Longitudinal imaging of the ageing mouse.

Several non-invasive imaging techniques are used to investigate the effect of pathologies and treatments over time in mouse models. Each preclinical in vivo technique provides longitudinal and quantitative measurements of changes in tissues and organs, which are fundamental for the evaluation of alterations in phenotype due to pathologies, interventions and treatments. However, it is still unclear how these imaging modalities can be used to study ageing with mice models. Almost all age related pathologies in mice such as osteoporosis, arthritis, diabetes, cancer, thrombi, dementia, to name a few, can be imaged in vivo by at least one longitudinal imaging modality. These measurements are the basis for quantification of treatment effects in the development phase of a novel treatment prior to its clinical testing. Furthermore, the non-invasive nature of such investigations allows the assessment of different tissue and organ phenotypes in the same animal and over time, providing the opportunity to study the dysfunction of multiple tissues associated with the ageing process. This review paper aims to provide an overview of the applications of the most commonly used in vivo imaging modalities used in mouse studies: micro-computed-tomography, preclinical magnetic-resonance-imaging, preclinical positron-emission-tomography, preclinical single photon emission computed tomography, ultrasound, intravital microscopy, and whole body optical imaging.

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.

Kinetic modeling of hyperpolarized (13)C pyruvate metabolism in tumors using a measured arterial input function.

Mathematical models are required to estimate kinetic parameters of [1-(13)C] pyruvate-lactate interconversion from magnetic resonance spectroscopy data. One- or two-way exchange models utilizing a hypothetical approximation to the true arterial input function (AIF), (e.g. an ideal 'box-car' function) have been used previously. We present a method for direct measurement of the AIF in the rat. The hyperpolarized [1-(13)C] pyruvate signal was measured in arterial blood as it was continuously withdrawn through a small chamber. The measured signal was corrected for T1 relaxation of pyruvate, RF pulses and dispersion of blood in the chamber to allow for the estimation of the direct AIF. Using direct AIF, rather than the commonly used box-car AIF, provided realistic estimates of the rate constant of conversion of pyruvate to lactate, kpl, the rate constant of conversion of lactate to pyruvate klp, the clearance rate constant of pyruvate from blood to tissue, Kip, and the relaxation rate of lactate T1la. Since no lactate signal was present in blood, it was possible to use a simple precursor-product relationship, with the tumor tissue pyruvate time-course as the input for the lactate time-course. This provided a robust estimate of kpl, similar to that obtained using a directly measured AIF.

Investigation of protein induction in tumour vascular targeted strategies by MALDI MSI.

Characterising the protein signatures in tumours following vascular-targeted therapy will help determine both treatment response and resistance mechanisms. Here, mass spectrometry imaging and MS/MS with and without ion mobility separation have been used for this purpose in a mouse fibrosarcoma model following treatment with the tubulin-binding tumour vascular disrupting agent, combretastatin A-4-phosphate (CA-4-P). Characterisation of peptides after in situ tissue tryptic digestion was carried out using Matrix-Assisted Laser Desorption/Ionisation-Mass Spectrometry (MALDI-MS) and Matrix-Assisted Laser Desorption/Ionisation-Ion Mobility Separation-Mass Spectrometry Imaging (MALDI IMS-MSI) to observe the spatial distribution of peptides. Matrix-Assisted Laser Desorption/Ionisation-Ion Mobility Separation-Tandem Mass Spectrometry (MALDI-IMS-MS/MS) of peaks was performed to elucidate any pharmacological responses and potential biomarkers. By taking tumour samples at a number of time points after treatment gross changes in the tissue were indicated by changes in the signal levels of certain peptides. These were identified as arising from haemoglobin and indicated the disruption of the tumour vasculature. It was hoped that the use of PCA-DA would reveal more subtle changes taking place in the tumour samples however these are masked by the dominance of the changes in the haemoglobin signals.

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.

Guidelines for the welfare and use of animals in cancer research.

Animal experiments remain essential to understand the fundamental mechanisms underpinning malignancy and to discover improved methods to prevent, diagnose and treat cancer. Excellent standards of animal care are fully consistent with the conduct of high quality cancer research. Here we provide updated guidelines on the welfare and use of animals in cancer research. All experiments should incorporate the 3Rs: replacement, reduction and refinement. Focusing on animal welfare, we present recommendations on all aspects of cancer research, including: study design, statistics and pilot studies; choice of tumour models (e.g., genetically engineered, orthotopic and metastatic); therapy (including drugs and radiation); imaging (covering techniques, anaesthesia and restraint); humane endpoints (including tumour burden and site); and publication of best practice.

In vivo characterization of horseradish peroxidase with indole-3-acetic acid and 5-bromoindole-3-acetic acid for gene therapy of cancer.

Gene-directed enzyme prodrug therapy is a form of targeted cancer therapy, in which an enzyme is used to convert a non-toxic prodrug to a cytotoxin within the tumor. Horseradish peroxidase (HRP) is able to convert the indole prodrugs indole-3-acetic acid (IAA) and the halogenated derivative 5-bromo-IAA (5Br-IAA) to toxic agents able to induce cell kill in vitro. This study characterized HRP-directed gene therapy in vivo. Human nasopharyngeal squamous cell carcinoma cells, FaDu, stably expressing HRP were grown as xenografts in SCID mice. Pharmacokinetic analysis of IAA and 5Br-IAA showed satisfactory drug profiles, and millimolar concentrations could be achieved in tumor tissue at non-toxic doses. HRP-expressing tumors showed a modest growth delay when treated with IAA compared with drug-vehicle controls. Treatment response could not be improved using different drug scheduling or drug vehicle, nor by combining HRP-directed gene therapy with fractionated radiotherapy.

Tumour vascular disrupting agents: combating treatment resistance.

A large group of tubulin-binding microtubule-depolymerizing agents act as tumour vascular disrupting agents (VDAs). Several members of this group are now in clinical trials in combination with conventional anticancer drugs and radiotherapy. Here we briefly update on the development of tubulin-binding combretastatins as VDAs, summarize what is known of their mechanisms of action and address issues relating to treatment resistance, using disodium combretastatin A-4 3-O-phosphate (CA-4-P) as an example. Characteristically, VDAs cause a rapid shutdown of blood flow to tumour tissue with much less effect in normal tissues. However, the tumour rim is relatively resistant to treatment. Hypoxia (or hypoxia reoxygenation) induces upregulation of genes associated with angiogenesis and drug resistance. It may be possible to take advantage of treatment-induced hypoxia by combining with drugs that are activated under hypoxic conditions. In summary, VDAs provide a novel approach to cancer treatment, which should effectively complement standard treatments, if treatment resistance is addressed by judicious combination treatment strategies.

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.

The endothelin B (ETB) receptor agonist IRL 1620 is highly vasoconstrictive in two syngeneic rat tumour lines: potential for selective tumour blood flow modification.

The vascular effects of the endothelin B (ET(B)) receptor agonist IRL 1620 were investigated in the rat P22 carcinosarcoma and a range of normal tissues in BDIX rats. Tissue blood flow rate was calculated from measurements of tissue uptake of radiolabelled iodoantipyrine. A comparison of vascular effects in the P22 tumour and the HSN sarcoma growing in CBH/CBi rats was made using laser Doppler flowmetry, showing similar effects of IRL 1620, with red cell flux rapidly decreasing by 50-60% and then returning to control levels within approximately 30 min. This corresponded to similar levels but different spatial organisation of ET(B) binding sites in the two tumours, as measured by autoradiography. The decrease in tumour blood flow and an increase in vascular resistance suggest that the vascular component of ET(B) receptors in the P22 tumour is localised on contractile elements rather than on endothelial cells. ET(A) receptors were also identified. Vasoconstriction occurred uniformly throughout the P22 tumour mass, consistent with a measured homogeneous distribution of ET(B) receptors. IRL 1620 caused vasoconstriction in normal skeletal muscle, kidney and small intestine of the BDIX rat as well as in tumour, but did not affect blood flow in other tissues. These effects could be useful for limiting toxicity of certain chemotherapeutic agents. Fully functional ET(B) receptors are clearly expressed on tumour vasculature and IRL 1620 shows promise for short-term modification of tumour blood flow. Expression levels of ET(B) receptors on the tumour vasculature could be useful for predicting which tumours are likely to respond to IRL 1620.

The assessment of antiangiogenic and antivascular therapies in early-stage clinical trials using magnetic resonance imaging: issues and recommendations.

Vascular and angiogenic processes provide an important target for novel cancer therapeutics. Dynamic contrast-enhanced magnetic resonance imaging is being used increasingly to noninvasively monitor the action of these therapeutics in early-stage clinical trials. This publication reports the outcome of a workshop that considered the methodology and design of magnetic resonance studies, recommending how this new tool might best be used.

Use of horseradish peroxidase for gene-directed enzyme prodrug therapy with paracetamol.

Gene therapy is a potential method of treating cancer with a greater degree of targeting than conventional therapies. In addition, therapy can be directed towards cells within the tumour population that are traditionally resistant to current treatment schedules. Horseradish peroxidase (HRP) can oxidise paracetamol to N-acetyl-p-benzoquinoneimine via a one-electron pathway. Incubation of human cells expressing HRP with 0.5-10 mM paracetamol reduced clonogenic survival, but had little effect on control cells. A small increase in apoptosis was seen and a decrease in the number of cells undergoing mitosis, consistent with reports in hepatocytes using higher paracetamol concentrations. The cytotoxicity was also seen under conditions of severe hypoxia (catalyst induced anoxia), indicating that the HRP/paracetamol combination may be suitable for hypoxia-targeted gene therapy.

Semi-automated software for the three-dimensional delineation of complex vascular networks.

The understanding of tumour angiogenesis is of great importance in cancer research, as is the tumour response to vascular-targeted drugs. This paper presents software aimed at aiding these investigations and other situations where linear or dendritic structures are to be delineated from three-dimensional (3D) data sets. This software application was written to analyse the data from 3D data sets by allowing the manual and semi-automated tracking and delineation of the vascular tree, including the measurement of vessel diameter. A new algorithm, CHARM, based on a compact Hough transform and the formation of a radial map, has been used to locate vessel centres and measure diameters automatically. The robustness of this algorithm to image smoothing and noise has been investigated.

Measuring tumour vascular response to antivascular and antiangiogenic drugs.

The tumour vasculature is an attractive target for therapy because of its accessibility to blood-borne anticancer agents and the reliance of most tumour cells on an intact vascular supply for their survival. For convenience, therapeutic targeting of the tumour vasculature can be divided into antiangiogenic approaches, which target the process of new blood vessel development and antivascular approaches, which target the established tumour vasculature. Many agents are now in clinical trial for the treatment of cancer by these methods. The main aim of this article is to describe the vascular effects of some of these agents and identify suitable end-points for measuring efficacy in early clinical trials. For drugs which are active below their maximum tolerated dose (MTD), measurement of vascular end-points is required to determine the most effective dosing/scheduling protocols. In addition, many of the current and developing antiangiogenic agents have additional mechanisms of action unrelated to angiogenesis per se, requiring measurement of vascular end-points to understand their mechanisms of action. Measurement of tumour microvascular density (MVD) from tumour biopsies is a common method for assessing the efficacy of antiangiogenic drugs. The limitations of this method and alternative end-points, which take into account vascular function, are discussed. Pre-clinical data regarding tumour response to the antivascular agent combretastatin A-4 3-0-phosphate (CA-4-P) are discussed in the context of guiding clinical trial planning. Finally, the accessibility of vascular end-points for clinical imaging is addressed.

Oxic and anoxic enhancement of radiation-mediated toxicity by horseradish peroxidase/indole-3-acetic acid gene therapy.

PURPOSE: To evaluate the interaction of horseradish peroxidase (HRP)/indole-3-acetic acid (IAA) gene therapy with therapeutically relevant doses of radiation. MATERIALS AND METHODS: Human T24 bladder and FaDu nasopharyngeal carcinoma cells were transiently transfected with the HRP cDNA using a non-viral delivery method. HRP expression was confirmed by immunostain and enzyme activity assay. The cells were exposed to IAA or the analogue 1-Me-IAA in conjunction with X-rays in air or in anoxic conditions, and cytotoxicity was determined by clonogenic assay. RESULTS: A significant and selective enhancement of radiation-mediated cytotoxicity was observed. Pre-incubation with the prodrugs induced sensitizer enhancement ratios (SER) ranging from 2.6 (0.1mM IAA) to 5.4 (0.5 mM IAA). Radiosensitization was also observed when prodrug exposure was performed immediately after irradiation (SER=2.1-5.6), or in anoxic conditions (SER=3.6). CONCLUSIONS: The use of gene therapy protocols to enhance the effect of ionizing radiation holds promise for anticancer therapy. The data suggest that the combination of HRP/IAA gene therapy with ionizing radiation could present therapeutic advantages in the treatment of solid malignancies, in particular to target the hypoxic population, which has been shown to correlate with poor outcome after radiotherapy.

Mechanisms associated with tumor vascular shut-down induced by combretastatin A-4 phosphate: intravital microscopy and measurement of vascular permeability.

The tumor vascular effects of the tubulin destabilizing agent disodium combretastatinA-4 3-O-phosphate (CA-4-P) were investigated in the rat P22 tumor growing in a dorsal skin flap window chamber implanted into BD9 rats. CA-4-P is in clinical trial as a tumor vascular targeting agent. In animal tumors, it can cause the shut-down of blood flow, leading to extensive tumor cell necrosis. However, the mechanisms leading to vascular shut-down are still unknown. Tumor vascular effects were visualized and monitored on-line before and after the administration of two doses of CA-4-P (30 and 100 mg/kg) using intravital microscopy. The combined effect of CA-4-P and systemic nitric oxide synthase (NOS) inhibition using N(omega)-nitro-L-arginine (L-NNA) was also assessed, because this combination has been shown previously to have a potentiating effect. The early effect of CA-4-P on tumor vascular permeability to albumin was determined to assess whether this could be involved in the mechanism of action of the drug. Tumor blood flow reduction was extremely rapid after CA-4-P treatment, with red cell velocity decreasing throughout the observation period and dropping to <5% of the starting value by 1 h. NOS inhibition alone caused a 50% decrease in red cell velocity, and the combined treatment of CA-4-P and NOS inhibition was approximately additive. The mechanism of blood flow reduction was very different for NOS inhibition and CA-4-P. That of NOS inhibition could be explained by a decrease in vessel diameter, which was most profound on the arteriolar side of the tumor circulation. In contrast, the effects of CA-4-P resembled an acute inflammatory reaction resulting in a visible loss of a large proportion of the smallest blood vessels. There was some return of visible vasculature at 1 h after treatment, but the blood in these vessels was static or nearly so, and many of the vessels were distended. The hematocrit within larger draining tumor venules tended to increase at early times after CA-4-P, suggesting fluid loss from the blood. The stacking of red cells to form rouleaux was also a common feature, coincident with slowing of blood flow; and these two factors would lead to an increase in viscous resistance to blood flow. Tumor vascular permeability to albumin was increased to approximately 160% of control values at 1 and 10 min after treatment. This could lead to an early decrease in tumor blood flow via an imbalance between intravascular and tissue pressures and/or an increase in blood viscosity as a result of increased hematocrit. These results suggest a mechanism of action of CA-4-P in vivo. Combination of CA-4-P with a NOS inhibitor has an additive effect, which it may be possible to exploit therapeutically.

Limitations of the reporter green fluorescent protein under simulated tumor conditions.

This paper reports a detailed analysis of the effect of low oxygen conditions (hypoxia) on the reporter green fluorescent protein (GFP). It questions the feasibility of using GFP for gene expression studies under tumor conditions. Hypoxia is a characteristic of both experimental and clinical tumors. Several important factors are pointed out which need to be considered when using GFP as reporter gene. GFP fluorescence is the final product of a long and complex pathway involving transcription, translation, and posttranslational modifications. All of these steps may be affected by the availability of oxygen. We show specifically that cellular GFP fluorescence decreased with reduced oxygenation, anoxia virtually eliminated fluorescence and protein levels, and fluorescence recovery after anoxia required 5-10 h of reoxygenation. In conclusion, GFP appears to be a good marker gene to study location or movement of proteins or cells but should be used with great caution as a reporter of gene expression under tumor conditions.

Measurement of tumor oxygenation: in vivo comparison of a luminescence fiber-optic sensor and a polarographic electrode in the p22 tumor.

Hypoxia is important in tumor biology and therapy. This study compared the novel luminescence fiber-optic OxyLite sensor with the Eppendorf polarographic electrode in measuring tumor oxygenation. Using the relatively well-oxygenated P22 tumor, oxygen measurements were made with both instruments in the same individual tumors. In 24 air-breathing animals, pooled electrode pO(2) readings lay in a range over twice that of sensor pO(2(5min)) values (-3.2 to 80 mm Hg and -0.1 to 34.8 mm Hg, respectively). However, there was no significant difference between the means +/- 2 SE of the median pO(2) values recorded by each instrument (11.0 +/- 3.3 and 8.1 +/- 1.9 mm Hg, for the electrode and sensor respectively, P = 0.07). In a group of 12 animals treated with carbon monoxide inhalation to induce tumor hypoxia, there was a small but significant difference between the means +/- 2 SE of the median pO(2) values reported by the electrode and sensor (1.7 +/- 0.9 and 2.9 +/- 0.7 mm Hg, respectively, P = 0.009). A variable degree of disparity was seen on comparison of pairs of median pO(2) values from individual tumors in both air-breathing and carbon monoxide-breathing animals. Despite the differences between the sets of readings made with each instrument from individual tumors, we have shown that the two instruments provide comparable assessments of tumor oxygenation in groups of tumors, over the range of median pO(2) values of 0.6 to 28.1 mm Hg.

Effects of combretastatin A4 phosphate on endothelial cell morphology in vitro and relationship to tumour vascular targeting activity in vivo.

BACKGROUND: Combretastatin A4 Phosphate (CA4P) is a tubulin binding agent which causes rapid tumour vascular shutdown. It has anti-proliferative and apoptotic effects on dividing endothelial cells after prolonged exposure, but these effects occur on a much longer time scale than the reduction in tumour blood flow. This study compared the time course of CA4P effects on endothelial cell shape and reduction in red cell velocity. METHODS: Endothelial cell area and form factor (1-4 pi x area x perimeter-2) were measured for proliferating and confluent HUVECs after CA4P treatment. Recovery of shape after CA4P and colchicine was compared. Window chamber studies of tumours were used to measure red cell velocity. Results 70% reduction in red cell velocity and 44% reduction in HUVEC form factor occurred by 10 minutes. Proliferating HUVECs underwent greater cell shape change after CA4P, which occurred at lower doses than for confluent cells. Cell shape recovered 24 hours after 30 minutes exposure to CA4P, but not after colchicine. CONCLUSIONS: The similar time course of cell shape change and red cell velocity reduction suggests endothelial cell shape change may be involved early in the in vivo events leading to vascular shutdown. Differences in the recovery from the shape changes induced by CA4P and colchicine could underlie the different toxicity profiles of these drugs.