdVEGF-C (E) infected ear is filled with small lymphatic sprouts. (G–I) Podoplanin stained tissue sections at 2 wk after adenoviral infection. Note the formation of a hyperplastic lymphatic network in response to adenoviral VEGF-C156S (G) and VEGF-C (H) compared with AdLacZ control (I). Sprouting and splitting of the vessels is especially clear in the AdVEGF-C infected ear (H). Scale bars: A–C, 60 μm; D–F, 220 μm; G–I, 70 μm.

Article Snippet: After 24–72 h, the cells were metabolically labeled for 8 h and subjected to immunoprecipitation with VEGF-C–specific antibodies or to a binding assay using soluble VEGFR-2-Ig (R&D Systems) and VEGFR-3-Ig ( ) fusion proteins.

Techniques: Infection, Staining, Control

Journal: The Journal of Experimental Medicine

Article Title: Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects

doi: 10.1084/jem.20020587

Figure Lengend Snippet: Lymphangiogen-esis in AAV-VEGF-C156S and AAV-VEGF-C infected adult skin. (A–F) Lymphatic vessels visualized with VEGFR-3 whole mount staining 6 wk after infection with the recombinant AAVs. Note the enlargement, sprouting (arrowheads), and splitting (arrows) of the lymphatic vessels in response to VEGF-C156S and VEGF-C. Lymphatic sprouting is more obvious in the AAV-VEGF-C infected ear (B and E) than in the AAV-VEGF-C156S infected ear (A and D). (G–I) Podoplanin staining of histological sections at the same time point. Note the hyperplastic lymphatic vessel network within the subcutis and muscle cell layers of the skin in the AAV-VEGF-C156S (G) and AAV-VEGF-C (H) infected samples. Scale bars: A–C, 150 μm; D–F, 60 μm; G–I, 70 μm.

Article Snippet: After 24–72 h, the cells were metabolically labeled for 8 h and subjected to immunoprecipitation with VEGF-C–specific antibodies or to a binding assay using soluble VEGFR-2-Ig (R&D Systems) and VEGFR-3-Ig ( ) fusion proteins.

Techniques: Infection, Staining, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects

doi: 10.1084/jem.20020587

Figure Lengend Snippet: Adenoviral VEGF-C156S does not affect blood vessel morphology. (A–C) Ears photographed 1 wk after adenoviral infection. Note the dilation and tortuosity of blood vessels in the AdVEGF-C infected skin (B) compared with the AdVEGF-C156S (A) and AdLacZ (C) infected skin. (D–F) Whole mount PECAM-1 staining of the ear skin blood vessels at the same time point. Compared with the AdLacZ control (F), the veins (V) and arteries (A) in the AdVEGF-C156S infected skin (D) appear morphologically normal, whereas in the AdVEGF-C infected ear (E) the veins are large and tortuous. Note weak staining of lymphatic vessels (L) in F. Scale bars: D–F, 150 μm.

Article Snippet: After 24–72 h, the cells were metabolically labeled for 8 h and subjected to immunoprecipitation with VEGF-C–specific antibodies or to a binding assay using soluble VEGFR-2-Ig (R&D Systems) and VEGFR-3-Ig ( ) fusion proteins.

Techniques: Infection, Staining, Control

Journal: The Journal of Experimental Medicine

Article Title: Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects

doi: 10.1084/jem.20020587

Figure Lengend Snippet: Adenoviral VEGF-C156S has a minimal effect on vascular permeability. (A) The difference in permeability between the treated versus the control ear measured by Evans Blue concentration ratio (ng/mg). (B and C) Mouse thoracic cavities photographed after systemic administration of AdVEGF-C156S or AdVEGF-C (1 × 10 9 pfu). Note the accumulation of pleural fluid in the AdVEGF-C infected mouse (C) but not in the AdVEGF-C156S infected mouse (B).

Article Snippet: After 24–72 h, the cells were metabolically labeled for 8 h and subjected to immunoprecipitation with VEGF-C–specific antibodies or to a binding assay using soluble VEGFR-2-Ig (R&D Systems) and VEGFR-3-Ig ( ) fusion proteins.

Techniques: Permeability, Control, Concentration Assay, Infection

Journal: The Journal of Experimental Medicine

Article Title: Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects

doi: 10.1084/jem.20020587

Figure Lengend Snippet: VEGF-C156S gene therapy in the Chy lymphedema mice. (A and B) AAV-mediated overexpression of VEGF-C156S (2 mo) and VEGF-C (8 mo) induces the formation and maintenance of a functional lymphatic vessel network, as analyzed by fluorescent microlymphangiography. C and D show comparison with noninfected Chy mouse ear and wild-type control ear, respectively. (E–H) VEGFR-3 whole mount staining of the ears 2 mo after AAV-infection. Note the formation of a tree-like network of lymphatic vessels in the skin of Chy mice in response to VEGF-C156S (E) and VEGF-C (F). Inset in E shows lymphatic sprouting and splitting (arrowheads) in the AAV-VEGF-C156S infected Chy mouse skin 5 wk after infection. The control Chy mouse skin (G) contains few abnormal VEGFR-3–positive vessel structures. (H) VEGFR-3 staining of wild-type mouse skin. Scale bars: A–D, 250 μm; E–H, 200 μm.

Article Snippet: After 24–72 h, the cells were metabolically labeled for 8 h and subjected to immunoprecipitation with VEGF-C–specific antibodies or to a binding assay using soluble VEGFR-2-Ig (R&D Systems) and VEGFR-3-Ig ( ) fusion proteins.

Techniques: Over Expression, Functional Assay, Comparison, Control, Staining, Infection

Journal: The Journal of Experimental Medicine

Article Title: Lymphangiogenic Gene Therapy With Minimal Blood Vascular Side Effects

doi: 10.1084/jem.20020587

Figure Lengend Snippet: Molecular markers of adult lymphatic vessels. (A and B) Visualization of cutaneous lymphatic vessels in VEGFR-3+/LacZ and VEGFR-2+/LacZ mice. Blood vessels are stained brown for biotinylated lectin while the blue staining marks the expression of the β galactosidase gene. Note that whereas VEGFR-2 is found in the collecting lymphatic vessels with valves (arrows), VEGFR-3 expression is strong in the initial lymphatic capillaries (arrowheads). (C and D) Lymphatic vessels on the membranous part and (E and F) on the muscular part of the diaphragm. (G and H) The collecting lymphatic vessels of a K14-VEGF-C × VEGFR-2+/LacZ mouse and a VEGFR-2+/LacZ littermate control. Scale bars: A and B, 320 μm; C and D, 250 μm; E and F, 120 μm; G and H, 180 μm.

Article Snippet: After 24–72 h, the cells were metabolically labeled for 8 h and subjected to immunoprecipitation with VEGF-C–specific antibodies or to a binding assay using soluble VEGFR-2-Ig (R&D Systems) and VEGFR-3-Ig ( ) fusion proteins.

Techniques: Staining, Expressing, 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 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

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