vegfr  (R&D Systems)

Journal: bioRxiv

Article Title: A tumor metabolism-angiogenesis-immune axis governs immunotherapy responses

doi: 10.64898/2026.02.23.707524

Figure Lengend Snippet: ( A ) Schema of neoadjuvant anti-VEGFR2 plus anti-CTLA-4 treatment in 4T1-Sc (glycolytic) tumor-bearing mice. ( B ) Metastasis progression after surgery by whole body BLI of Fluc (n=5/group) and overall survival (n=10/group from 2 independent experiments). ( C ) Tumor weight at the time of surgery after treatment as in (A). ( D ) Vasculature leakiness by Evans blue assay in mice treated as in (A). ( E ) Flow cytometry quantification of % CD31 + ECs, angiogenic PCs, HEVs and PD-L1 expression by MFI in ECs. ( F ) Quantification of the indicated soluble angiogenic and immune factors by Luminex beads-based immunoassay in TIF from tumors treated as in (A) and ( G ) flow cytometry quantification of intratumor CD45 + leucocyte and T-cell absolute numbers per mg of tumors in the same samples. Data are mean ± SEM of n=5-10 mice/group from 2 independent experiments. 2-sided unpaired t test: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

Article Snippet: Antibody treatments were performed by intraperitoneal injection of 100 μg anti-CTLA-4 (clone 9D9 IgG2b, BioXcell), 200 μg anti-VEGFR2 (clone DC101, BioXcell), their combination, or isotype controls (clone MPC-11 and clone HRPN, respectively, BioXcell) 3 days apart for three administrations as indicated in the figures.

Techniques: Evans Blue Assay, Flow Cytometry, Expressing, Luminex

Journal: bioRxiv

Article Title: A tumor metabolism-angiogenesis-immune axis governs immunotherapy responses

doi: 10.64898/2026.02.23.707524

Figure Lengend Snippet: ( A ) WT Balb/c mice were orthotopically injected in the m.f.p. with 4T1-Sc or 4T1-KD (1×10 6 cells) and treated with neoadjuvant anti-CTLA-4 4 ± anti-VEGFR2 or isotype control as in . Primary tumors were surgically resected on day 10 and 13 for 4T1-Sc and 4T1-KD respectively to ensure similar tumor burden was reached between the two models at the time of surgery. Mice were then followed for metastases by whole body BLI, and metastasis-free survival is reported for mice enrolled in 3 independent surgery experiments (pooled n=14-20/group). ( B-D ) WT C57BL/6 mice were orthotopically injected i.d. with B16-Sc or B16-KD (0.3×10 6 cells) and treated with anti-CTLA-4 4 ± anti-VEGFR2 or isotype control as indicated by the arrows, and (B) primary tumor growth (n=5-10), (C) overall survival of combo-treated groups (n=10), and (D) circulating tumor cells at the end of treatment (n=5/each) were evaluated. ( E-K ) 4T1 tumors from mice treated as in (A) were processed for: flow cytometry quantification of frequencies of the indicated vascular cells (E), (F) expression (by MFI) of the indicated markers in ECs, and (G) frequencies of ECs expressing the indicated markers; TIF analyses by Luminex bead-based immunoassays to measure the concentration of the indicated hem/lymphangiogenic factors (H) (n=5-10/group); SCENITH analyses of ECs (I) and (J) HEVs (puromycin incorporation was used as a readout of ATP production under oligomycin treatment, where puromycin⁺ cells indicate glycolytic dependence and puromycin⁻ cells indicate mitochondrial dependence; n=8/group in 1 of 2 independent experiments); flow cytometry quantification of CD45 + leucocytes and tumor infiltrating lymphocytes (TILs: CD8 + , CD4 + Foxp3 - Teff, CD4 + Foxp3 + Tregs) per mg of tumor (K; n=5-10/group, from 1 of 3 independent experiments). ( L ) Schema of treatment in Kaede mice implanted with contralateral B16-Sc and B16-KD tumors photoconverted to measure T-cell egress to dLN depending on the glycolytic state and treatment and flow cytometry quantification of ratio photoconverted CD8 + T cells, gp100-specific CD8 + T cells and terminally exhausted CD8 + T cells (Tex term ; by surface staining of PD-1, CD69, and LY108 expression for compatibility with Kaede protein detection by surface staining) in dLN relative to tumor (TUM) in control vs. combo-treated mice (combined from 2 independent experiments with 2-3 and 4 mice/condition each, with similar results). Data are mean ± SEM. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Article Snippet: Antibody treatments were performed by intraperitoneal injection of 100 μg anti-CTLA-4 (clone 9D9 IgG2b, BioXcell), 200 μg anti-VEGFR2 (clone DC101, BioXcell), their combination, or isotype controls (clone MPC-11 and clone HRPN, respectively, BioXcell) 3 days apart for three administrations as indicated in the figures.

Techniques: Injection, Control, Flow Cytometry, Expressing, Luminex, Concentration Assay, Staining

Journal: bioRxiv

Article Title: A tumor metabolism-angiogenesis-immune axis governs immunotherapy responses

doi: 10.64898/2026.02.23.707524

Figure Lengend Snippet: 4T1-bearing mice were treated as in and ( A ) frequencies of CD8 + T cells expressing IFN-γ ± TNF, and ( B ) frequencies of antigen-experienced CD44 + CD8 + T cells expressing the exhaustion marker Tox were quantified by flow cytometry in tumors after treatment (n=5-10/group, from 1 of 3 independent experiments). ( C ) Representative flow cytometry plot of CD62L and CD44 in CD8 + TILs and quantification of CD8 + CD62L + CD44 - naïve, CD62L + CD44 + , central memory (Tcm), and CD62L - CD44 + effector (Tem) cell frequencies and related absolute numbers per mg of tumors in 4T1-Sc and 4T1-KD tumors treated with anti-CTLA-4 ± anti-VEGFR2 or isotype control as in . ( D ) Tumor growth profiles of glycolysis-high B16-Sc implanted in WT mice and treated with anti-CTLA-4 + anti-VEGFR2 (combo) alone or with a CD8 depleting (n=5/group) or a MECA-79 blocking antibody (n=5/group) in comparison with matched isotype control (n=5). Arrows indicate treatment time points. ( E-I ) Flow cytometry analyses of tumor-infiltrating CD8 + Tcm and Tem cells from 4T1-Sc and 4T1-KD tumors treated with anti-CTLA-4 ± anti-VEGFR2 or isotype control as in : (E) quantification of granzyme B (GzmB) + cell frequencies in CD8 + Tcm and Tem; (F) representative flow plots and quantification of CD8 + Tcm and Tem cell fractions expressing VEGFR2 ± CTLA-4; (G) quantification of GzmB + cell frequencies within VEGFR2 + CTLA-4 - CD8 + Tcm and Tem cell subsets and (I) representative flow plots of GzmB expression in VEGFR2 ± CTLA-4-expressing CD8 + Tcm subsets upon IgG control or combo treatment in 4T1-Sc and 4T1-KD tumors; ( I ) SCENITH analyses of CD8 + Tcm (left) and Tem (right) (puromycin incorporation was used as a readout of ATP production under oligomycin treatment, where puromycin⁺ cells indicate glycolytic dependence and puromycin⁻ cells indicate mitochondrial dependence). Data are mean ± SEM (n=5-10/group in one of 2-3 independent experiments). *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

Article Snippet: Antibody treatments were performed by intraperitoneal injection of 100 μg anti-CTLA-4 (clone 9D9 IgG2b, BioXcell), 200 μg anti-VEGFR2 (clone DC101, BioXcell), their combination, or isotype controls (clone MPC-11 and clone HRPN, respectively, BioXcell) 3 days apart for three administrations as indicated in the figures.

Techniques: Expressing, Marker, Flow Cytometry, Control, Blocking Assay, Comparison

Journal: Signal Transduction and Targeted Therapy

Article Title: Extraocular delivery of bioswitchable tri-miR-22-loaded tetrahedral DNA nanostructures for intraocular neovascular and neurodegenerative repair

doi: 10.1038/s41392-025-02566-4

Figure Lengend Snippet: Subconjunctival BiRDS suppressed retinal neovascularization and promoted healthy angiogenesis in the OIR mouse model. a Schematic description of the establishment of the OIR model and the design of the animal experiments. b Schematic representation of retinal flat-mount lesions (neovascularized and nonperfused areas) in OIR model mice and key morphological hallmarks of healthy angiogenesis (filopodia, tip cells, and stalk cells). c Upper: Retinal flat mounts after OIR and drug-treated OIR mice. Scale bar = 1 mm. Lower: higher-magnification images of pathological neovascular tufts. Scale bar = 50 μm. d Avascular area measured for quantification, as indicated by the dotted yellow lines. Scale bar = 1 mm. e Higher magnification images of pathological vessels sprouting from veins, as indicated by the dotted green line. Scale bar = 100 μm. f Upper: representative images of tip cells; the yellow arrowhead indicates tip cells. Scale bar = 50 μm. Lower: representative images of filopodia; yellow arrows indicate filopodia. Scale bar = 10 μm. g Retinal cryosections and immunofluorescence staining of drug-treated OIR mice. Scale bar = 50 μm. White: IB4 (vessels); red: CD31 (neovasculature); green: VEGFR; blue: DAPI (nuclei). h ‒ n Quantification of neovascular areas ( h ), avascular areas ( i ), sprouting areas ( j ), and tip cells ( k ) or counts of filopodia ( l ) and counts of neovascular cell nuclei anterior to the ILM ( m ) or vascular tube of the DCP ( n ). SCP, superficial capillary plexus; DCP, deep capillary plexus. Mean ± SD. n = 6. *** p < 0.001, ** p < 0.01

Article Snippet: The following antibodies were used for immunofluorescence: Isolectin GS-IB4, Alexa FluorTM 568( I21412 , Thermo Fisher Scientific, Massachusetts, USA), CD31(SC-376764, Santa Cruz Biotechnology, Dallas, TX, USA), VEGFR (#AF644; R&D Systems, Minneapolis, MN, USA), Tuj1 (#4466; Cell Signaling Technology, Danvers, MA, USA), GFAP (#12389; Cell Signaling Technology, Danvers, MA, USA), PKC-α (#sc-8393; Santa Cruz Biotechnology, Dallas, TX, USA), Rhodopsin (#ab221664; Abcam, Cambridge, UK), Calbindin (#ab82812; Abcam, Cambridge, UK), Alexa Fluor 488-labeled goat anti-rabbit IgG (#4412S; Cell Signaling Technology, Danvers, MA, USA), and Alexa Fluor 488-labeled goat anti-mouse IgG (#4408S; Cell Signaling Technology, Danvers, MA, USA), Alexa Fluor 555-conjugated goat anti-rabbit IgG (#25363S, Cell Signaling Technology, Danvers, MA, USA), Alexa Fluor 555-conjugated goat anti-mouse IgG (#37459S, Cell Signaling Technology, Danvers, MA, USA).

Techniques: Immunofluorescence, Staining

Journal: JCI Insight

Article Title: NF- κ B–driven lymphangiogenesis affects kidney function via a VEGFR-3–mediated pathway

doi: 10.1172/jci.insight.198992

Figure Lengend Snippet: ( A ) WT mice were crossbred with VEGFR-3 Cre-expressing mice to generate Flt 4Cr exRel Aflox transgenic animals. Upon successful Cre-mediated recombination, exon 1 of the RelA gene, the neomycin resistance cassette, and promoter region were excised, resulting in subsequent GFP expression. The dotted box represents the components deleted upon recombination. Black and gray triangles denote loxP and frt sites, respectively. WT and its recombined form were adapted from Heise et al. . ( B ) Visualization of renal lymph nodes (white arrow) and lymphatic vessels (yellow arrow) with associated lumbar lymph nodes (green arrow) following 5% Evan’s blue dye injection via the footpad, prior to surgical resection. ( C ) Hepatic eGFP RNA expression analysis relative to that of WT mice, presented as fold change, and normalized to Gapdh confirming successful gene knockout efficiency in tamoxifen-treated knockout mice ( N = 3) compared with tamoxifen-treated WT controls ( N = 4). ( D ) Real-time quantitative PCR validation of target gene knockout using primers spanning from exon 1 to exon 3. The observed reduction in RNA expression indicates successful exon 1 excision following Cre-mediated recombination. RelA transcript levels were normalized to Gapdh expression and are presented as fold change relative to control WT mice (WT, N = 4; KO, N = 3). ( E ) Experimental timeline depicting sequential tamoxifen and cisplatin treatments, with tissue collection occurring 3 days after high-dose cisplatin (20 mg/kg) or saline administration. The washout period was 10 to 11 days. PGK, phosphoglycerate kinase promoter; Neo pA, neomycin resistance gene-polyadenylation; WT, RelA fl/fl ; KO, VEGFR-3 RelA–/– . * P < 0.0332. C is unpaired t test (2 tailed). D is 2-way ANOVA.

Article Snippet: After a series of methanol dehydration/rehydration, delipidation with organic solvents (e.g., dichloromethane), permeabilization, blocking, and washing, kidneys were immunolabeled with LYVE-1 (1:200; Novus Biologicals, NB-600-1008) and VEGFR-3 (1:200; R&D Systems, AF743) for 96 hours at 37°C.

Techniques: Expressing, Transgenic Assay, Injection, RNA Expression, Gene Knockout, Knock-Out, Real-time Polymerase Chain Reaction, Biomarker Discovery, Control, Saline

Journal: JCI Insight

Article Title: NF- κ B–driven lymphangiogenesis affects kidney function via a VEGFR-3–mediated pathway

doi: 10.1172/jci.insight.198992

Figure Lengend Snippet: ( A ) Glomerular filtration rate (GFR) in control and cisplatin-treated knockout and WT mice (WT control, N = 4; KO control, N = 7; WT cisplatin, N = 15; KO cisplatin, N = 17). ( B ) Serum creatinine measurements in control and cisplatin-treated knockout and WT mice. Kidney function analysis included data from at least 4 independent knockout cohorts and 3 WT control cohorts (WT control, N = 13; KO control, N = 12; WT cisplatin, N = 18; KO cisplatin, N = 21). ( C ) Representative images of PAS-stained kidney sections from the cortices in knockout and WT mice. Black arrows indicate loss of brush border; white arrows indicate cortical casts. Scale bar: 100 μm. ( D ) Tubular casts, tubular necrosis, and brush border loss were scored in a blinded fashion from 0 (low injury) to 4 (high injury) using an area-based system. Sample sizes (WT control, N = 4; KO control, N = 6; WT cisplatin, N = 11; KO cisplatin, N = 10). Statistical significance ( P < 0.05) was determined using 2-way ANOVA. ** P < 0.0021, *** P < 0.002, **** P < 0.0001. WT, RelA fl/fl ; KO, VEGFR-3 RelA–/– .

Article Snippet: After a series of methanol dehydration/rehydration, delipidation with organic solvents (e.g., dichloromethane), permeabilization, blocking, and washing, kidneys were immunolabeled with LYVE-1 (1:200; Novus Biologicals, NB-600-1008) and VEGFR-3 (1:200; R&D Systems, AF743) for 96 hours at 37°C.

Techniques: Filtration, Control, Knock-Out, Staining

Journal: JCI Insight

Article Title: NF- κ B–driven lymphangiogenesis affects kidney function via a VEGFR-3–mediated pathway

doi: 10.1172/jci.insight.198992

Figure Lengend Snippet: ( A ) Flow cytometry gating strategy for analyzing CD45 + leukocyte populations in kidneys from a cisplatin-treated knockout mouse (20 mg/kg), including neutrophils, kidney-resident macrophages (KRMs), infiltrating macrophages (IMs), and inflammatory Ly6c hi IMs. ( B ) Quantification of KRMs (CD11b int , F4/80 hi ) as a percentage of total CD45 + cells in RelA-knockout and WT control mice. ( C ) Quantification of neutrophils (Ly6G + ) as a percentage of total CD45 + cells. ( D ) Quantification of IMs (CD11b hi , F4/80 int ) as a percentage of total CD45 + cells. ( E ) Quantification of inflammatory Ly6c hi IM cells as a percentage of total IMs (CD11b hi , F4/80 int ) (WT control, N = 9; KO control, N = 9; WT cisplatin, N = 10; KO cisplatin, N = 7). Cisplatin-treated mice with serum creatinine levels at least twice those of saline controls were included in this analysis. Graphs include data from 4 independent cohorts. * P < 0.0332, ** P < 0.0021, **** P < 0.0001. WT, RelA fl/fl ; KO, VEGFR-3 RelA–/– .

Article Snippet: After a series of methanol dehydration/rehydration, delipidation with organic solvents (e.g., dichloromethane), permeabilization, blocking, and washing, kidneys were immunolabeled with LYVE-1 (1:200; Novus Biologicals, NB-600-1008) and VEGFR-3 (1:200; R&D Systems, AF743) for 96 hours at 37°C.

Techniques: Flow Cytometry, Knock-Out, Control, Saline

Journal: JCI Insight

Article Title: NF- κ B–driven lymphangiogenesis affects kidney function via a VEGFR-3–mediated pathway

doi: 10.1172/jci.insight.198992

Figure Lengend Snippet: ( A ) Lymphatic associated genes’ and ( B ) NF-κB and Vcam1 genes’ expression analyses from kidney lysate, presented as fold change relative to tamoxifen+saline WT controls. Analysis included data from 4 independent knockout cohorts and 3 WT control cohorts (WT control, N = 11; KO control, N = 7; WT cisplatin, N = 9; KO cisplatin, N = 15 for Flt4, Lyve-1, Ccl21a, Prox-1 , and Pdpn ; N = 12 for Vcam1 ; N = 14 for p50, Vegfc , and p52 ). All transcript levels were normalized to Gapdh . Statistical significance ( P < 0.05) was determined using 2-way ANOVA. * P < 0.0332, ** P < 0.0021, *** P < 0.0002, **** P < 0.0001. WT, RelA fl/fl ; KO, VEGFR-3 RelA–/– .

Article Snippet: After a series of methanol dehydration/rehydration, delipidation with organic solvents (e.g., dichloromethane), permeabilization, blocking, and washing, kidneys were immunolabeled with LYVE-1 (1:200; Novus Biologicals, NB-600-1008) and VEGFR-3 (1:200; R&D Systems, AF743) for 96 hours at 37°C.

Techniques: Expressing, Saline, Knock-Out, Control

Journal: JCI Insight

Article Title: NF- κ B–driven lymphangiogenesis affects kidney function via a VEGFR-3–mediated pathway

doi: 10.1172/jci.insight.198992

Figure Lengend Snippet: ( A ) Analysis of lymphatic marker protein expression in kidney lysate by Western blotting. Protein expression levels were quantified using densitometry and normalized to either β-actin or GAPDH as loading controls. Representative blots are shown with corresponding quantification. Densitometric analysis includes data from 3 independent knockout cohorts and 3 WT cohorts (WT control, N = 11; KO control, N = 8; WT cisplatin, N = 9; KO cisplatin, N = 15). Statistical significance ( P < 0.05) was determined using 2-way ANOVA. ** P < 0.0021, *** P < 0.0002, **** P < 0.0001. ( B ) Visualization of lymphatic structures in cleared kidney tissue using confocal microscopy. Images show quarter-sectioned, optically cleared kidneys from both a WT control and knockout mouse. Lymphatic vessels are labeled with two markers: LYVE-1 (red) and VEGFR-3 (green). Images shown are maximum intensity projections that have been processed with denoising and deconvolution algorithms using NIS-Elements. Original magnification, ×10. WT, RelA fl/fl ; KO, VEGFR-3 RelA–/– .

Article Snippet: After a series of methanol dehydration/rehydration, delipidation with organic solvents (e.g., dichloromethane), permeabilization, blocking, and washing, kidneys were immunolabeled with LYVE-1 (1:200; Novus Biologicals, NB-600-1008) and VEGFR-3 (1:200; R&D Systems, AF743) for 96 hours at 37°C.

Techniques: Marker, Expressing, Western Blot, Knock-Out, Control, Confocal Microscopy, Labeling

Journal: JCI Insight

Article Title: NF- κ B–driven lymphangiogenesis affects kidney function via a VEGFR-3–mediated pathway

doi: 10.1172/jci.insight.198992

Figure Lengend Snippet: ( A ) Representative confocal microscopy images (maximum intensity projections) of quarter kidney sections immunolabeled for VEGFR-3 (green, Alexa 647) and LYVE-1 (red, Alexa 594). Images compare VEGFR-3–specific RelA-deficient mice to floxed controls under baseline conditions and after cisplatin-induced AKI. 10× objective. ( B and C ) Quantitative analysis of lymphatic vessel morphology for ( B ) VEGFR-3 + and ( C ) LYVE-1 + vessels. Measured parameters include branch point density, total filament volume (μm 3 ), and total filament length (μm). Analysis was performed using Imaris Filaments module following image processing (denoising, deconvolution, thresholding). All measurements were normalized to imaging volume based on image field dimensions: x axis (2,038 pixels), y axis (2,038 pixels), and z axis (section thickness in μm), with a pixel size of 1.24 μm/pixel (WT control, N = 3; KO control, N = 3; WT cisplatin, N = 4; KO cisplatin, N = 5). WT, RelA fl/fl ; KO, VEGFR-3 RelA–/– .

Article Snippet: After a series of methanol dehydration/rehydration, delipidation with organic solvents (e.g., dichloromethane), permeabilization, blocking, and washing, kidneys were immunolabeled with LYVE-1 (1:200; Novus Biologicals, NB-600-1008) and VEGFR-3 (1:200; R&D Systems, AF743) for 96 hours at 37°C.

Techniques: Confocal Microscopy, Immunolabeling, Imaging, Control