Correlation between morphological parameters and IBA-1 expression.

<p>Correlation plots between IBA-1 expression and CD68 expression or the various morphometric parameters, under control and 4 mg/kg LPS conditions (n = 7 and 5 animals/group, respectively). Cell populations from individual mice are plotted in different symbols. The numbers indicates the mean linear correlation coefficient (R²) value per comparison derived from linear fits to each animal dataset within a condition.</p

Longitudinal assessment of microglia activation <i>in vivo</i> by tracking changes in soma size.

<p>(<b>A</b>) Quantification of soma size, IBA-1 and CD68 expression relative to control conditions 24 or 48 hours post LPS injection. Asterisks indicate significance for each condition compared to control (0 mg/kg LPS, 24 h), horizontal line represents statistical comparisons between indicated time points, n = 5, 6, 6, 6 and 6 animals/group, respectively. (<b>B</b> and <b>C</b>) Top: representative images of microglia (green) and blood vessels (red) imaged <i>in vivo</i> at indicated time points. Bottom: Outlines of segmented microglia from the corresponding image above color-coded to indicate soma size per cell under control (<b>B</b>) and LPS (2 mg/kg) (<b>C</b>) conditions; red corresponds to microglia with soma size >65 µm², thus activated. (<b>D</b>) Quantification of soma size measured <i>in vivo</i> as a function of time and LPS dose. Data from one control animal, and one LPS stimulated animal, are shown. Scale bar equals 50 µm.</p

VEGF-C signaling promotes structural remodeling of established lymphatic vessels in metastatic disease model.

<p>(A) Representative epi-fluorescence image (left) of an Ing to Ax lymphatic vessel labeled <i>via</i> 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.</p

Inhibiting VEGF-C, but not VEGF-A, reduces secondary metastatic spread to the lung in a model of adjuvant therapy.

<p>(A) Schematic of the adjuvant therapy model. (B) Representative images of lungs collected 14 days post primary tumor resection, circles indicate metastatic lesions. Animals were dosed weekly with the indicated antibodies starting at day 12, the day of ear resection. Anti-VEGF-C clone VC4.5 was used for two experiments, VC1.12 for the third, in all cases the results were comparable. (C and D) Quantification of the average number of secondary metastatic lesions per animal or the frequency of animals containing at least one secondary metastatic lesion, respectively, plotted per experiment normalized to IgG control (O) and as the average across experiments (+) per treatment condition. (E and F) Relative size of primary and secondary metastatic lesion size, respectively, plotted per experiment normalized to IgG control (O) and as the average across experiments (+) per treatment condition. As plots represent multiple experiments, the mean (–) and 95% confidence intervals (- -) are plotted, instead of SEM, for comparison.</p

Lymph transport is increased distal to primary tumors and prevented with antagonism VEGF-C.

<p>(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.</p

Lymph pulsatile movement increases with metastatic disease progression.

<p>(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.</p

Longitudinal measurement of lymph pulsatile movement.

<p>(<b>A</b>) 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. (<b>○</b>) represents the region of interest (ROI) where fluorescence intensity is quantified to detect lymph pulsatile movement. (<b>B</b>) Time series illustrating a bolus of probe-laden lymph being propelled through the lymphatic vessel. (<b>C</b>) 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. (<b>D</b>) 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 <b>A</b> and 1 mm in <b>B</b>.</p

Inhibition of VEGF family members prevent up-regulation of pulsatile lymph movement.

<p>(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 mm³, n = 5 per group. Bar graphs represent mean ± SEM.</p

Tumor-induced lymphatic modulation is specific to tumor-associated lymphatics.

<p>(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.</p

Intervention dosing with anti-VEGF-C antibody mitigates up-regulation of lymph transport.

<p>(A) Dosing schedule for intervention study design. Arrowheads denote the timing of dose(s) per schedule relative to C6 tail xenograft tumor implantation and imaging sessions. Imaging occurred at 1 week intervals initiated after tumor implantation, while dosing was offset +2 days relative to tumor implantation and weekly imaging, as indicated. (B) Average lymphatic pulsatile frequency and tumor volume ± SEM measured longitudinally in IgG control and anti-VEGF-C (VC4.5) treated cohorts as a function of dosing scheme, n = 5 animals per condition. (C) Average lymphatic pulsatile frequency and tumor volume ± SEM measured longitudinally in IgG control and anti-VEGF-A treated cohorts as a function of dosing scheme, n = 5 animals per condition. Asterisks indicate comparison of treatment group to IgG control at each time point.</p