The supplementary data file contains the following data and figures: Fig S1- CD31 and CD34 co-localize to the vasculature in fs120-LS and fs188-LS subcutaneous tumors. Fig S2 - VEGFR1 activity has no effect on tumor growth or vascular density of subcutaneous fs120-LS or fs188-LS tumors. Fig S3 - fs120-LS and fs188-LS cells metastasize to the lung early during subcutaneous tumor growth. Fig S4 - fs120-LS cells express higher levels of PlGF2 than fs188-LS cells in vitro, with no difference in levels of PLGF2 detected in subcutaneous tumors. Fig S5 - Expression of laminin and collagen-I in lysates of fs120-LS and fs188-LS subcutaneous tumors. Fig S6 - Quantification of single cell migration using live video microscopy. Fig S7 - Localization of CD11b cells with respect to intravenously injected fibrosarcoma cells within the lung. Fig S8 - Summary of results showing key differences between fibrosarcomas expressing VEGF120 and VEGF188 and the effects of B20-4.1.1 and stromal VEGFR1 activity. Fig S9 - Effect of cediranib on survival of fs120-LS cells in the lung 48 h after iv injection.

The supplementary methods file contains additional detailed information regarding: 1. The production of cell lines stably expressing luciferase. 2. Mouse strains and pre-clinical models of tumor growth and metastasis. 3. Acquisition and analysis of bioluminescence data from preclinical and in vitro studies. 4. Acquisition and analysis of in vitro migration data. 5. Antibodies and immuno-techniques used within this study.

Dissolution dynamic nuclear polarisation (dDNP) of 13 C-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 13 C-labelled pyruvate (13 C1 -pyruvate) and urea (13 C-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 T1 s 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.

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.

This work explores synthesis of novel cholesterol derivative for the preparation of cationic liposomes and its interaction with Paclitaxel (PTX) within liposome membrane using molecular dynamic (MD) simulation and in-vitro studies.

The combretastatins have attracted significant interest as small-molecule therapies for cancer due to their ability to function as vascular disrupting agents. We have successfully prepared a range of combretastatin analogues that are based on a novel sydnone heterocycle core, and their potential as tubulin binders has been assessed in vitro and in vivo. The most potent candidate was found to disrupt microtubules and affect cellular morphology at sub-micromolar levels. Moreover, it was found to bind reversibly to tubulin and significantly increase endothelial cell monolayer permeability, in a similar manner to combretastatin A4. Surprisingly, the compound did not exhibit efficacy in vivo, possibly due to rapid metabolism.

The Cover Feature shows a novel sydnone-based analog of the combretastatins which target and disrupt microtubules, affecting cellular morphology, even at submicromolar concentrations. These compounds offer a new platform for developing hydrophilic, configurationally stable analogs of combretastatins, and a new arsenal of weapons for the fight against cancer. More information can be found in the Full Paper by Gillian M. Tozer, Joseph P. A. Harrity, Chryso Kanthou et al. on page 2618 in Issue 24, 2018 (DOI: 10.1002/cmdc.201800567).

The development of blood vessels by the process of angiogenesis underpins the growth and metastasis of many tumour types. Various angiogenesis inhibitors targeted against vascular endothelial growth factor A (VEGF-A) and its receptors have entered the clinic more than a decade ago. However, despite substantial clinical improvements, their overall efficacy proved to be significantly lower than many of the pre-clinical studies had predicted. Antiangiogenic agents have been combined with chemotherapy, radiotherapy and more recently immunotherapy in many pre-clinical and clinical studies in an effort to improve their efficacy. To date, only their use alongside chemotherapy is approved as part of standard treatment protocols. Most pre-clinical studies have reported improved tumour control from the addition of antiangiogenic therapies to radiotherapy and progress has been made in unravelling the complex mechanisms through which VEGF inhibition potentiates radiotherapy responses. However, the efficacy of this combination is variable, and many questions still remain as to how best to administer the two modalities to achieve optimal response and minimal toxicity. One important limiting factor is that, unlike some other targeted therapies, antiangiogenic agents are not administered to selected patient populations, since biomarkers for identifying responders have not yet been established. Here, we outline VEGF biology and review current approaches that aim to identify biomarkers for stratifying patients for treatment with angiogenesis inhibitors. We also discuss current progress in elucidating mechanisms of interaction between radiotherapy and VEGF inhibitors. Ongoing clinical trials will determine whether these combinations will ultimately improve treatment outcomes for cancer patients.

Subcutaneously implanted experimental tumors in mice are commonly used in cancer research. Despite their superficial location, they remain a challenge to image non-invasively at sufficient spatial resolution for microvascular studies. Here we evaluate the capabilities of optical coherence tomography (OCT) angiography for imaging such tumors directly through the murine skin in-vivo. Data sets were collected from mouse tumors derived from fibrosarcoma cells genetically engineered to express only single splice variant isoforms of vascular endothelial growth factor A (VEGF); either VEGF120 or VEGF188 (fs120 and fs188 tumors respectively). Measured vessel diameter was found to be significantly (p<0.001) higher for fs120 tumors (60.7 ± 4.9μm) compared to fs188 tumors (45.0 ± 4.0μm). The fs120 tumors also displayed significantly higher vessel tortuosity, fractal dimension and density. The ability to differentiate between tumor types with OCT suggests that the visible abnormal vasculature is representative of the tumor microcirculation, providing a robust, non-invasive method for observing the longitudinal dynamics of the subcutaneous tumor microcirculation.

This work describes the topological analysis of the vasculature of tumours. The analysis is performed with a scale-space technique, which traces the centrelines of vessels as topological ridges of the image intensities and then obtains a series of measurements, which are used to compare the vasculatures. Besides the measurements directly associated with the centrelines, the scales obtained allow the estimation of width and thus area covered with vessels. Tumours of SW1222 human colorectal carcinoma xenografts were observed when growing in dorsal skin-fold window chambers in mice. Three variants of the tumours expressing either endogenous levels of angiopoietins (WT) or over-expressing either angiopoietin-1 (Ang-1) or angiopoietin-2 (Ang-2) were assessed with/without vascular targeted therapy. The scale-space technique was able to discriminate between the vasculatures of the three different tumour types prior to treatment. Results also suggested that over-expression of Ang-2 was associated with susceptibility of the tumour vasculature to the vascular disrupting agent, combretastatin A4 phosphate (CA4P). Substantiation of this finding would point to the potential of tumour Ang-2 expression as a predictive bio-marker for response to CA4P.

CD68+ tumor-associated macrophages (TAMs) are pro-tumorigenic, pro-angiogenic and are associated with decreased survival rates in patients with cancer, including breast cancer. Non-specific models of macrophage ablation reduce the number of TAMs and limit the development of mammary tumors. However, the lack of specificity and side effects associated with these models compromise their reliability. We hypothesized that specific and controlled macrophage depletion would provide precise data on the effects of reducing TAM numbers on tumor development. In this study, the MacLow mouse model of doxycycline-inducible and selective CD68+ macrophage depletion was crossed with the murine mammary tumor virus (MMTV)-Polyoma virus middle T antigen (PyMT) mouse model of spontaneous ductal breast adenocarcinoma to generate the PyMT-MacLow line. In doxycycline-treated PyMT-MacLow mice, macrophage numbers were decreased in areas surrounding tumors by 43%. Reducing the number of macrophages by this level delayed tumor progression, generated less proliferative tumors, decreased the vascularization of carcinomas and down-regulated the expression of many pro-angiogenic genes. These results demonstrate that depleting CD68+ macrophages in an inducible and selective manner delays the development of mammary tumors and that the PyMT-MacLow model is a useful and unique tool for studying the role of TAMs in breast cancer.

Abstract Elevated plasma concentrations of soluble VEGFA isoforms are associated with poor prognosis in parallel with improved response to treatment with the anti-VEGFA antibody bevacizumab. To uncover the underlying mechanism to these observations, we administered anti-VEGFA therapy to mice bearing luminescent mouse fibrosarcomas expressing single VEGFA isoforms or their wild-type counterparts expressing all isoforms (fs120, fs164, fs188, or fsWT). Expression of the more soluble isoforms conferred an advantage for lung metastasis from subcutaneous tumors (fs120/164 vs. fs188/WT); fs120 cells also produced more lung colonies than fs188 cells when injected intravenously. Metastasis from subcutaneous fs120 tumors was more sensitive than fs188 to treatment with the anti-VEGFA antibody B20-4.1.1. Despite elevated plasma levels of VEGFA in fs120 tumor-bearing mice and a dependence on VEGF receptor 1 activity for metastasis to the lung, B20-4.1.1 did not affect survival in the lung on intravenous injection. B20-4.1.1 inhibited subcutaneous tumor growth and decreased vascular density in both fs120 and fs188 tumors. However, migration of fs120, but not fs188 cells, in vitro was inhibited by B20-4.1.1. The greater survival of fs120 cells in the lung was associated with VEGFR1-dependent accumulation of CD11b-positive myeloid cells and higher expression of the VEGFR1 ligand, PlGF2, by the fs120 cells in vitro and in the plasma and lungs of fs120 tumor-bearing mice. We conclude that soluble VEGFA isoform expression increases fibrosarcoma metastasis through multiple mechanisms that vary in their sensitivity to anti-VEGF/VEGFR inhibition, with VEGFA-targeted therapy suppressing metastasis through effects on the primary tumor rather than the metastatic site. Cancer Res; 77(10); 2633–46. ©2017 AACR.

Solid tumours vary in sensitivity to the vascular disrupting agent combretastatin A-4 3-O-phosphate (CA4P), but underlying factors are poorly understood. The signaling sphingolipid, sphingosine-1-phosphate (S1P), promotes vascular barrier integrity by promoting assembly of VE-cadherin/β-catenin complexes. We tested the hypothesis that tumour pre-treatment with S1P would render tumours less susceptible to CA4P. S1P (1μM) pretreatment attenuated an increase in endothelial cell (HUVEC) monolayer permeability induced by 10μM CA4P. Intravenously administered S1P (8mg/kg/hr for 20 minutes then 2mg/kg/hr for 40 minutes), reduced CA4P-induced (30mg/kg) blood flow shut-down in fibrosarcoma tumours in SCID mice (n≥7 per group), as measured by tumour retention of an intravenously administered fluorescent lectin. A trend towards in vivo protection was also found using laser Doppler flowmetry. Immunohistochemical staining of tumours ex vivo revealed disrupted patterns of VE-cadherin in vasculature of mice treated with CA4P, which were decreased by pretreatment with S1P. S1P treatment also stabilized N-cadherin junctions between endothelial cells and smooth muscle cells in culture, and stabilized tubulin filaments in HUVEC monolayers. We conclude that the rapid shutdown of tumour microvasculature by CA4P is due in part to disruption of adherens junctions and that S1P has a protective effect on both adherens junctions and the endothelial cell cytoskeleton.

PurposeA rat model was developed to enable direct administration of hyperpolarized 13C‐labeled molecules into a tumor‐supplying artery for magnetic resonance spectroscopy (MRS) studies of tumor metabolism.MethodsRat P22 sarcomas were implanted into the right inguinal fat pad of BDIX rats such that the developing tumors received their principle blood supply directly from the right superior epigastric artery. Hyperpolarized 13C‐molecules were either infused directly to the tumor through the epigastric artery or systemically through the contralateral femoral vein. Spectroscopic data were obtained on a 7 Tesla preclinical scanner.ResultsIntra‐arterial infusion of hyperpolarized 13C‐pyruvate increased the pyruvate tumor signal by a factor of 4.6, compared with intravenous infusion, despite an approximately 7 times smaller total dose to the rat. Hyperpolarized glucose signal was detected at near‐physiological systemic blood concentration. Pyruvate to lactate but not glucose to lactate metabolism was detected in the tumor. Hyperpolarized 13C‐labeled combretastatin A1 diphosphate, a tumor vascular disrupting agent, showed an in vivo signal in the tumor.ConclusionsThe model maximizes tumor substrate/drug delivery and minimizes T1 relaxation signal losses in addition to systemic toxicity. Therefore, it permits metabolic studies of hyperpolarized substrates with relatively short T1 and opens up the possibility for preclinical studies of hyperpolarized drug molecules. Magn Reson Med 78:2116–2126, 2017. © 2017 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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 beta-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.

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.

The rate of blood flow through a tissue (F) is a critical parameter for assessing the functional efficiency of a blood vessel network following angiogenesis. This chapter aims to provide the principles behind the estimation of F, how F relates to other commonly used measures of tissue perfusion, and a practical approach for estimating F in laboratory animals, using small readily diffusible and metabolically inert radio-tracers. The methods described require relatively nonspecialized equipment. However, the analytical descriptions apply equally to complementary techniques involving more sophisticated noninvasive imaging.Two techniques are described for the quantitative estimation of F based on measuring the rate of tissue uptake following intravenous administration of radioactive iodo-antipyrine (or other suitable tracer). The Tissue Equilibration Technique is the classical approach and the Indicator Fractionation Technique, which is simpler to perform, is a practical alternative in many cases. The experimental procedures and analytical methods for both techniques are given, as well as guidelines for choosing the most appropriate method.