Quantified measurements of Vascular Dysfunction in tumours treated with TPZ in reduced Oxygen conditions.

<p>Please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076832#pone-0076832-g006" target="_blank">Figure 6</a> for statistical analyses. PF  =  perfused fraction; SD  =  standard deviation; VF  =  viable fraction; VDS  =  vascular dysfunction score; VDS_min  =  (mean control VDS) + (2× SD); 60TPZ  =  tirapazamine at 60 mg/kg.</p

TPZ mediates damage to endothelial tube structures in a concentration, time and oxygen dependent manner.

<p>HMECs seeded onto Matrigel-coated plates formed tubular structures by 24 h and were then treated with TPZ in (A) 2% or (B) 0.2% oxygen conditions for indicated times. Whole wells (top; scale bars 500 µm) and magnified representative areas (bottom; scale bars 150 µm) are shown.</p

Vascular function and tumour microenvironment in HCT116 and HT29 colorectal xenografts.

<p>HCT116 & HT29 tumours were harvested at 2 or 20 min after intravenous injection of 500 kDa FITC-dextran (A). Fluorescence intensity of FITC-labeled dextran is displayed as a function of distance from nearest vasculature in HCT116 (i) and HT29 (ii) xenografts. Images show FITC fluorescence (black) and CD31 stained vasculature (red). Quantitative analysis of the tumour microenvironment (B) shows incorporated BrdUrd staining as a marker for S-phase cells as whole tumour means (B, right) or as a function of distance from nearest vessel (B, middle). Tumour hypoxia is reflected by pimonidazole labeling as a function of distance from vasculature (B, left). Magnified tumour map images reflect representative staining patterns of HCT116 and HT29; staining shows unperfused vasculature (CD31, red), perfused blood vessels (CD31 overlapped with DiOC₇(3), blue), perfusion dye (DiOC₇(3), cyan), hypoxia (pimonidazole, green) and s-phase (BrdUrd, black) overlaid on hematoxylin background staining (grey). Scale bars 150 µm.</p

Hypoxia enhances anti-vascular effects of TPZ in HCT116 and HT29 xenografts.

<p>HCT116 and HT29 colorectal xenografts were treated with 60/kg TPZ in room air or low oxygen conditions. (A) VDS is reported for individual (i) HCT116 and (ii) HT29 colorectal xenografts (bars) from control and treatment groups in indicated oxygen conditions; horizontal lines represent group means. (B) Staining data shows an increase in hypoxia, via pimonidazole labeling, in low oxygen conditions. (C) Tumour maps of HT29 colorectal xenografts show staining for unperfused vasculature (CD31, red), perfused vasculature (DiOC7 (3) +ve CD31, blue), S-phase cells (BrdUrd, black) and hypoxia (pimonidazole, green). Tumours representative of central vascular dysfunction effects observed within indicated treatment groups are shown for each group; scale bars 150 µm; (*p<0.05) (**p<0.01) (***p<0.001).</p

Quantified measurements of Vascular Dysfunction in tumours treated with TPZ and NOS inhibitor L-NNA.

<p>Please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076832#pone-0076832-g004" target="_blank">Figures 4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076832#pone-0076832-g005" target="_blank">5</a> for statistical analyses. PF  =  perfused fraction; SD  =  standard deviation; VF  =  viable fraction; VDS  =  vascular dysfunction score; VDS_min  =  (mean control VDS) + (2× SD); 60TPZ  =  tirapazamine at 60 mg/kg; 40TPZ  =  tirapazamine at 40 mg/kg; 180L-NNA  = l-nitro-l-arginine at 180 mg/kg.</p

Vascular architecture of HCT116 and HT29 colorectal xenografts.

<p>Differences are observed between HCT116 and HT29 colorectal xenografts for vessel density (A, left) and perfused fraction (A, right). The amounts of smooth muscle (SMA, B) and basal lamina (CIV, C) are assessed as the fraction of vessels dual labeled for the marker as well as the amount of staining as a function of distance from nearest vessels. Nitric Oxide Synthase (NOS) staining (D) is shown as the average amount of universal NOS (uNOS, grey) in all viable tissue or as the fraction of vessels (CD31 alone, red; overlapped with eNOS, black) dual labeled for endothelial NOS (eNOS) (overlapped with CD31, black). Whole tumour images and higher resolution inserts illustrate the micro-regional heterogeneity in HCT116 & HT29 tumours; scale bars 150 µm; (**p<0.01).</p

NOS inhibition enhances anti-vascular effects of TPZ in HCT116 xenografts.

<p>HCT116 colorectal xenografts were treated with TPZ at 40/kg or 60 mg/kg alone or in combination with 180 mg/kg NOS inhibitor L-NNA. (A) VDS is reported for individual tumours (bars); horizontal lines represent group means. (B) graph shows staining intensity of pimonidazole as a function of distance from vasculature, showing micro regional differences in hypoxia for control and L-NNA treated tumours, with no significant differences. (C) Tumour growth rate data for control and single or combination treated groups; each point represents mean ± SE. (D) Tumour maps show staining for unperfused vasculature (CD31, red), perfused vasculature (DiOC7 (3)+ve CD31, blue), S-phase cells (BrdUrd, black) and hypoxia (pimonidazole, green). Tumours representative of central vascular dysfunction effects observed within indicated treatment groups are shown for each group; (*p<0.05) (**p<0.01); scale bars 150 µm.</p

Melanoma growth is reduced in <i>Cd34^−/−</i> animals at day 14, with CD34 expression on vasculature.

<p>A, Average tumor mass 14 days after subcutaneous injection of 5×10⁵ B16F1-OVA cells. (Pooled from three experiments, <i>Cd34^+/+</i> n = 12; <i>Cd34^−/−</i> n = 15). B, Average number of lung metastases 12 days after intravenous injection of 3×10⁵ B16F1-OVA cells. (<i>Cd34^+/+</i> n = 5, <i>Cd34^−/−</i> n = 6, *represents p<0.05; Error bars = SEM). C, Representative photomicrographs from s.c.-injected tumor sections stained for CD34 and CD31 or carbocyanine fluorescence, as indicated (CD31-red, CD34-blue, carbocyanine-green).</p

Reduced subcutaneous tumor size at day 14 correlates with loss of CD34 on non-hematopoietic cells.

<p>A, Average tumor mass and B, volume at day 14 in <i>Cd34^+/+</i> or <i>Cd34^−/−</i> animals reconstituted with wildtype Ly5.1 bone marrow. (Pooled from two experiments, <i>Cd34^+/+</i> n = 13, <i>Cd34^−/−</i> n = 14). C, Average tumor mass at day 14, in reciprocal reconstitutions of wildtype Ly5.1 animals with <i>Cd34^+/+</i> or <i>Cd34^−/−</i> bone marrow (n = 9, *represents p<0.05; Error bars = SEM).</p

Increased carbocyanine leakage and altered vessel morphology in subcutaneously-injected tumors from <i>Cd34^−/−</i> animals.

<p>Carbocyanine fluorescence intensity quantified A, proximal to CD31⁺ vessels (at distance 0 µm) and B, with increasing distance from the nearest vessel (CD31⁺), in day 14 tumors. C, Average distance of each pixel within the tumor tissue from the nearest CD31⁺ staining pixel. D, Perfused vessels (%), corresponding to the percentage of CD31⁺ pixels colocalized with carbocyanine staining. For carbocyanine data, one outlier per genotype was removed prior to analysis. (n = 5 for carbocyanine, n = 6 for average distance, *represents p<0.05; **represents p<0.01; Error bars = SEM). Representative CT images of tumors excised 14 days post-injection and perfused with Microfil contrast reagent from E, <i>Cd34^+/+</i> and F, <i>Cd34^−/−</i> mice. Scale bars, 2 mm (n = 3).</p