The response of tumour vasculature to angiotensin II revealed by its systemic and local administration to 'tissue-isolated' tumours

A tissue-isolated preparation of the P22 rat carcinosarcoma was used to investigate the tumour vascular response to angiotensin II (ATII). In particular, the relative importance of systemic and local tumour factors was assessed by comparing tumour vascular resistance during systemic administration of ATII and during administration directly into the tumour-supplying artery. The effect of hypervolaemia on tumour vascular resistance was determined as well as the effect of ATII on oxygen metabolism. Tumour vascular resistance was increased by ATII in a dose-dependent manner. The response was biphasic with an initial peak in resistance followed by a lower plateau phase. Systemic administration of ATII was more effective in increasing tumour vascular resistance than direct administration. This suggests that systemic administration is not causing any reopening of previously collapsed tumour blood vessels. Further evidence for this is that hypervolaemia caused no reduction in tumour vascular resistance and that there was no difference in oxygen extraction by tumours between groups treated with systemically and directly administered ATII. A heterogeneous distribution of ATII receptors in the P22 tumour is a more likely explanation for the known heterogeneity of blood flow response to ATII

The relationship between regional variations in blood flow and histology in a transplanted rat fibrosarcoma

The regional distribution of blood flow to the LBDS1 fibrosarcoma, transplanted into the subcutaneous site in rats, was investigated using the readily diffusible compound 14C-iodo-antipyrine (14C-IAP). Quantitative autoradiography was used to establish absolute values of specific blood flow F for 100 X 100 X 20 microns adjacent tissue volumes of the unperturbed tumour. Mean blood flow to whole tumours was found to decrease with increase in tumour size. This relationship was abolished if blood flow was only measured in sections cut from the periphery of the tumours. Detailed analysis of a sub-group of tumours showed that blood flow to individual tumours was heterogeneous. The range of blood flow was large, indicating that mean blood flow to a whole tumour is a poor reflection of the blood perfusion pattern of that tumour. Necrotic tumour regions were usually very poorly perfused. With the exception of the smallest tumours studied, blood flow was lower in the centre of tumours than in the periphery. Necrosis also tended to develop centrally. However, the peripheral to central gradient of blood flow was apparent even when densely cellular, viable tumour regions and necrotic regions were analysed separately. The decrease in blood flow with tumour size was also apparent in densely cellular, viable tumour regions when analysed separately. Qualitative comparison of tumour histology and regional blood flow showed that there were areas of very low blood flow associated with viable tumour regions. Less common were areas of rather high blood flow associated with necrotic tumour regions. A complicated relationship exists between tumour histology and blood flow. The quantitative autoradiography technique is suitable for investigating the most poorly perfused and the most well perfused viable fractions of animal tumours which may limit the efficacy of different types of therapy

Synthesis of [3-C-13]-2,3-dihydroxy-4-methoxybenzaldehyde

An efficient synthesis of [3-13C]-2,3-dihydroxy-4-methoxybenzaldehyde, an isotopically labelled probe of a common intermediate used in the synthesis of a number of biologically relevant molecules, has been achieved in 9 steps from an acyclic, non-aromatic precursor. A 13C label for molecular imaging was introduced in a linear synthesis from the reaction of [13C]-labelled methyl iodide with glutaric monomethyl ester chloride. Cyclisation then aromatisation gave 1,3-dimethoxybenzene and an additional methoxy group was introduced by a formylation/Baeyer–Villiger/hydrolysis/methylation sequence. Subsequent ortho-formylation and selective demethylation yielded the desired [3-13C]-2,3-dihydroxy-4-methoxybenzaldehyde

Characterisation of tumour blood flow using a 'tissue-isolated' preparation

Tumour blood flow was characterised in a 'tissue-isolated' rat tumour model, in which the vascular supply is derived from a single artery and vein. Tumours were perfused in situ and blood flow was calculated from simultaneous measurement of (1) venous outflow from the tumour and (2) uptake into the tumour of radiolabelled iodo-antipyrine (IAP). Comparison of results from the two measurements enabled assessment of the amount of blood 'shunted' through the tumours with minimal exchange between blood and tissue. Kinetics of IAP uptake were also used to determine the apparent volume of distribution (VDapp) for the tracer and the equilibrium tissue-blood partition coefficient (lambda). lambda was also measured by in vitro techniques and checks were made for binding and metabolism of IAP using high-pressure liquid chromatography. VDapp and lambda were used to calculate the perfused fraction (alpha) of the tumours. Tumour blood flow, as measured by IAP (TBFIAP), was 94.8 +/- 4.4% of the blood flow as measured by venous outflow, indicating only a small amount of non-exchanging flow. This level of shunting is lower than some previous estimates in which the percentage tumour entrapment of microspheres was used. The unperfused fraction ranged from 0 to 20% of the tumour volume in the majority of tumours. This could be due to tumour necrosis and/or acutely ischaemic tumour regions. For practical purposes, measurement of the total venous outflow of tumours is a reasonable measure of exchangeable tumour blood flow in this system and allows for on-line measurements. Tracer methods can be used to obtain additional information on the distribution of blood flow within tumours

Sydnone Cycloaddition Route to Pyrazole-Based Analogs of Combretastatin A4.

The combretastatins are an important class of tubulin-binding agents. Of this family, a number of compounds are potent tumor Vascular Disrupting Agents (VDAs) and have shown promise in the clinic for cancer therapy. We have developed a modular synthetic route to combretastatin analogs based on a pyrazole core through highly-regioselective alkyne cycloaddition reactions of sydnones. These compounds show modest to high potency against human umbilical vein endothelial cell proliferation. Moreover, evidence is presented that these novel VDAs have the same mode of action as CA4P and bind reversibly to β-tubulin - believed to be a key feature in avoiding toxicity. The most active compound from in vitro studies was taken forward to an in vivo model and instigated an increase in tumor cell necrosis

The influence of hypoxia and energy depletion on the response of endothelial cells to the vascular disrupting agent combretastatin A-4-phosphate

Combretastatin A-4 phosphate (CA4P) is a microtubule-disrupting tumour-selective vascular disrupting agent (VDA). CA4P activates the actin-regulating RhoA-GTPase/ ROCK pathway, which is required for full vascular disruption. While hypoxia renders tumours resistant to many conventional therapies, little is known about its influence on VDA activity. Here, we found that active RhoA and ROCK effector phospho-myosin light chain (pMLC) were downregulated in endothelial cells by severe hypoxia. CA4P failed to activate RhoA/ROCK/pMLC but its activity was restored upon reoxygenation. Hypoxia also inhibited CA4P-mediated actinomyosin contractility, VE-cadherin junction disruption and permeability rise. Glucose withdrawal downregulated pMLC, and coupled with hypoxia, reduced pMLC faster and more profoundly than hypoxia alone. Concurrent inhibition of glycolysis (2-deoxy-D-glucose, 2DG) and mitochondrial respiration (rotenone) caused profound actin filament loss, blocked RhoA/ROCK signalling and rendered microtubules CA4P-resistant. Withdrawal of the metabolism inhibitors restored the cytoskeleton and CA4P activity. The AMP-activated kinase AMPK was investigated as a potential mediator of pMLC downregulation. Pharmacological AMPK activators that generate AMP, unlike allosteric activators, downregulated pMLC but only when combined with 2DG and/or rotenone. Altogether, our results suggest that Rho/ROCK and actinomyosin contractility are regulated by AMP/ATP levels independently of AMPK, and point to hypoxia/energy depletion as potential modifiers of CA4P response

Kinetic modelling of dissolution dynamic nuclear polarisation 13C magnetic resonance spectroscopy data for analysis of pyruvate delivery and fate in tumours

Dissolution dynamic nuclear polarisation (dDNP) of 13C-labelled pyruvate in magnetic resonance spectroscopy/imaging (MRS/MRSI) has the potential for monitoring tumour progression and treatment response. Pyruvate delivery, its metabolism to lactate and efflux were investigated in rat P22 sarcomas following simultaneous intravenous administration of hyperpolarised 13C-labelled pyruvate (13C1-pyruvate) and urea (13C-urea), a nonmetabolised marker. A general mathematical model of pyruvate-lactate exchange, incorporating an arterial input function (AIF), enabled the losses of pyruvate and lactate from tumour to be estimated, in addition to the clearance rate of pyruvate signal from blood into tumour, Kip, and the forward and reverse fractional rate constants for pyruvate-lactate signal exchange, kpl and klp. An analogous model was developed for urea, enabling estimation of urea tumour losses and the blood clearance parameter, Kiu. A spectral fitting procedure to blood time-course data proved superior to assuming a gamma-variate form for the AIFs. Mean arterial blood pressure marginally correlated with clearance rates. Kiu equalled Kip, indicating equivalent permeability of the tumour vasculature to urea and pyruvate. Fractional loss rate constants due to effluxes of pyruvate, lactate and urea from tumour tissue into blood (kpo, klo and kuo, respectively) indicated that T1s and the average flip angle, θ, obtained from arterial blood were poor surrogates for these parameters in tumour tissue. A precursor-product model, using the tumour pyruvate signal time-course as the input for the corresponding lactate signal time-course, was modified to account for the observed delay between them. The corresponding fractional rate constant, kavail, most likely reflected heterogeneous tumour microcirculation. Loss parameters, estimated from this model with different TRs, provided a lower limit on the estimates of tumour T1 for lactate and urea. The results do not support use of hyperpolarised urea for providing information on the tumour microcirculation over and above what can be obtained from pyruvate alone. The results also highlight the need for rigorous processes controlling signal quantitation, if absolute estimations of biological parameters are required

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

Gene delivery to hypoxic cells in vitro

Hypoxia in solid tumours has been correlated with poor prognosis and resistance to radiation and chemotherapy. Hypoxia is also a strong stimulus for gene expression. We previously proposed a gene therapy approach which exploits the presence of severe hypoxia in tumours for the induction of therapeutic genes. Hypoxic cells are known to have a reduced metabolic rate, transcription and translation. These facts may prevent gene transfer and therefore warranted further investigation. In this paper the feasibility of gene delivery in vitro under tumour conditions was demonstrated. DNA was delivered in vitro using a peptide-mediated non-viral system. Across a range of oxygen tensions and mammalian cell lines (including human tumour and endothelial cells) it was shown that hypoxic cells could be transfected. Transfection efficiencies varied depending on the level of hypoxia, cell characteristics and gene promoters used. An in vitro model of hypoxia/reoxygenation, designed to mimic the variable nature of tumour hypoxia, showed that hypoxic preconditioning and reoxygenation alone did not reduce transfection efficiency significantly; only chronic anoxia reduced transfection. The fact that neither intermediate hypoxia nor intermittent anoxia significantly reduced transfection is promising for future hypoxia-targeted gene therapy strategies