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: iScience

Article Title: Induced retinal pigment epithelial cells with anti-epithelial-to-mesenchymal transition ability delay retinal degeneration

doi: 10.1016/j.isci.2022.105050

Figure Lengend Snippet: iRPE cells have similar phenotype and functions as iPSC-RPE cells (A) Schematic for the transforming process. A cocktail of TF-expressing retroviruses was used to transfect De-iPSC-RPE cells. After seven days, iRPE clone was observed in the culture and picked out for subculturing. (B and C) RPE-specific and EMT-associated markers detected by (B) immunostaining and (C) western blotting after cells were cultured for 8 days. The expression pattern of these markers in iRPE cells is more similar to that in iPSC-RPE cells. Scale bar = 50 μm. (D) Electron micrographs of iPSC-RPE cells, De-iPSC-RPE cells, and iRPE cells demonstrated that quite a few microvilli were on the surface of iRPE and iPSC-RPE cells. Scale bar = 0.5 μm. (E and F) The bound and phagocyted POSs (pointed by arrows) in iPSC-RPE, De-iPSC-RPE, and iRPE cells (E) and quantification of phagocytosis (F) as determined by the number of bound and phagocyted POS per field. Scale bar = 50 μm. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n ≥ 5. (G and H) TER analysis (G) and HRP permeability assay (H) showed that iRPE cells maintained the same epithelial integrity as iPSC-RPE cells. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n = 6. (I) Expression levels of PEDF and VEGF from upper and lower chambers were determined by ELISA. iRPE cells and iPSC-RPE cells demonstrated similar secretion patterns. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n = 3.

Article Snippet: PEDF and VEGF were quantified by PEDF ELISA kit (Elabscience, Wuhan, China) and VEGF ELISA kit (Proteintech).

Techniques: Expressing, Subculturing Assay, Immunostaining, Western Blot, Cell Culture, Permeability, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Induced retinal pigment epithelial cells with anti-epithelial-to-mesenchymal transition ability delay retinal degeneration

doi: 10.1016/j.isci.2022.105050

Figure Lengend Snippet: BMP7 and FOXF2 are critical regulators of EMT in iRPE cells (A) The higher level of BMP7 secreted by iRPE cells compared with De-iPSC-RPE cells was determined by ELISA. Data are mean ± SD, unpaired two-sided t-tests, n = 4. (B and C) The reduced expression level of FOXF2 in iRPE compared with that in De-iPSC-RPE cells was determined by (B) immunostaining and (C) western blotting. Scale bar = 50 μm. (D) The efficiency of bmp7 knockdown was determined by qRT-PCR; shBmp7-1 was slightly more efficient at reducing the mRNA level of bmp7 than shBmp7-2; therefore, it was selected for subsequent experiments. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n = 4. (E) The shBmp7 construct contained the ZsGreen expression element to indicate successful transfection. Scale bar = 50 μm. (F and G) FLAG-FOXF2 overexpression (ov-FOXF2) in (F) iRPE cells (G) shBmp7-iRPE cells. Scale bar = 50 μm. (H and I) The expression levels of RPE-specific markers and EMT markers were determined by (H) western blotting and (I) quantitative analysis. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n = 3. (J) BMP7 expression levels in shCont-iRPE, shBmp7-iRPE, ov-FOXF2- iRPE, and shBmp7 + ov-FOXF2-iRPE. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n = 4. (K) The immunostaining of RPE-specific markers and EMT markers. Overexpression of FOXF2 exhibited similar effects to knockdown of bmp7 in iRPE cells by downregulating RPE markers and upregulating EMT marker. Scale bar = 50 μm.

Article Snippet: PEDF and VEGF were quantified by PEDF ELISA kit (Elabscience, Wuhan, China) and VEGF ELISA kit (Proteintech).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Immunostaining, Western Blot, Knockdown, Quantitative RT-PCR, Construct, Transfection, Over Expression, Marker

Journal: iScience

Article Title: Induced retinal pigment epithelial cells with anti-epithelial-to-mesenchymal transition ability delay retinal degeneration

doi: 10.1016/j.isci.2022.105050

Figure Lengend Snippet: Four TFs transcriptionally regulate bmp7 , foxf2 , lin7a , pard6b , and ppm1a in a direct or indirect manner (A) ELISA analysis demonstrated that BMP7 levels was reduced in 4TFs−nr2e1-RPE, 4TFs−mitf-a-RPE, 4TFs−c-myc-RPE, and 4TFs−crx-RPE cells compared with that in iRPE cells. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n = 4. (B and C) The levels of FOXF2, LIN7A, PARD6B, and PPM1A were determined by (B) western blotting and (C) quantitative analysis. Data are mean ± SD, one-way ANOVA and post hoc Bonferroni test, n = 3. (D) Generation of FLAG-MITF-A-iRPE, FLAG-CRX-iRPE, FLAG-NR2E1-iRPE, and FLAG-C-MYC-iRPE cells. Scale bar = 50 μm. (E–I) Enriched peaks of CRX binding to the target genes and fold enrichment of CRX immunoprecipitation compared with IgG control, as determined by qRT-PCR. Data are mean ± SD, unpaired two-sided t-tests, n = 3. (J–M) Enriched peaks of MITF-A and NR2E1 binding to the target gene lin7a and fold enrichment of MITF-A and NR2E1 immunoprecipitation compared with IgG control, as determined by qRT-PCR. Data are mean ± SD, unpaired two-sided t-tests, n = 3. (N) Schematic model for the regulation of EMT and MET processes by the four TFs. CRX, MITF-A, NR2E1, and C-MYC directly or indirectly regulated the expression of bmp7 , lin7a , pard6b, ppm1a , and foxf2 to inhibit EMT and promote MET.

Article Snippet: PEDF and VEGF were quantified by PEDF ELISA kit (Elabscience, Wuhan, China) and VEGF ELISA kit (Proteintech).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Binding Assay, Immunoprecipitation, Control, Quantitative RT-PCR, Expressing

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

Article Title: Expression of TP53, BCL-2, and VEGFA Genes in Esophagus Carcinoma and its Biological Significance

doi: 10.12659/MSM.894640

Figure Lengend Snippet: Expression of VEGF proteins by IHC staining. ( A ) Control group: ( B ) Model group at 4 weeks; ( C ) Model group at 10 weeks; ( D ) Model group at 20 weeks; ( E ) Model group at 30 weeks. Magnification, ×200.

Article Snippet: After dewaxing and rehydration, mouse anti-p53 monoclonal antibody (1:100, Boster Biotech, China), mouse anti-Bcl-2 monoclonal antibody (1:60, Boster Biotech, China), or mouse anti-VEGF monoclonal antibody (1:100, Boster Biotech, China) were added for overnight incubation.

Techniques: Expressing, Immunohistochemistry, Control