Simon Akerman

Blood vessel maturation and response to vascular-disrupting therapy in single vascular endothelial growth factor-A isoform-producing tumors

Tubulin-binding vascular-disrupting agents (VDA) are currently in clinical trials for cancer therapy but the factors that influence tumor susceptibility to these agents are poorly understood. We evaluated the consequences of modifying tumor vascular morphology and function on vascular and therapeutic response to combretastatin-A4 3-O-phosphate (CA-4-P), which was chosen as a model VDA. Mouse fibrosarcoma cell lines that are capable of expressing all vascular endothelial growth factor (VEGF) isoforms (control) or only single isoforms of VEGF (VEGF120, VEGF164, or VEGF188) were developed under endogenous VEGF promoter control. Once tumors were established, VEGF isoform expression did not affect growth or blood flow rate. However, VEGF188 was uniquely associated with tumor vascular maturity, resistance to hemorrhage, and resistance to CA-4-P. Pericyte staining was much greater in VEGF188 and control tumors than in VEGF120 and VEGF164 tumors. Vascular volume was highest in VEGF120 and control tumors (CD31 staining) but total vascular length was highest in VEGF188 tumors, reflecting very narrow vessels forming complex vascular networks. I.v. administered 40 kDa FITC-dextran leaked slowly from the vasculature of VEGF188 tumors compared with VEGF120 tumors. Intravital microscopy measurements of vascular length and RBC velocity showed that CA-4-P produced significantly more vascular damage in VEGF120 and VEGF164 tumors than in VEGF188 and control tumors. Importantly, this translated into a similar differential in therapeutic response, as determined by tumor growth delay. Results imply differences in signaling pathways between VEGF isoforms and suggest that VEGF isoforms might be useful in vascular-disrupting cancer therapy to predict tumor susceptibility to VDAs.

Systemic antiangiogenic activity of cationic poly-L-lysine dendrimer delays tumor growth

This study describes the previously unreported intrinsic capacity of poly-L-lysine (PLL) sixth generation (G6) dendrimer molecules to exhibit systemic antiangiogenic activity that could lead to solid tumor growth arrest. The PLL-dendrimer-inhibited tubule formation of SVEC4-10 murine endothelial cells and neovascularization in the chick embryo chick chorioallantoic membrane (CAM) assay. Intravenous administration of the PLL-dendrimer molecules into C57BL/6 mice inhibited vascularisation in Matrigel plugs implanted subcutaneously. Antiangiogenic activity was further evidenced using intravital microscopy of tumors grown within dorsal skinfold window chambers. Reduced vascularization of P22 rat sarcoma implanted in the dorsal window chamber of SCID mice was observed following tail vein administration (i.v.) of the PLL dendrimers. Also, the in vivo toxicological profile of the PLL-dendrimer molecules was shown to be safe at the dose regime studied. The antiangiogenic activity of the PLL dendrimer was further shown to be associated with significant suppression of B16F10 solid tumor volume and delayed tumor growth. Enhanced apoptosis/necrosis within tumors of PLL-dendrimer-treated animals only and reduction in the number of CD31 positive cells were observed in comparison to protamine treatment. This study suggests that PLL-dendrimer molecules can exhibit a systemic antiangiogenic activity that may be used for therapy of solid tumors, and in combination with their capacity to carry other therapeutic or diagnostic agents may potentially offer capabilities for the design of theranostic systems.

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 μm s−1, with minimum and maximum track velocities 7 and 1212 μm 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.

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.

Vascular effects dominate solid tumor response to treatment with combretastatin A-4-phosphate

Vascular‐targeted therapeutics are increasingly used in the clinic. However, less is known about the direct response of tumor cells to these agents. We have developed a combretastatin‐A‐4‐phosphate (CA4P) resistant variant of SW1222 human colorectal carcinoma cells to examine the relative importance of vascular versus tumor cell targeting in the ultimate treatment response. SW1222Res cells were generated through exposure of wild‐type cells (SW1222WT) to increasing CA4P concentrations in vitro. Increased resistance was confirmed through analyses of cell viability, apoptosis and multidrug‐resistance (MDR) protein expression. In vivo, comparative studies examined tumor cell necrosis, apoptosis, vessel morphology and functional vascular end‐points following treatment with CA4P (single 100 mg/kg dose). Tumor response to repeated CA4P dosing (50 mg/kg/day, 5 days/week for 2 weeks) was examined through growth measurement, and ultimate tumor cell survival was studied by ex vivo clonogenic assay. In vitro, SW1222Res cells showed reduced CA4P sensitivity, enhanced MDR protein expression and a reduced apoptotic index. In vivo, CA4P induced significantly lower apoptotic cell death in SW1222Res versus SW1222WT tumors indicating maintenance of resistance characteristics. However, CA4P‐induced tumor necrosis was equivalent in both lines. Similarly, rapid CA4P‐mediated vessel disruption and blood flow shut‐down were observed in both lines. Cell surviving fraction was comparable in the two tumor types following single dose CA4P and SW1222Res tumors were at least as sensitive as SW1222WT tumors to repeated dosing. Despite tumor cell resistance to CA4P, SW1222Res response in vivo was not impaired, strongly supporting the view that vascular damage dominates the therapeutic response to this agent.

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

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.

Influence of soluble or matrix-bound isoforms of vascular endothelial growth factor-A on tumor response to vascular-targeted strategies

Antiangiogenic therapy based on blocking the actions of vascular endothelial growth factor‐A (VEGF) can lead to “normalization” of blood vessels in both animal and human tumors. Differential expression of VEGF isoforms affects tumor vascular maturity, which could influence the normalization process and response to subsequent treatment. Fibrosarcoma cells expressing only VEGF120 or VEGF188 isoforms were implanted either subcutaneously (s.c.) or in dorsal skin‐fold “window” chambers in SCID mice. VEGF120 was associated with vascular fragility and hemorrhage. Tumor‐bearing mice were treated with repeat doses of SU5416, an indolinone receptor tyrosine kinase inhibitor with activity against VEGFR‐2 and proven preclinical ability to induce tumor vascular normalization. SU5416 reduced vascularization in s.c. implants of both VEGF120 and VEGF188 tumors. However, in the window chamber, SU5416 treatment increased red cell velocity in VEGF120 (representing vascular normalization) but not VEGF188 tumors. SU5416 treatment had no effect on growth or necrosis levels in either tumor type but tended to counteract the increase in interstitial fluid pressure seen with growth of VEGF120 tumors. SU5416 pretreatment resulted in the normally fragile blood vessels in VEGF120‐expressing tumors becoming resistant to the vascular damaging effects of the tubulin‐binding vascular disrupting agent (VDA), combretastatin A4 3‐O‐phosphate (CA4P). Thus, vascular normalization induced by antiangiogenic treatment can reduce the efficacy of subsequent VDA treatment. Expression of VEGF120 made tumors particularly susceptible to vascular normalization by SU5416, which in turn made them resistant to CA4P. Therefore, VEGF isoform expression may be useful for predicting response to both antiangiogenic and vascular‐disrupting therapy.

Measuring Red Blood Cell Velocity with a Keyhole Tracking Algorithm

A tracking algorithm is proposed to measure the velocity of red blood cells traveling through microvessels of tumors growing in skin flaps implanted on mice. The tracking is based on a keyhole model that describes the probable movement of a segmented cell between contiguous frames in a video sequence. When a history of movements exists, past, present and a predicted landing position define two regions of probability with a keyhole shape. This keyhole is used to de- termine if cells in contiguous frames should be linked to form tracks. Pre-processing segments cells from background and post-processing joins tracks and discards links that could have been formed due to noise or uncertainty. The algorithm pre- sents several advantages over traditional methods such as kymographs or particle image velocimetry: manual interven- tion is restricted to the thresholding, several vessels can be analyzed simultaneously, algorithm is robust to noise and a wealth of statistical measures can be obtained. Two tumors with different geometries were analyzed; average velocities were 211±136 [μm/s] (mean±std) with a range 15.9-797 [μm/s], and 89±62 [μm/s] with a range 5.5-300 [μm/s] respec- tively, which are consistent with previous results in the litera- ture.