Journal: Molecular cancer therapeutics

Article Title: Preclinical Characterization of XL092, a Novel Receptor Tyrosine Kinase Inhibitor of MET, VEGFR2, AXL, and MER.

doi: 10.1158/1535-7163.MCT-22-0262

Figure Lengend Snippet: Figure 1. XL092 inhibits MET, AXL, and VEGFR2 activation in vivo. A, Structure of XL092. pMET/MET levels were evaluated in NCI-H441 (B) and SNU-5 (C) tumors from mice treated with either 3 (red) or 10 (blue) mg/kg XL092 or vehicle (black) orally daily for 14 days (n ¼ 10, pooled for each treatment group). D, pVEGFR2 and VEGFR2 levels were evaluated in hVEGF-treated Hs 746T tumor-bearing mouse lung tissue following single oral doses of 30 or 100 mg/kg XL092 (n ¼ 5, pooled for each treatment group) or vehicle (n ¼ 6, pooled). E, Mean pMET/MET and pAXL/AXL ratios SEM were measured in Hs 746T tumor-bearing mice treated with single oral doses of either 30 or 100 mg/kg XL092 or vehicle (n ¼ 6 for each treatment group). Significance valueswere determined using a nonparametric Mann–Whitney U test with significance levels indicated using , P < 0.01.

Article Snippet: 2022 TheAuthors; Publishedby theAmericanAssociation for CancerResearch AACRJournals.org | 179 D ow nloaded from http://aacrjournals.org/m ct/article-pdf/22/2/179/3266267/179.pdf by guest on 28 O ctober 2024 immunoprecipitation performed for Western blot analysis, antibodies used were from R&D Systems [Goat-anti-mouse antibody, VEGFR2 (#AF644) and AXL (#AF154)].

Techniques: Activation Assay, In Vivo, MANN-WHITNEY

Journal: Cells

Article Title: Fibrotic Changes and Endothelial-to-Mesenchymal Transition Promoted by VEGFR2 Antagonism Alter the Therapeutic Effects of VEGFA Pathway Blockage in a Mouse Model of Choroidal Neovascularization

doi: 10.3390/cells9092057

Figure Lengend Snippet: Primary human retinal endothelial cells (hREC) can be used to model endothelial-to-mesenchymal transition (EndoMT) in vitro. ( a ) Expression levels for the genes encoding the EndoMT-associated proteins SNAI1, SNAI2, α-SMA, FSP-1, vimentin, fibronectin, COL1A2, and COL3A1; and the endothelial differentiation markers VE-cadherin, CD31 and vascular endothelial growth factor receptor-2 (VEGFR2), are shown relative to the housekeeping gene HPRT1 and normalized to the control hREC as assessed by qPCR on day 6 of EndoMT induction. Data = mean ± SEM, ** p < 0.01, *** p < 0.001 compared to the control hREC by unpaired 2-tail t-test, n = 4 (VE-cadherin), 6 (collagen type III alpha 1 chain (COL3A1)), 8 (SNAI1, α-smooth muscle actin (α-SMA), fibroblast-specific protein 1 (FSP-1), CD31), 9 (vimentin), 10 (fibronectin, VEGFR2), and 11 (SNAI2, COL3A1) independent wells per group. ( b ) Histological analysis of EndoMT cells on day 6 of EndoMT induction. Phase contract microscopy and phalloidin staining (green) in the top four panels on the left illustrate the distinctions in cellular morphology between hREC and EndoMT cells. Alterations in expression and localization of endothelial differentiation markers CD31 and VE-cadherin and the mesenchymal markers vimentin, α-SMA, fibronectin and SNAI1 between hREC and EndoMT cells are also shown. Note the nuclear localization of SNAI1 (green, arrows) in EndoMT cells whereas the control hREC do not display SNAI1 expression. Isotype-matched IgG staining controls are shown on the bottom rows. Scale bars = 100 µm.

Article Snippet: Diluted DMSO vehicle control, 5 nmoles of the p38MAPK inhibitor SB203580 (Abcam), 2.5 or 5.0 μg of anti-VEGFR2 neutralizing antibody (MAB4431; R&D Systems, Minneapolis, MN, USA), or a combination of SB203580 plus anti-VEGFR2 neutralizing antibody was injected into both eyes.

Techniques: In Vitro, Expressing, Control, Microscopy, Staining

Journal: Cells

Article Title: Fibrotic Changes and Endothelial-to-Mesenchymal Transition Promoted by VEGFR2 Antagonism Alter the Therapeutic Effects of VEGFA Pathway Blockage in a Mouse Model of Choroidal Neovascularization

doi: 10.3390/cells9092057

Figure Lengend Snippet: Treatment with VEGFA at a high concentration significantly reduces expression of genes associated with EndoMT and restores gene expression associated with endothelial cell differentiation. After EndoMT induction using IL-1β (0.1 ng/mL), TNF-α (5.0 ng/mL), and TGF-β2 (5.0 ng/mL), EndoMT cells were cultured for seven days in medium containing 5% FBS and 1% penicillin/streptomycin in the presence or absence of VEGFA165 (50 ng/mL). Expression levels for the genes encoding the EndoMT-associated proteins SNAI2, FSP-1, vimentin, and COL3A1, as well as the endothelial differentiation markers CD31 and VEGFR2, are normalized to the housekeeping gene HPRT1 and shown relative to the hREC control as assessed by qPCR. ( n = 3 independent wells per group). Data = mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, 2-tail unpaired t-test.

Article Snippet: Diluted DMSO vehicle control, 5 nmoles of the p38MAPK inhibitor SB203580 (Abcam), 2.5 or 5.0 μg of anti-VEGFR2 neutralizing antibody (MAB4431; R&D Systems, Minneapolis, MN, USA), or a combination of SB203580 plus anti-VEGFR2 neutralizing antibody was injected into both eyes.

Techniques: Concentration Assay, Expressing, Gene Expression, Cell Differentiation, Cell Culture, Control

Journal: Cells

Article Title: Fibrotic Changes and Endothelial-to-Mesenchymal Transition Promoted by VEGFR2 Antagonism Alter the Therapeutic Effects of VEGFA Pathway Blockage in a Mouse Model of Choroidal Neovascularization

doi: 10.3390/cells9092057

Figure Lengend Snippet: Phospho-p38 MAPK inhibitors prevent and reverse EndoMT-associated gene expression in vitro. ( a ) To study possible prevention of EndoMT, hREC were treated with the p38 MAPK inhibitor SB203580 or SB202190 (10 μM) during EndoMT induction. Expression levels for the genes encoding the EndoMT-associated proteins SNAI1, SNAI2, α-SMA, FSP-1, vimentin, fibronectin, COL1A2 and COL3A1; and the endothelial differentiation markers VE-cadherin, CD31 and VEGFR2 were assessed by qPCR on day 6 of EndoMT induction. Data = mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001 by 2-tail unpaired t-test relative to the untreated EndoMT controls, n = 7 (COL3A1, VE-cadherin), 9 (α-SMA, FSP-1, vimentin), 10 (SNAI1, SNAI2, fibronectin, COL1A2, CD31), 12 (VEGFR2) independent wells. ( b ) To study reversal of EndoMT, EndoMT cells were treated with SB203580 (10 μM) for 9 days after EndoMT induction. Expression levels for the genes encoding EndoMT-associated proteins, and endothelial differentiation markers were assessed by qPCR after 9 days of treatment. Data = mean ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001 by 2-tail unpaired t-test, relative to the untreated EndoMT controls; n = 3 (COL1A2) and n = 6 (the rest of the genes) independent wells.

Article Snippet: Diluted DMSO vehicle control, 5 nmoles of the p38MAPK inhibitor SB203580 (Abcam), 2.5 or 5.0 μg of anti-VEGFR2 neutralizing antibody (MAB4431; R&D Systems, Minneapolis, MN, USA), or a combination of SB203580 plus anti-VEGFR2 neutralizing antibody was injected into both eyes.

Techniques: Gene Expression, In Vitro, Expressing

Journal: Cells

Article Title: Fibrotic Changes and Endothelial-to-Mesenchymal Transition Promoted by VEGFR2 Antagonism Alter the Therapeutic Effects of VEGFA Pathway Blockage in a Mouse Model of Choroidal Neovascularization

doi: 10.3390/cells9092057

Figure Lengend Snippet: Inhibition of VEGFR2 as a solo treatment promotes EndoMT, whereas anti-VEGFR2 therapy in combination with inhibition of EndoMT/fibrosis reduces CNV area and CNV number in a mouse model of wet AMD. ( a ) Eyes of P24 JR5558 mice were treated with a single intravitreal injection of vehicle, SB203580 (10 nmoles), anti-VEGFR2 neutralizing antibody (5 μg), or a combination of SB203580 and anti-VEGFR2 antibody. RPE–choroid eyecups with retina removed were harvested seven days post-injection, prepared as flat-mounts, and immunostained for the EndoMT marker vimentin (white) in addition to the vascular marker IB4 (green) in the CNV (left panels). Luminosity of the vimentin staining was quantified and normalized to CNV lesion area (right panel). ( b ) Eyes of P24 JR5558 mice were treated with a single intravitreal injection of vehicle, SB203580 (10 nmoles), anti-VEGFR2 neutralizing antibody (2.5 μg), or a combination of SB203580 and anti-VEGFR2 antibody. RPE–choroid eyecups were harvested seven days post-injection, prepared as flat-mounts, and immunostained for isolectin B4 (IB4, green) to visualize the vasculature in the CNV (top panels). Micrographs of the IB4-stained eyecups were used to quantify total CNV area per eye and the number of CNV lesions per eye (bottom panels). For micrographs in A, scale bars = 100 μm. For graphs, data = mean ± SEM. # p < 0.05, ### p < 0.001 by one-way ANOVA with Bunnett’s multiple comparison test compared to the vehicle control. * p < 0.05, ** p < 0.01, *** p < 0.001 by 2-tail unpaired t -test. n = 240 (Vehicle), 95 (SB), 83 (Anti-VEGFR2), and 186 (SB+Anti-VEGFR2) individual CNV per group in (a), and 28 (Vehicle), 16 (SB), 11 (Anti-VEGFR2), and 10 (SB+Anti-VEGFR2) individual eyes per group in ( b ). All dosing in animals was performed in a masked fashion and all image analysis was performed by a trained investigator who was masked to the identity of the treatment groups.

Article Snippet: Diluted DMSO vehicle control, 5 nmoles of the p38MAPK inhibitor SB203580 (Abcam), 2.5 or 5.0 μg of anti-VEGFR2 neutralizing antibody (MAB4431; R&D Systems, Minneapolis, MN, USA), or a combination of SB203580 plus anti-VEGFR2 neutralizing antibody was injected into both eyes.

Techniques: Inhibition, Injection, Marker, Staining, Comparison, Control

Journal: bioRxiv

Article Title: Inhibition of peripheral VEGF signaling rapidly reduces leucocyte obstructions in brain capillaries and increases cortical blood flow in an Alzheimer’s disease mouse model

doi: 10.1101/2021.03.05.433976

Figure Lengend Snippet: ELISA measurements of VEGF1R receptor ( A ), VEGF2R receptor ( B ) from brain lysates, and soluble VEGF1R receptor ( C ), and soluble VEGF2R receptor ( D ) from plasma samples after 1 week of anti-VEGF-A treatment or saline injections in APP/PS1 and WT mice (n=3 mice per group). Kruskal-Wallis test with multiple comparison correction to compare across groups. In all graphs each point represents the ELISA measurement from one mouse, red horizontal line represents the median; * indicates p = 0.05.

Article Snippet: After the measuring the concentrations ELISAs were performed following the manufacture’s protocol (VEGF1R (MVR100) and VEGF2R (MVR200B), R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Saline, Comparison

Journal: Journal for Immunotherapy of Cancer

Article Title: Designed ankyrin repeat proteins are effective targeting elements for chimeric antigen receptors

doi: 10.1186/s40425-015-0099-4

Figure Lengend Snippet: DARPin28z induces murine CAR-T cell cytokine production upon HER2 stimulation. a 10 6 scFv28z, DARPin28z, or CAR –’ve transduced murine T cells were stimulated with HER2 (HER2Fc fusion protein) or an unrelated target (KDRFc fusion protein) for four hours at 37 °C in a 96-well plate. Production of IFN-γ and TNF-α was measured by intracellular cytokine staining (ICS) and subsequent flow cytometry. Data from CD8 + Thy1.1 + T cells is presented as mean of n = 3 experiments (CAR –’ve) and n = 5 experiments (scFv28z and DARPin28z) ± SD. Bracketed numbers are quantitative of the representative plots shown. b Visual comparison of cytokine production data from a. c Cytokine expression data expressed relative to CAR-positivity of transduced cells where CAR + transformant = % CAR + /% Thy1.1 + . Error bars = SD. * = p < 0.05 ** = p < 0.005 *** = p < 0.001

Article Snippet: 10 6 murine or human CAR-T cells were stimulated in round bottom tissue culture treated 96-well plates coated with 200 ng HER2Fc chimeric protein (Cat No. 1129-ER-050, R&D Systems) or 200 ng KDRFc chimeric protein (Cat No. 443-KD, R&D Systems) for 4 h at 37 °C.

Techniques: Staining, Flow Cytometry, Comparison, Expressing

Journal: Journal for Immunotherapy of Cancer

Article Title: Designed ankyrin repeat proteins are effective targeting elements for chimeric antigen receptors

doi: 10.1186/s40425-015-0099-4

Figure Lengend Snippet: DARPin28z induces human CAR-T cell cytokine production upon HER2 stimulation. 10 6 scFv28z, DARPin28z, or CAR –’ve transduced human T cells were stimulated with HER2 (HER2Fc fusion protein) or an unrelated target (KDRFc fusion protein) for four hours at 37 °C in a 96-well plate. a Production of IFN-γ and TNF-α was measured by ICS and subsequent flow cytometry. Data from CD8 + NGFR + T cells is presented as mean ± SD. Bracketed numbers are quantitative of the representative plots shown. b Production of IL-2 by CD8 + NGFR + T cells as measured by ICS. c Production of CD107a by CD8 + NGFR + T cells as measured by ICS. d Pie graphs capturing the distribution of single and multi-functional CAR-T cells as produced with SPICE software. Pie arcs indicate functional populations represented by pie wedges. Error bars = SD. * = p < 0.05 ** = p < 0.005 *** = p < 0.001. All data from n = 3 experiments repeated with T cells from two donors

Article Snippet: 10 6 murine or human CAR-T cells were stimulated in round bottom tissue culture treated 96-well plates coated with 200 ng HER2Fc chimeric protein (Cat No. 1129-ER-050, R&D Systems) or 200 ng KDRFc chimeric protein (Cat No. 443-KD, R&D Systems) for 4 h at 37 °C.

Techniques: Flow Cytometry, Functional Assay, Produced, Software

Journal: Frontiers in Cell and Developmental Biology

Article Title: Tissue-resident macrophages co-develop with myocardial tissue in human induced pluripotent stem cell-derived organoids

doi: 10.3389/fcell.2025.1629988

Figure Lengend Snippet: Cardiac Organoids. (A) Schematic representation of the cardiac organoid differentiation protocol displaying experimental timing, cell culture media as well as small molecules and cytokines used to induce cardiac tissue. The image was created in BioRender (Wörsdörfer, P. (2025) https://BioRender.com/rz8x29g ). (B) Semiquantitative RT-PCR detecting mesodermal ( TBXT , KDR , GATA4 , RUNX1 ), cardiac ( NKX2.5 , TBX5 , ISL1 , TNNT2 , DES , RYR2 ), and endothelial ( PECAM1 ) marker gene expression at days 2, 4 and 8 of differentiation. (C) H&E staining of an organoid section at day 2. (D,D′) Immunofluorescence analysis of the organoid shown in C detecting early mesodermal markers TBXT and VIM at day 2 of organoid development. (E) Immunofluorescence analysis of the organoid shown in C detecting early mesodermal markers GATA6 and KDR at day 2 of organoid development. (F) H&E staining showing hollow cavity-like structures in cardiac organoids at day 7. (G,G′) Immunofluorescence analysis of the organoid shown in F detecting mesenchymal cells (VIM + ) in close association with cardiomyocytes (TNNT2 + ) at day 7 of organoid development. (H) H&E staining of organoid sections at day 35 of development. (I) Immunofluorescence analysis of the organoid shown in H showing close interaction of fibroblasts (VIM + ) and cardiomyocytes (TNNT2 + ) at day 35 of organoid development. (J) Cardiac organoids progressively increase in size (day 0 = 712.2 ± 6.8 µm, n = 72; day 7 = 1,231 ± 20.3 µm, n = 70; day 25 = 1,613 ± 45.4 µm, n = 41. ***p < 0.001, ****p < 0.0001. (K) Contraction frequency of organoids increases over time, from 25 ± 1.9 bpm at day 7–57 ± 10.5 bpm at day 35 (n = 8). **p < 0.01, ****p < 0.0001.

Article Snippet: Primary antibodies against PECAM1 (CD31) (DAKO, M0823), VIM (Invitrogen, MA5-16409), ACTN1 (Abcam, Ab68167), KDR (Miltenyi Biotec, 130-125-988), TBXT (R&D Systems, AF2085), TNNT2 (Life Technologies, MA512960 ), CD34 (Life Technologies, MA5-32059), AIF1 (WAKO/Fuji Film, 019-19741) and PTPRC (CD45) (Life Technologies, 14-0451-82) were diluted in NBS blocking solution and incubated overnight at 4 °C.

Techniques: Cell Culture, Reverse Transcription Polymerase Chain Reaction, Marker, Gene Expression, Staining, Immunofluorescence