Journal: PLoS ONE

Article Title: Inhibition of VEGF-C Modulates Distal Lymphatic Remodeling and Secondary Metastasis

doi: 10.1371/journal.pone.0068755

Figure Lengend Snippet: ( A ) Schematic (left) and near-infrared fluorescence image (right) showing injection sites of fluorophore and lymphatic drainage pathway to Inguinal (Ing) and Axillary (Ax) lymph nodes (LNs). (□) denotes the region imaged to capture dynamic lymph transport. ( ○ ) represents the region of interest (ROI) where fluorescence intensity is quantified to detect lymph pulsatile movement. ( B ) Time series illustrating a bolus of probe-laden lymph being propelled through the lymphatic vessel. ( C ) Normalized fluorescence intensity plotted as a function of time within a ROI measured on three consecutive weeks. Pulsatile events are detected as peaks in the trace. ( D ) Pulsatile frequency per animal and as the mean across animals per week for three consecutive weeks: week 1, 4.78±0.13; week 2, 4.97±0.10; week 3, 4.85±0.13 pulsations/minute ± SEM, n = 12. Scale bar equals 1 cm in A and 1 mm in B .

Article Snippet: When visualized by whole animal near-infrared fluorescence imaging (Kodak 400 FX Pro, 630 nm excitation, 700 nm emission, 1 sec exposure, 2× binning), fluorescent signal is detected at the injection site and within lymph vessels associated with the inguinal to axillary drainage path.

Techniques: Fluorescence, Injection

Journal: PLoS ONE

Article Title: Inhibition of VEGF-C Modulates Distal Lymphatic Remodeling and Secondary Metastasis

doi: 10.1371/journal.pone.0068755

Figure Lengend Snippet: (A) Average volume of C6 tail xenograft tumors at indicated times post-implantation. Bar graph represents mean ± SEM, n = 6 animals. (B) Near-infrared fluorescence image following intratumoral injection of probe confirming tumor lymph drainage through the inguinal lymph node (Ing LN). Scale bar equals 1 cm. (C) Representative traces of normalized fluorescence intensity measured from labeled Ing to Ax lymph vessels in tumor-bearing animals at indicated weeks post-implantation. (D) Pulsatile frequency per animal and as the mean across animals per week after tumor implantation: week 1, 4.77±0.09; week 2, 5.60±0.27; week 3, 7.13±0.30 pulsations/minute ± SEM, n = 6 animals. (E) Relative individual tumor volume per week plotted as a function of pulsatile frequency. Relative tumor volumes were calculated by dividing individual tumor volumes by the mean tumor volume for the corresponding week. (F) Pulsatile frequency in naïve versus 66c14 tail xenograft tumor bearing animals five weeks post implantation: 4.84±0.17 versus 7.04±0.17 pulsations/minute, n = 5 animals per group, bar graph represents mean ± SEM.

Article Snippet: When visualized by whole animal near-infrared fluorescence imaging (Kodak 400 FX Pro, 630 nm excitation, 700 nm emission, 1 sec exposure, 2× binning), fluorescent signal is detected at the injection site and within lymph vessels associated with the inguinal to axillary drainage path.

Techniques: Fluorescence, Injection, Labeling, Tumor Implantation

Journal: PLoS ONE

Article Title: Inhibition of VEGF-C Modulates Distal Lymphatic Remodeling and Secondary Metastasis

doi: 10.1371/journal.pone.0068755

Figure Lengend Snippet: (A) Representative traces of normalized fluorescence intensity measured from labeled inguinal to axillary lymph vessels in naïve and C6 tumor-bearing animals three weeks post xenograph tumor implantation at indicated locations. (B) Average pulsation frequency in naïve versus tumor location: naïve; 4.75±0.17; tail, 7.08±0.24; back, 7.24±0.31; ear 4.96±0.31 pulsations/minute. Bar graphs represent mean ± SEM, n = 4 and 5 animals per group for naïve and tumor-bearing groups, respectively.

Article Snippet: When visualized by whole animal near-infrared fluorescence imaging (Kodak 400 FX Pro, 630 nm excitation, 700 nm emission, 1 sec exposure, 2× binning), fluorescent signal is detected at the injection site and within lymph vessels associated with the inguinal to axillary drainage path.

Techniques: Fluorescence, Labeling, Tumor Implantation

Journal: PLoS ONE

Article Title: Inhibition of VEGF-C Modulates Distal Lymphatic Remodeling and Secondary Metastasis

doi: 10.1371/journal.pone.0068755

Figure Lengend Snippet: (A) Representative normalized fluorescence intensity traces collected from the Ing to Ax lymph vessel in naïve or C6 tail xenograft tumor-bearing animals three weeks post tumor implantation. Treated animals were dosed weekly with control or function blocking antibodies against NRP2, VEGF-C or VEGF-A starting two days after xenograft implantation. (B) Pulsatile frequency per tumor and treatment condition: naïve, 4.68±0.10; control IgG, 7.28±0.19; anti-NRP2 B , 5.28±0.49; anti-VEGF-C (VC1.12), 3.72±0.57; anti-VEGF-A, 4.08±0.34 pulsations/minute, n = 5 per group. (C) Average pulsatile frequency in naïve animals treated with anti-NRP2 B or anti-VEGF-C (VC4.5), n = 4 and 6 animals, respectively. Animals were dosed and imaged weekly for three weeks with the first imaging session starting five days after the first dose. Dashed line represents the average pulsatile frequency, 4.64 pulsations/minute, observed in naïve animals across multiple experiments (n = 30 animals, 6 experiments). (D) Average tail xenograft tumor volume three weeks after implantation in animals dosed once a week for three weeks with indicated antibody: control IgG, 263.30±9.90; anti-NRP2 B , 255.59±14.89; anti-VEGF-C (VC1.12), 267.78±11.75; anti-VEGF-A, 205.49±7.01 mm 3 , n = 5 per group. Bar graphs represent mean ± SEM.

Article Snippet: When visualized by whole animal near-infrared fluorescence imaging (Kodak 400 FX Pro, 630 nm excitation, 700 nm emission, 1 sec exposure, 2× binning), fluorescent signal is detected at the injection site and within lymph vessels associated with the inguinal to axillary drainage path.

Techniques: Fluorescence, Tumor Implantation, Control, Blocking Assay, Imaging

Journal: PLoS ONE

Article Title: Inhibition of VEGF-C Modulates Distal Lymphatic Remodeling and Secondary Metastasis

doi: 10.1371/journal.pone.0068755

Figure Lengend Snippet: (A) Representative near-infrared fluorescence images of C6 tail xenograft tumors from indicated groups imaged 24 hours post 100 µL I.V. injection of AngioSense680. Animals were dosed weekly with indicated antibody starting two days post xenograft implantation, then imaged three weeks post implantation. Scale bar equals 5 mm. (B) Average fluorescence intensities within xenograft tumors across treatment conditions: control IgG, 4,746.75±388.66; anti-VEGF-C (VC4.5), 4,888.58±282.69; Anti-VEGF-A, 3,005.78±193.09. Bar graphs represent mean ± SEM, n = 6 animals per group.

Article Snippet: When visualized by whole animal near-infrared fluorescence imaging (Kodak 400 FX Pro, 630 nm excitation, 700 nm emission, 1 sec exposure, 2× binning), fluorescent signal is detected at the injection site and within lymph vessels associated with the inguinal to axillary drainage path.

Techniques: Fluorescence, Injection, Control

Journal: PLoS ONE

Article Title: Inhibition of VEGF-C Modulates Distal Lymphatic Remodeling and Secondary Metastasis

doi: 10.1371/journal.pone.0068755

Figure Lengend Snippet: (A) Representative near-infrared fluorescence images of inguinal lymph nodes (Ing LN) at indicated times post initiation of constant (5 µL/min for 15 minutes) intradermal infusion of fluorescent probe. Naïve or C6 tail xenograft tumor-bearing animals were dosed weekly for three weeks with either control IgG or anti-VEGF-C (VC4.5) antibodies starting two days after xenograft implantation. Scale bar equals 5 mm. (B) Quantification of fluorescence intensities within a region of interest containing the inguinal LN reveals that the average time to peak in fluorescence intensity in tumor-bearing animals is roughly half of that required in naïve animals. Anti-VEGF-C dosing reduced the rate of probe accumulation in tumor-bearing animals. Thin lines represent time series data from individual animals, dashed line represents average across the respective group, n = 6 animals per group.

Article Snippet: When visualized by whole animal near-infrared fluorescence imaging (Kodak 400 FX Pro, 630 nm excitation, 700 nm emission, 1 sec exposure, 2× binning), fluorescent signal is detected at the injection site and within lymph vessels associated with the inguinal to axillary drainage path.

Techniques: Fluorescence, Control

Journal: PLoS ONE

Article Title: Inhibition of VEGF-C Modulates Distal Lymphatic Remodeling and Secondary Metastasis

doi: 10.1371/journal.pone.0068755

Figure Lengend Snippet: (A) Representative epi-fluorescence image (left) of an Ing to Ax lymphatic vessel labeled via intradermal injection of FITC-Lectin. Right, mean valve density ± SEM per C6 tail xenograft tumor and treatment condition: naïve, 0.259±0.013; control IgG, 0.264±0.020; anti-NRP2 B , 0.286±0.030; anti-VEGF-C (VC1.12), 0.247±0.018; anti-VEGF-A, 0.260±0.021 valves/100 µm vessel length, n = 5 animals per group. (B) Representative micrographs of αSMA-positive LSMCs along the Ing to Ax vessel. (C) Left, mean Ing to Ax lymph vessel diameter ± SEM measured mid-point between valves: naïve, 125.0±10.5; control IgG, 178.3±8.1; anti-NRP2 B , 142.1±6.9; anti-VEGF-C (VC1.12), 148.2±7.5; anti-VEGF-A, 149.4±7.6 µm, n = 14, 12, 13, 12, 14 animals per group, respectively. Right, mean LSMC density ± SEM estimated between lymph valves along the Ing to Ax vessel per condition: naïve, 7.20±0.34; control IgG, 8.41±0.43; anti-NRP2 B , 6.95±0.18; anti-VEGF-C (VC1.12), 6.63±0.51; anti-VEGF-A, 8.71±0.44 LSMC/100 µm vessel, n = 7, 5, 6, 8, 7 animals per group, respectively. (D) Model of tumor-associated structural remodeling of distal lymphatics. Scale bar equals 100 µm in A and B. For indicated treatment conditions, tumor-bearing animals were dosed weekly for three weeks starting two days after xenograft implantation.

Article Snippet: When visualized by whole animal near-infrared fluorescence imaging (Kodak 400 FX Pro, 630 nm excitation, 700 nm emission, 1 sec exposure, 2× binning), fluorescent signal is detected at the injection site and within lymph vessels associated with the inguinal to axillary drainage path.

Techniques: Fluorescence, Labeling, Injection, Control