An in vivo role for Rho kinase activation in the tumour vascular disrupting activity of combretastatin A-4 3-O-phosphate.

BACKGROUND AND PURPOSE: Combretastatin A-4 3-O-phosphate (CA4P) is in clinical trial as a tumour vascular disrupting agent (VDA) but the cause of blood flow disruption is unclear. We tested the hypothesis that activation of Rho/Rho kinase (ROCK) is fundamental to the effects of this drug in vivo. EXPERIMENTAL APPROACH: Mouse models of human colorectal carcinoma (SW1222 and LS174T) were used. Effects of the ROCK inhibitor, Y27632, alone or in combination with CA4P, on ROCK activity, vascular function, necrosis and immune cell infiltration in solid tumours were determined. Mean arterial BP (MABP) was measured to monitor systemic interactions and the vasodilator, hydralazine, was used to control for the hypotensive effects of Y27632. KEY RESULTS: Y27632 caused a rapid drop in blood flow in SW1222 tumours, with recovery by around 3 h, which was paralleled by MABP changes. Y27632 pretreatment reduced CA4P-induced ROCK activation and partially blocked CA4P-induced tumour vascular effects, in both tumour types. Y27632 also partially inhibited CA4P-induced tumour necrosis and was associated with reduced immune cell infiltration in SW1222 tumours. Hydralazine caused a similar hypotensive effect as Y27632 but had no protective effect against CA4P treatment. CONCLUSIONS AND IMPLICATIONS: These results demonstrate that ROCK activity is critical for full manifestation of the vascular activity of CA4P in vivo, providing the evidence for pharmacological intervention to enhance the anti-tumour efficacy of CA4P and related VDAs.

An automatic algorithm for the segmentation and morphological analysis of microvessels in immunostained histological tumour sections.

A fully automatic segmentation and morphological analysis algorithm for the analysis of microvessels from CD31 immunostained histological tumour sections is presented. Development of the algorithm exploited the distinctive hues of stained vascular endothelial cells, cell nuclei and background, to provide the seeds for a 'region-growing' method for object segmentation in the 3D hue, saturation, value (HSV) colour model. The segmented objects, identified as microvessels by CD31 immunostaining, were post-processed with three morphological tasks: joining separate objects that were likely to belong to a single vessel, closing objects that had a narrow gap around their periphery, and splitting objects with multiple lumina into individual vessels. The automatic segmentation was validated against a hand-segmented set of 44 images from three different SW1222 human colorectal carcinomas xenografted into mice. 96.3 ± 0.9% of pixels were found to be correctly classified. Automated segmentation was carried out on a further 53 images from three histologically distinct mouse fibrosarcomas (MFs) for morphological comparison with the SW1222 tumours. Four morphometric measurements were calculated for each segmented vessel: vascular area (VA), ratio of lumen area to vascular area (lu/VA), eccentricity (e), and roundness (ro). In addition, the total vascular area relative to tumour tissue area (rVA) was calculated. lu/VA, e and ro were found to be significantly smaller in MF tumours than in SW1222 tumours (p < 0.05; unpaired t-test). The algorithm is available through the website http://www.caiman.org.uk where images can be uploaded, processed and sent back to users. The output from CAIMAN consists of the original image with boundaries of segmented vessels overlaid, the calculated parameters and a Matlab file, which contains the segmentation that the user can use to derive further results.

Measuring the velocity of fluorescently labelled red blood cells with a keyhole tracking algorithm.

In this paper we propose a tracking algorithm to measure the velocity of fluorescently labelled red blood cells travelling through microvessels of tumours, growing in dorsal skin flap window chambers, implanted on mice. Preprocessing removed noise and artefacts from the images and then segmented cells from background. The tracking algorithm is based on a 'keyhole' model that describes the probable movement of a segmented cell between contiguous frames of a video sequence. When a history of cell movement exists, past, present and a predicted landing position of the cells will define two regions of probability that resemble the shape of a keyhole. This keyhole model was used to determine if cells in contiguous frames should be linked to form tracks and also as a postprocessing tool to join split tracks and discard links that could have been formed due to noise or uncertainty. When there was no history, a circular region around the centroid of the parent cell was used as a region of probability. Outliers were removed based on the distribution of the average velocities of the tracks. Since the position and time of each cell is recorded, a wealth of statistical measures can be obtained from the tracks. The algorithm was tested on two sets of experiments. First, the vasculatures of eight tumours with different geometries were analyzed; average velocities ranged from 86 to 372 microm s(-1), with minimum and maximum track velocities 7 and 1212 microm s(-1), respectively. Second, a longitudinal study of velocities was performed after administering a vascular disrupting agent to two tumours and the time behaviour was analyzed over 24 h. In one of the tumours there is a complete shutdown of the vasculature whereas in the other there is a clear decrease of velocity at 30 min, with subsequent recovery by 6 h. The tracking algorithm enabled the simultaneous measurement of red blood cell velocity in multiple vessels within an intravital video sequence, enabling analysis of heterogeneity of flow and response to treatment in mouse models of cancer.