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: Pharmacological inhibition of KDR disrupts PMN formation in vitro and in vivo. A) Schematic of assessing KDR inhibitor effect in micro‐organ chip in vitro co‐culture system (Top) and orthotopic 4T1 breast cancer in vivo model (Bottom) . B) Left : Representative fluorescence images showing 4T1‐GFP+ cell colonization in tumor‐co‐cultured lung tissue clusters after 3 days continuous treatment with either Cabozantinib or vehicle control. Right : Normalized colonization density (cells per clusters) in KDR inhibitor‐ versus vehicle‐treated groups. (Scale bars: 500 µm; *p < 0.05, **p < 0.01, ***p < 0.001 by two‐tailed t‐test). C) Left : Immunofluorescence analysis of PMN markers in micro‐organ chip cultured lung tissues. Nuclear staining (DAPI, blue), Vascular remodeling (CD31+, yellow, EMCN, red), KDR (green), ECM remodeling (Fibronectin, green; Vimentin, yellow). (Scale bars: 30 µm). Right : Quantitative analysis of CD31+, CD31+ and EMCN+, KDR+, Fibronectin, and Vimentin deposition. (n=3; *p < 0.05, **p < 0.01, ***p < 0.001 by two‐tailed t‐test). D) Representative longitudinal bioluminescence imaging (foci 10–15 days post 4T1‐GL injection) and quantitative fluorescence analysis of pulmonary metastases at 3–15 days post‐4T1‐GL cells injection. (n=4, p = 0.0140, Fisher's LSD post hoc test). E) IF analysis of lung tissues from orthotopic tumor‐bearing mice. Representative images of CD31 and EMCN (angiogenesis), KDR (target engagement), and Fibronectin (ECM remodeling) in lung sections and quantification results. (Scale bars: 60 µm 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: Inhibition, In Vitro, In Vivo, Co-Culture Assay, Fluorescence, Cell Culture, Control, Two Tailed Test, Immunofluorescence, Staining, Imaging, Injection, 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: 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 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: Molecular & Cellular Proteomics : MCP

Article Title: Monitoring Cell-surface N -Glycoproteome Dynamics by Quantitative Proteomics Reveals Mechanistic Insights into Macrophage Differentiation *

doi: 10.1074/mcp.M116.063859

Figure Lengend Snippet: THP-1 differentiation in presence of dasatinib causes changes in cellular morphology, cell-surface marker presentation and phagocytotic activity. A, morphological differences between monocytic THP-1 cells after cultivation or PMA differentiation in the presence or absence of 1 μm dasatinib for 48 h monitored by light microscopy. Differentiation in the presence of dasatinib results in a less adherent and more dendritic cell shape. Addition of dasatinib to already differentiated cells has no influence on the morphology. B, immunoblots for cell-surface markers regulated during differentiation of THP-1 cells by PMA (M: molecular weight marker). C, proteins with significantly altered abundance in THP-1 cells differentiated in presence or absence of dasatinib. D, heat map representation of quantified plasma membrane-associated proteins during differentiation of monocytic THP-1 to macrophage-like cells in presence or absence of dasatinib and sunitinib. Colors represent log2 relative abundance to untreated controls. E, phagocytosis assay in differentiated THP-1 cells in absence or presence of 2 μm cytochalasin D. Mean of four replicates; error bars represent standard deviations. See also supplemental Fig. S4.

Article Snippet: Phorbol 12-myristate 13-acetate (PMA) and imatinib (Gleevec, STI-571) were purchased from Sigma-Aldrich (St. Louis, MO); dasatinib (Sprycel, BMS-354825) and sunitinib (Sutent, SU11248) were from Santa Cruz Biotechnology (Santa Cruz, CA), and vitamin D 3 was from Cayman Chemicals (Biomol, Hamburg, Germany).

Techniques: Marker, Activity Assay, Light Microscopy, Western Blot, Molecular Weight, Phagocytosis Assay

Journal: eLife

Article Title: A zebrafish embryo screen utilizing gastrulation identifies the HTR2C inhibitor pizotifen as a suppressor of EMT-mediated metastasis

doi: 10.7554/eLife.70151

Figure Lengend Snippet: A list of the drugs that interfere with epiboly progression in zebrafish. Related to Figure 1 . A list of positive ‘hit’ drugs that interfered with epiboly progression. Gastrulation defects or status of each of the zebrafish embryos that were treated with either 10 or 50 μM concentrations are indicated.

Article Snippet: Pizotifen (sc-201143) and S(-)eticlopride hydrochloride (E101) were purchased from Santa Cruz (Dallas, TX) and Sigma-Aldrich (St Louis, MO).

Techniques:

Journal: eLife

Article Title: A zebrafish embryo screen utilizing gastrulation identifies the HTR2C inhibitor pizotifen as a suppressor of EMT-mediated metastasis

doi: 10.7554/eLife.70151

Figure Lengend Snippet: ( A ) Effect of the epiboly-interrupting drugs on cell motility and invasion of MBA-MB-231 cells. MBA-MB-231 cells were treated with vehicle or each of the epiboly-interrupting drugs and then subjected to Boyden chamber assays. Fetal bovine serum (1% v/v) was used as the chemoattractant in both assays. Each experiment was performed at least twice. ( B ) Western blot analysis of HTR2C levels (top) in a non-metastatic human cancer cell line, MCF7 (breast) and highly metastatic human cancer cell lines, MDA-MB-231 (breast), MDA-MB-435 (melanoma), PC9 (lung), MIA-PaCa2 (pancreas), PC3 (prostate), and SW620 (colon); GAPDH loading control is shown (bottom). ( C ) Effect of pizotifen on cell motility and invasion of MBA-MB-231, MDA-MB-435, and PC9 cells. Either vehicle or pizotifen treated the cells were subjected to Boyden chamber assays. Fetal bovine serum (1% v/v) was used as the chemoattractant in both assays. Each experiment was performed at least twice. ( D ) and ( E ) Representative images of dissemination of 231R, shLacZ 231R or shHTR2C 231R cells in zebrafish xenotransplantation model. The fish larvae that were inoculated with 231R cells were treated with either vehicle (top left) or the drug (lower left) ( D ). The fish larvae that were inoculated with either shLacZ 231R or shHTR2C 231R cells (lower left) ( E ). White arrows head indicate disseminated 231R cells. The images were shown in 4× magnification. Scale bar, 100 µm. The mean frequencies of the fish showing head, trunk, or end-tail dissemination were counted (graph on right). Each value is indicated as the mean ± SEM of two independent experiments. Statistical analysis was determined by Student’s t test.

Article Snippet: Pizotifen (sc-201143) and S(-)eticlopride hydrochloride (E101) were purchased from Santa Cruz (Dallas, TX) and Sigma-Aldrich (St Louis, MO).

Techniques: Western Blot, Control

Journal: eLife

Article Title: A zebrafish embryo screen utilizing gastrulation identifies the HTR2C inhibitor pizotifen as a suppressor of EMT-mediated metastasis

doi: 10.7554/eLife.70151

Figure Lengend Snippet: Primary targets of the identified drugs.

Article Snippet: Pizotifen (sc-201143) and S(-)eticlopride hydrochloride (E101) were purchased from Santa Cruz (Dallas, TX) and Sigma-Aldrich (St Louis, MO).

Techniques: Membrane

Journal: eLife

Article Title: A zebrafish embryo screen utilizing gastrulation identifies the HTR2C inhibitor pizotifen as a suppressor of EMT-mediated metastasis

doi: 10.7554/eLife.70151

Figure Lengend Snippet: ( A ) Representative images of dissemination of 231R cells in zebrafish xenotransplantation model. The fish larvae that were inoculated with 231R cells were treated with either vehicle (top left, bottom left) or pizotifen (top right, bottom right). ( B ) Representative images of dissemination of MIA-PaCa2 cells in zebrafish xenotransplantation model. The fish were inoculated with MIA-PaCa2 cells, and treated with either vehicle (top left) or drug (lower left). White arrow heads indicate disseminated MIA-PaCa2 cells. The images were shown in 4× magnification. Scale bar, 100 μm. The mean frequencies of the fish showing head, trunk, or end-tail dissemination were tabulated (right). Each value is indicated as the mean ± SEM of two independent experiments. Statistical analysis was determined by Student’s t test.

Article Snippet: Pizotifen (sc-201143) and S(-)eticlopride hydrochloride (E101) were purchased from Santa Cruz (Dallas, TX) and Sigma-Aldrich (St Louis, MO).

Techniques:

Journal: eLife

Article Title: A zebrafish embryo screen utilizing gastrulation identifies the HTR2C inhibitor pizotifen as a suppressor of EMT-mediated metastasis

doi: 10.7554/eLife.70151

Figure Lengend Snippet: Effects of pharmacological inhibition of HTR2C on metastatic dissemination of MDA-MB-231 cells in zebrafish xenografted models. Related to Figure 2D . The numbers and frequencies of the fish showing the dissemination patterns in vehicle- or pizotifen-treated group were indicated. The fish showed both patterns of dissemination were redundantly counted in this analysis.

Article Snippet: Pizotifen (sc-201143) and S(-)eticlopride hydrochloride (E101) were purchased from Santa Cruz (Dallas, TX) and Sigma-Aldrich (St Louis, MO).

Techniques: Inhibition

Journal: eLife

Article Title: A zebrafish embryo screen utilizing gastrulation identifies the HTR2C inhibitor pizotifen as a suppressor of EMT-mediated metastasis

doi: 10.7554/eLife.70151

Figure Lengend Snippet: Effects of pharmacological inhibition of HTR2C on metastatic dissemination of Mia-PaCa2 cells in zebrafish xenografted models. Related to Figure 4 . The numbers and frequencies of the fish showing the dissemination patterns in vehicle- or pizotifen-treated group were indicated. The fish showed both patterns of dissemination were redundantly counted in this analysis.

Article Snippet: Pizotifen (sc-201143) and S(-)eticlopride hydrochloride (E101) were purchased from Santa Cruz (Dallas, TX) and Sigma-Aldrich (St Louis, MO).

Techniques: Inhibition

Journal: eLife

Article Title: A zebrafish embryo screen utilizing gastrulation identifies the HTR2C inhibitor pizotifen as a suppressor of EMT-mediated metastasis

doi: 10.7554/eLife.70151

Figure Lengend Snippet: ( A ) Mean volumes (n = 10 per group) of 4T1 primary tumors formed in the mammary fat pad of either vehicle- or pizotifen-treated mice at day 10 post injection. ( B ) Ki67 expression level in 4T1 primary tumors formed in the mammary fat pad of either vehicle- or pizotifen-treated mice at day 10 post injection. The mean expression levels of Ki67 (n = 10 mice per group) were determined and were calculated as the mean ration of Ki67-positive cells to 4’,6-diamidino-2-phenylindole (DAPI) area. ( C ) Representative images of primary tumors on day 10 post injection (top panels) and metastatic burden on day 70 post injection (bottom panels) taken using an IVIS Imaging System. ( D ) Representative images of the lungs from either vehicle- (top) or pizotifen-treated mice (bottom) at 70 days post tumor inoculation. Number of metastatic nodules in the lung of either vehicle- or pizotifen-treated mice (right). ( E ) Representative hematoxylin and eosin (H&E) staining of the lung (top) and liver (bottom) from either vehicle- or pizotifen-treated mice. Black arrow heads indicate metastatic 4T1 cells. ( F ) The mean number of metastatic lesions in step sections of the lungs from the mice that were inoculated with 4T1-12B cells expressing short hairpin RNA (shRNA) targeting for either LacZ or HTR2C. ( G ) Representative H&E staining of the lung and liver from the mice that were inoculated with 4T1-12B cells expressing shRNA targeting for either LacZ or HTR2C. Black arrow heads indicate metastatic 4T1 cells. Each value is indicated as the mean ± SEM. Statistical analysis was determined by Student’s t test.

Article Snippet: Pizotifen (sc-201143) and S(-)eticlopride hydrochloride (E101) were purchased from Santa Cruz (Dallas, TX) and Sigma-Aldrich (St Louis, MO).

Techniques: Injection, Expressing, Imaging, Staining, shRNA

Journal: eLife

Article Title: A zebrafish embryo screen utilizing gastrulation identifies the HTR2C inhibitor pizotifen as a suppressor of EMT-mediated metastasis

doi: 10.7554/eLife.70151

Figure Lengend Snippet: Cleaved caspase 3 expression level in 4T1 primary tumors formed in the mammary fat pad of either vehicle- or pizotifen-treated mice at day 10 post injection.

Article Snippet: Pizotifen (sc-201143) and S(-)eticlopride hydrochloride (E101) were purchased from Santa Cruz (Dallas, TX) and Sigma-Aldrich (St Louis, MO).

Techniques: Expressing, Injection

Journal: eLife

Article Title: A zebrafish embryo screen utilizing gastrulation identifies the HTR2C inhibitor pizotifen as a suppressor of EMT-mediated metastasis

doi: 10.7554/eLife.70151

Figure Lengend Snippet: ( A ) Immunofluorescence (IF) staining of E-cadherin in either vehicle- or pizotifen-treated MDA-MB-231 cells. ( B ) Surface expression of E-cadherin in either vehicle (black)- or pizotifen (red)-treated MDA-MB-231 cells by FACS analysis. Non-stained controls are shown in gray. ( C ) Protein expressions levels of E-cadherin, ZEB1, and β-catenin in the cytoplasm and nucleus of 4T1 primary tumors from either vehicle- or pizotifen-treated mice are shown; Luciferase, histone H3, and β-tubulin are used as loading control for whole cell, nuclear, or cytoplasmic lysate, respectively. ( D ) Protein expression levels of epithelial and mesenchymal markers and ZEB1 in either vehicle- or pizotifen-treated MDA-MB-231 cells or E-cadherin-positive (E-cad + ) cells in pizotifen-treated MDA-MB-231 cells are shown. ( E ) IF staining of β-catenin in the MCF7 cells expressing either vector control (top left, bottom left) or HTR2C (top right, bottom right). ( F ) Expressions of β-catenin in the cytoplasm and nucleus of MCF7 cells. ( G ) IF staining of β-catenin in either vehicle (top left, bottom left) or pizotifen-treated MDA-MB-231 cells (top right, bottom right). ( H ) Expressions of β-catenin in the cytoplasm and nucleus of MDA-MB-231 cells and the E-cad + cells.

Article Snippet: Pizotifen (sc-201143) and S(-)eticlopride hydrochloride (E101) were purchased from Santa Cruz (Dallas, TX) and Sigma-Aldrich (St Louis, MO).

Techniques: Immunofluorescence, Staining, Expressing, Luciferase, Control, Plasmid Preparation

Journal: eLife

Article Title: A zebrafish embryo screen utilizing gastrulation identifies the HTR2C inhibitor pizotifen as a suppressor of EMT-mediated metastasis

doi: 10.7554/eLife.70151

Figure Lengend Snippet: Protein expression levels of Twist1 in either vehicle- or pizotifen-treated MDA-MB-231 cells are shown (middle): GAPDH loading control is shown (bottom). Protein expression levels of Snail and Twist1 of 4T1 primary tumors from either vehicle- or pizotifen-treated mice are shown (right); luciferase is used as loading control for whole cell. Luciferase control was obtained in the same experiment from .

Article Snippet: Pizotifen (sc-201143) and S(-)eticlopride hydrochloride (E101) were purchased from Santa Cruz (Dallas, TX) and Sigma-Aldrich (St Louis, MO).

Techniques: Expressing, Control, Luciferase

Journal: eLife

Article Title: A zebrafish embryo screen utilizing gastrulation identifies the HTR2C inhibitor pizotifen as a suppressor of EMT-mediated metastasis

doi: 10.7554/eLife.70151

Figure Lengend Snippet:

Article Snippet: Pizotifen (sc-201143) and S(-)eticlopride hydrochloride (E101) were purchased from Santa Cruz (Dallas, TX) and Sigma-Aldrich (St Louis, MO).

Techniques: Luciferase, Recombinant, Gene Expression, Plasmid Preparation, Software