Validation of VDA with a human cell line.

<p>(<b>A</b>) Bioluminescence images taken just prior to DMXAA treatment, as well as at 6 and 24 hrs post treatment of NIH-III mice bearing subcutaneous tumors composed of luciferase-expressing MDA-MB-231 breast cancer cells. (<b>B</b>) Quantification of the photon flux from representative tumors demonstrates a large drop in bioluminescent signal by 6 hours. (<b>C</b>) 3-D micro-CT renderings of tumors (gray), and embedded vasculature (red), of the MDA-MB-231-Luc2 tumors. (<b>D</b>) Quantification of the binarized images demonstrating vessel volume (VV), and vessel density (VV/TV). Data represents averages ± s.e.m. (N = 3), asterisks indicate p<0.05.</p

Microfil combined with micro-CT.

<p>(<b>A</b>) Image of a Microfil-perfused tumor grown subcutaneously in a mouse (top). The Microfil polymer is yellow, allowing one to visualize the effectiveness of the perfusion. Images of a non-perfused tumor (bottom left), and of the perfused dermis without a tumor (bottom right) are included as a control. (<b>B</b>) 3-D reconstructions of the tumor in (<b>A</b>) showing a surface rendering of the vasculature (top left), the thickness of the vessels demonstrated by a heat map and the intravascular distances (top right), or vessel separation demonstrated with a sphere-filling model (bottom left – full view, bottom right – cross-section through centre of tumor). Note that the vessels are not shown in the sphere-filled images, only the inter-vessel spaces.</p

Quantification of vessel thickness.

<p>(<b>A</b>) 3-D renderings of vasculature (red  =  vessel diameters of 0.2 mm or greater) showing the organized kidney vasculature versus the disorganized, irregular vessel diameters in the NSCLC tumors (344SQ, 7417PF and H1299). (<b>B</b>) Quantification of vessel thickness of two human and two murine cell lines over the entire spectrum of vessel diameters, showing very significant differences between normal and tumor vasculature, with most of the tumors only having vessels up to 0.5 mm, while the kidneys had vessels up to 1.25 mm in diameter (note the log scale on Y axis).</p

Assessing the effects of a vascular disrupting agent.

<p>(<b>A</b>) Bioluminescence images of luciferase-expressing 344SQ-EL cell line-derived subcutaneous tumors. Images were taken prior to treatment, and again at 6 and 24 hrs post DMXAA treatment. This agent led to a dramatic loss of bioluminescence. (<b>B</b>) Quantification of changes in photon emission rates of the DMXAA-treated, and control tumors (data represent averages ± s.e.m., N = 20). (<b>C</b>) 3-D micro-CT surface renderings (gray and red) and cross-sectional maximal-spheres filling model rendering (red  =  vessel diameters of 2 mm or greater) for representative subcutaneous tumors treated with either DMXAA or vehicle control, and perfused with Microfil after 24 hrs. The images demonstrate a large increase in the necrotic regions following DMXAA treatment. (<b>D</b>) Quantification of the vessel volume, density, and separation of DMXAA-treated tumors. There was a decrease in vessel volume and density, and in well-vascularized areas, as indicated by the drop in spheres present with sizes between 0–100 µm. This result was consistent with an increase in the size of the necrotic areas in the DMXAA-treated tumors. Data indicate averages ± s.e.m., N = 6 per sample. Asterisks indicate: * p<0.05, ** p<0.01.</p

Quantitative <em>Ex-Vivo</em> Micro-Computed Tomographic Imaging of Blood Vessels and Necrotic Regions within Tumors

<div><p>Techniques for visualizing and quantifying the microvasculature of tumors are essential not only for studying angiogenic processes but also for monitoring the effects of anti-angiogenic treatments. Given the relatively limited information that can be gleaned from conventional 2-D histological analyses, there has been considerable interest in methods that enable the 3-D assessment of the vasculature. To this end, we employed a polymerizing intravascular contrast medium (Microfil) and micro-computed tomography (micro-CT) in combination with a maximal spheres direct 3-D analysis method to visualize and quantify <em>ex-vivo</em> vessel structural features, and to define regions of hypoperfusion within tumors that would be indicative of necrosis. Employing these techniques we quantified the effects of a vascular disrupting agent on the tumor vasculature. The methods described herein for quantifying whole tumor vascularity represent a significant advance in the 3-D study of tumor angiogenesis and evaluation of novel therapeutics, and will also find potential application in other fields where quantification of blood vessel structure and necrosis are important outcome parameters.</p> </div

3-D renderings and quantification of tumor vasculature.

<p>(<b>A</b>) 3-D micro-CT surface renderings of a Microfil-perfused kidney (left) and three subcutaneous tumors derived from NSCLC cell lines: 344SQ (2^nd from left), 7417PF (3^rd from left) and H1299 (right). The entire tissue or tumor mass is in gray, and the vasculature in red. Using this imaging analysis in combination with 3-D measurement techniques, voxel-counting and maximal spheres analysis, two murine NSCLC cell lines (344SQ and 7417PF), and two human cell lines (the NSCLC cell line H1299 and the breast cancer line, MDA-MB-231) were examined. For comparison purposes, the kidney, being similar in size to the subcutaneous tumors, was also examined. (<b>B</b>) Quantification yielded representative average measurements for total volume (TV), vessel volume (VV), vessel density (VV/TV), vessel number (V.N), vessel thickness (V.Th), vessel separation (V.Sp), vessel connectivity or branching (V.ConnD), and vessel surface area (V.SA). Data are shown as the average ± s.e.m. (N = 3–6 per cell line). All of the cell lines (apart from H1299) demonstrated significant variability in vessel parameters compared with the kidney. Asterisks indicate: * p<0.05, ** p<0.01, *** p<0.001.</p

Imaging and quantification of avascular necrosis.

<p>(<b>A</b>) 3-D renderings from the kidney and NSCLC tumors following application of the maximal sphere-filling model; intact sample views above and cross-sectional images below (red indicating 2 mm or greater diameter of sphere). The presence of red (avascular) areas can be seen in all the tumor cell lines tested. (<b>B</b>) Histogram quantification of the maximal-spheres of two human and two murine cell lines by evaluating the total number of spheres present at increasing ranges of diameters (from 10 µm to 5 mm) (note the log scale on Y axis). Considerable variability is evident within both the vascularised and avascular areas within the tumors, with significantly more avascular areas being present in tumors, in contrast to the well-vascularized kidneys. Data indicate averages ± s.e.m. (N = 3–6 tumors per cell line). Asterisks indicate: * p<0.05, ** p<0.01, *** p<0.001.</p