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AIM: The adapter protein p130Cas, encoded by the Bcar1 gene, is a key regulator of cell movement, adhesion, and cell cycle control in diverse cell types. Bcar1 constitutive knockout mice are embryonic lethal by embryonic days (E) 11.5-12.5, but the role of Bcar1 in embryonic development remains unclear. Here, we investigated the role of Bcar1 specifically in cardiovascular development and defined the cellular and molecular mechanisms disrupted following targeted Bcar1 deletions. METHODS AND RESULTS: We crossed Bcar1 floxed mice with Cre transgenic lines allowing for cell-specific knockout either in smooth muscle and early cardiac tissues (SM22-Cre), mature smooth muscle cells (smMHC-Cre), endothelial cells (Tie2-Cre), second heart field cells (Mef2c-Cre), or neural crest cells (NCC) (Pax3-Cre) and characterised these conditional knock outs using a combination of histological and molecular biology techniques.Conditional knockout of Bcar1 in SM22-expressing smooth muscle cells and cardiac tissues (Bcar1SM22KO) was embryonically lethal from E14.5-15.5 due to severe cardiovascular defects, including abnormal ventricular development and failure of outflow tract (OFT) septation leading to a single outflow vessel reminiscent of persistent truncus arteriosus. SM22-restricted loss of Bcar1 was associated with failure of OFT cushion cells to undergo differentiation to septal mesenchymal cells positive for SMC-specific α-actin, and disrupted expression of proteins and transcription factors involved in epithelial-to-mesenchymal transformation (EMT). Furthermore, knockout of Bcar1 specifically in NCC (Bcar1PAX3KO) recapitulated part of the OFT septation and aortic sac defects seen in the Bcar1SM22KO mutants, indicating a cell-specific requirement for Bcar1 in NCC essential for OFT septation. In contrast, conditional knockouts of Bcar1 in differentiated smooth muscle, endothelial cells, and second heart field cells survived to term and were phenotypically normal at birth and post-natally. CONCLUSIONS: Our work reveals a cell-specific requirement for Bcar1 in NCC, early myogenic and cardiac cells, essential for OFT septation, myocardialisation and EMT/cell cycle regulation and differentiation to myogenic lineages. TRANSLATIONAL PERSPECTIVE: The molecular pathways coordinating cardiogenesis and the remodelling of the OFT are complex, and dysregulation of these pathways causes human heart defects. Our findings highlight a specific requirement for Bcar1 essential for cardiogenesis. Furthermore, the failure of OFT septation in Bcar1SM22KO mice resembles persistent truncus arteriosus (PTA), a feature of several human congenital heart diseases, including DiGeorge Syndrome. Our findings have implications for the mechanisms underlying the pathogenesis of congenital heart disease, and suggest that mice with conditional Bcar1 deletions may be useful models for dissecting mechanisms involved in the pathogenesis of human heart defects.

Objectives Effects of liver disease on portal venous (PV), hepatic arterial (HA), total liver blood flow (TLBF), and cardiac function are poorly understood. Terlipressin modulates PV flow but effects on HA, TLBF, and sepsis/acute-on-chronic liver failure (ACLF)-induced haemodynamic changes are poorly characterised. In this study, we investigated the effects of terlipressin and sepsis/ACLF on hepatic haemodynamics and cardiac function in a rodent cirrhosis model using caval subtraction phase-contrast (PC) MRI and cardiac cine MRI. Methods Sprague-Dawley rats (n = 18 bile duct–ligated (BDL), n = 16 sham surgery controls) underwent caval subtraction PCMRI to estimate TLBF and HA flow and short-axis cardiac cine MRI for systolic function at baseline, following terlipressin and lipopolysaccharide (LPS) infusion, to model ACLF. Results All baseline hepatic haemodynamic/cardiac systolic function parameters (except heart rate and LV mass) were significantly different in BDL rats. Following terlipressin, baseline PV flow (sham 181.4 ± 12.1 ml/min/100 g; BDL 68.5 ± 10.1 ml/min/100 g) reduced (sham − 90.3 ± 11.1 ml/min/100 g, p < 0.0001; BDL − 31.0 ± 8.0 ml/min/100 g, p = 0.02), sham baseline HA flow (33.0 ± 11.3 ml/min/100 g) increased (+ 92.8 ± 21.3 ml/min/100 g, p = 0.0003), but BDL baseline HA flow (83.8 ml/min/100 g) decreased (− 34.4 ± 7.5 ml/min/100 g, p = 0.11). Sham baseline TLBF (214.3 ± 16.7 ml/min/100 g) was maintained (+ 2.5 ± 14.0 ml/min/100 g, p > 0.99) but BDL baseline TLBF (152.3 ± 18.7 ml/min/100 g) declined (− 65.5 ± 8.5 ml/min/100 g, p = 0.0004). Following LPS, there were significant differences between cohort and change in HA fraction (p = 0.03) and TLBF (p = 0.01) with BDL baseline HA fraction (46.2 ± 4.6%) reducing (− 20.9 ± 7.5%, p = 0.03) but sham baseline HA fraction (38.2 ± 2.0%) remaining unchanged (+ 2.9 ± 6.1%, p > 0.99). Animal cohort and change in systolic function interactions were significant only for heart rate (p = 0.01) and end-diastolic volume (p = 0.03). Conclusions Caval subtraction PCMRI and cardiac MRI in a rodent model of cirrhosis demonstrate significant baseline hepatic haemodynamic/cardiac differences, failure of the HA buffer response post-terlipressin and an altered HA fraction response in sepsis, informing potential translation to ACLF patients. Key Points Caval subtraction phase-contrast and cardiac MRI demonstrate: • Significant differences between cirrhotic/non-cirrhotic rodent hepatic blood flow and cardiac systolic function at baseline. • Failure of the hepatic arterial buffer response in cirrhotic rodents in response to terlipressin. • Reductions in hepatic arterial flow fraction in the setting of acute-on-chronic liver failure.

Introduction To combine numerical simulations, in vitro and in vivo experiments to evaluate the feasibility of measuring diffusion exchange across the cell membrane with diffusion exchange spectroscopy (DEXSY). Methods DEXSY acquisitions were simulated over a range of permeabilities in nerve tissue and yeast substrates. In vitro measurements were performed in a yeast substrate and in vivo measurements in mouse tumor xenograft models, all at 9.4 T. Results Diffusion exchange was observed in simulations over a physiologically relevant range of cell permeability values. In vitro and in vivo measures also provided evidence of diffusion exchange, which was quantified with the Diffusion Exchange Index (DEI). Conclusions Our findings provide preliminary evidence that DEXSY can be used to make in vivo measurements of diffusion exchange and cell membrane permeability.

Nanoparticles have the potential to increase the efficacy of anticancer drugs whilst reducing off-target side effects. However, there remain uncertainties regarding the cellular uptake kinetics of nanoparticles which could have implications for nanoparticle design and delivery. Polymersomes are nanoparticle candidates for cancer therapy which encapsulate chemotherapy drugs. Here we develop a mathematical model to simulate the uptake of polymersomes via endocytosis, a process by which polymersomes bind to the cell surface before becoming internalised by the cell where they then break down, releasing their contents which could include chemotherapy drugs. We focus on two in vitro configurations relevant to the testing and development of cancer therapies: a well-mixed culture model and a tumour spheroid setup. Our mathematical model of the well-mixed culture model comprises a set of coupled ordinary differential equations for the unbound and bound polymersomes and associated binding dynamics. Using a singular perturbation analysis we identify an optimal number of ligands on the polymersome surface which maximises internalised polymersomes and thus intracellular chemotherapy drug concentration. In our mathematical model of the spheroid, a multiphase system of partial differential equations is developed to describe the spatial and temporal distribution of bound and unbound polymersomes via advection and diffusion, alongside oxygen, tumour growth, cell proliferation and viability. Consistent with experimental observations, the model predicts the evolution of oxygen gradients leading to a necrotic core. We investigate the impact of two different internalisation functions on spheroid growth, a constant and a bond dependent function. It was found that the constant function yields faster uptake and therefore chemotherapy delivery. We also show how various parameters, such as spheroid permeability, lead to travelling wave or steady-state solutions.

Noninvasive measurements of liver perfusion and fibrosis in cirrhotic small animals can help develop treatments for haemodynamic complications of liver disease. Here, we measure liver perfusion in cirrhotic rodents using flow‐sensitive alternating inversion recovery arterial spin labelling (FAIR ASL), evaluating agreement with previously validated caval subtraction phase‐contrast magnetic resonance imaging (PCMRI) total liver blood flow (TLBF). Baseline differences in cirrhotic rodents and the haemodynamic effects of acute inflammation were investigated using FAIR ASL and tissue T1. Sprague–Dawley rats (nine bile duct ligated [BDL] and ten sham surgery controls) underwent baseline hepatic FAIR ASL with T1 measurement and caval subtraction PCMRI (with two‐dimensional infra‐/supra‐hepatic inferior vena caval studies), induction of inflammation with intravenous lipopolysaccharide (LPS) and repeat liver FAIR ASL with T1 measurement after ~90 minutes. The mean difference between FAIR ASL hepatic perfusion and caval subtraction PCMRI TLBF was −51 ± 30 ml/min/100 g (Bland–Altman 95% limits‐of‐agreement ±258 ml/min/100 g). The FAIR ASL coefficient of variation was smaller than for caval subtraction PCMRI (29.3% vs 50.1%; P = .03). At baseline, FAIR ASL liver perfusion was lower in BDL rats (199 ± 32 ml/min/100 g vs sham 316 ± 24 ml/min/100 g; P = .01) but liver T1 was higher (BDL 1533 ± 50 vs sham 1256 ± 18 ms; P = .0004). Post‐LPS FAIR ASL liver perfusion response differences were observed between sham/BDL rats (P = .02), approaching significance in sham (+78 ± 33 ml/min/100 g; P = .06) but not BDL rats (−49 ± 40 ml/min/100 g; P = .47). Post‐LPS differences in liver tissue T1 were nonsignificant (P = .35). FAIR ASL hepatic perfusion and caval subtraction PCMRI TLBF agreement was modest, with significant baseline FAIR ASL liver perfusion and tissue T1 differences in rodents with advanced cirrhosis compared with controls. Following inflammatory stress, differences in hepatic perfusion response were detected between cirrhotic/control animals, but liver T1 was unaffected. Findings underline the potential of FAIR ASL in the assessment of vasoactive treatments for patients with chronic liver disease and inflammation., Flow‐sensitive alternating inversion recovery arterial spin labelling (FAIR ASL) was used to measure liver perfusion and T1 at baseline and following inflammatory stress in cirrhotic and control rats. Baseline agreement of FAIR ASL liver perfusion with previously validated caval subtraction phase‐contrast MRI was modest. Following inflammatory stress, significant differences in hepatic perfusion response were detected between cirrhotic and control animals but liver T1 was unaffected, highlighting the potential of FAIR ASL in the assessment of chronic liver disease and inflammation.

Understanding the uptake of a drug by diseased tissue, and the drug's subsequent spatiotemporal distribution, are central factors in the development of effective targeted therapies. However, the interaction between the pathophysiology of diseased tissue and individual therapeutic agents can be complex, and can vary across tissue types and across subjects. Here, we show that the combination of mathematical modelling, high-resolution optical imaging of intact and optically cleared tumour tissue from animal models, and in vivo imaging of vascular perfusion predicts the heterogeneous uptake, by large tissue samples, of specific therapeutic agents, as well as their spatiotemporal distribution. In particular, by using murine models of colorectal cancer and glioma, we report and validate predictions of steady-state blood flow and intravascular and interstitial fluid pressure in tumours, of the spatially heterogeneous uptake of chelated gadolinium by tumours, and of the effect of a vascular disrupting agent on tumour vasculature.

Several distinct fluid flow phenomena occur in solid tumors, including intravascular blood flow and interstitial convection. Interstitial fluid pressure is often raised in solid tumors, which can limit drug delivery. To probe low-velocity flow in tumors resulting from raised interstitial fluid pressure, we developed a novel MRI technique named convection-MRI, which uses a phase-contrast acquisition with a dual-inversion vascular nulling preparation to separate intra- and extravascular flow. Here, we report the results of experiments in flow phantoms, numerical simulations, and tumor xenograft models to investigate the technical feasibility of convection-MRI. We observed a significant correlation between estimates of effective fluid pressure from convection-MRI with gold-standard, invasive measurements of interstitial fluid pressure in mouse models of human colorectal carcinoma. Our results show how convection-MRI can provide insights into the growth and responsiveness to vascular-targeting therapy in colorectal cancers. Significance: A noninvasive method for measuring low-velocity fluid flow caused by raised fluid pressure can be used to assess changes caused by therapy. Cancer Res; 78(7); 1859–72. ©2018 AACR.

The neurovascular mechanisms underpinning the local regulation of cerebral blood flow (CBF) and oxygen transport remain elusive. In this study we have combined novel in vivo imaging of cortical microvascular and mural cell architecture with mathematical modelling of blood flow and oxygen transport, to provide new insights into CBF regulation that would be inaccessible in a conventional experimental context. Our study implicates vasomotion of smooth muscle actin-covered vessels, rather than pericyte-covered capillaries, as the main mechanism for modulating tissue oxygenation. We also resolve seemingly paradoxical observations in the literature around reduced blood velocity in response to arteriolar constrictions and deduce the cause to be propagation of constrictions to upstream penetrating arterioles. We provide support for pericytes acting as signalling conduits for upstream smooth muscle activation, and erythrocyte deformation as a complementary regulatory mechanism. Finally, we caution against the use of blood velocity as a proxy measurement for flow. Our combined imaging-modelling platform complements conventional experimentation allowing cerebrovascular physiology to be probed in unprecedented detail.

Heterogeneity of lymphatic vessels during embryogenesis is critical for organ-specific lymphatic function. Little is known about lymphatics in the developing kidney, despite their established roles in pathology of the mature organ. We performed three-dimensional imaging to characterize lymphatic vessel formation in the mammalian embryonic kidney at single-cell resolution. In mouse, we visually and quantitatively assessed the development of kidney lymphatic vessels, remodeling from a ring-like anastomosis under the nascent renal pelvis; a site of VEGF-C expression, to form a patent vascular plexus. We identified a heterogenous population of lymphatic endothelial cell clusters in mouse and human embryonic kidneys. Exogenous VEGF-C expanded the lymphatic population in explanted mouse embryonic kidneys. Finally, we characterized complex kidney lymphatic abnormalities in a genetic mouse model of polycystic kidney disease. Our study provides novel insights into the development of kidney lymphatic vasculature; a system which likely has fundamental roles in renal development, physiology and disease., eLife digest In most organs in the body, fluid tends to build up in the spaces between cells, especially if the organs become inflamed. Each organ has a ‘waste disposal system’; a set of specialized tubes called lymphatic vessels, to clear away this excess fluid and keep a check on inflammation. Defects in these tubes have been linked to a wide range of diseases including heart attacks, obesity, dementia and cancer. The kidneys are responsible for filtering blood and balancing many of the body’s chemical processes. Polycystic kidney disease (PKD) is the most common genetic kidney disorder and it results in cysts filled with fluid building up in the kidney. The growth of cysts in PKD may be due to a problem with the lymphatic vessels. However, compared to other organs, how lymphatic vessels first form within the kidney and what they do is not well understood. Now, Jafree et al. have used three-dimensional imaging to study how lymphatic vessels form in the kidneys of mice and humans. The experiments showed that lymphatic vessels first appear when mouse kidneys are about half developed, and start to grow rapidly when the kidneys are thought to begin filtering blood. Clusters of cells that may help lymphatic vessels to grow were also found hidden deep within the kidneys of mouse embryos. Treating the kidneys with a factor that stimulates the growth of lymphatic vessels increased the numbers of these clusters. Jafree et al. found similar clusters of cells in human kidneys, suggesting that lymphatic vessels in the kidneys of different mammals may develop in the same way. Further experiments showed that the lymphatic vessels of kidneys in mice with PKD become distorted early on in the disease, when cysts are still small and before the mice develop symptoms. In the future, identifying drugs that target kidney lymphatic vessels may lead to more effective treatments for patients with PKD and other kidney diseases.

Cancers exhibit spatially heterogeneous, unique vascular architectures across individual samples, cell-lines and patients. This inherently disorganised collection of leaky blood vessels contribute significantly to suboptimal treatment efficacy. Preclinical tools are urgently required which incorporate the inherent variability and heterogeneity of tumours to optimise and engineer anti-cancer therapies. In this study, we present a novel computational framework which incorporates whole, realistic tumours extracted ex vivo to efficiently simulate vascular blood flow and interstitial fluid transport in silico for validation against in vivo biomedical imaging. Our model couples Poiseuille and Darcy descriptions of vascular and interstitial flow, respectively, and incorporates spatially heterogeneous blood vessel lumen and interstitial permeabilities to generate accurate predictions of tumour fluid dynamics. Our platform enables highly-controlled experiments to be performed which provide insight into how tumour vascular heterogeneity contributes to tumour fluid transport. We detail the application of our framework to an orthotopic murine glioma (GL261) and a human colorectal carcinoma (LS147T), and perform sensitivity analysis to gain an understanding of the key biological mechanisms which determine tumour fluid transport. Finally we mimic vascular normalization by modifying parameters, such as vascular and interstitial permeabilities, and show that incorporating realistic vasculatures is key to modelling the contrasting fluid dynamic response between tumour samples. Contrary to literature, we show that reducing tumour interstitial fluid pressure is not essential to increase interstitial perfusion and that therapies should seek to develop an interstitial fluid pressure gradient. We also hypothesise that stabilising vessel diameters and permeabilities are not key responses following vascular normalization and that therapy may alter interstitial hydraulic conductivity. Consequently, we suggest that normalizing the interstitial microenvironment may provide a more effective means to increase interstitial perfusion within tumours., Author summary The structure of tumours varies widely, with dense and chaotically-formed networks of blood vessels that differ between each individual tumour and even between different regions of the same tumour. This atypical environment can inhibit the delivery of anti-cancer therapies. Computational tools are urgently required which facilitate a deeper understanding of the relationship between blood vessel architectures and therapeutic response. We have developed a computational framework which integrates the complex tumour vascular architecture to predict fluid transport across all lengths scales in whole tumours. We apply our model to two tumour cell-lines and show that differences in their inherent vascular structures influence flow through cancerous tissue. We also use our platform to predict the fluid dynamic response following vascular normalization therapy in realistic, static tumour networks and show that the response is dependent on tumour vascular architecture. We hypothesise that therapy may alter the permeability of interstitial tissue to fluid transport and show that lowering interstitial fluid pressure is not a necessary therapeutic outcome to increase tumour perfusion.

OBJECTIVES: Caval subtraction phase-contrast magnetic resonance imaging (PCMRI) noninvasive measurements of total liver blood flow (TLBF) and hepatic arterial (HA) flow have been validated in animal models and translated into normal volunteers, but not patients. This study aims to demonstrate its use in patients with liver cirrhosis, evaluate measurement consistency, correlate measurements with portal hypertension severity, and invasively validate TLBF measurements. MATERIALS AND METHODS: Local research ethics committee approval was obtained. Twelve patients (mean, 50.8 ± 3.1 years; 10 men) with histologically confirmed cirrhosis were recruited prospectively, undergoing 2-dimensional PCMRI of the portal vein (PV) and the infrahepatic and suprahepatic inferior vena cava. Total liver blood flow and HA flow were estimated by subtracting infrahepatic from suprahepatic inferior vena cava flow and PV flow from estimated TLBF, respectively. Invasive hepatic venous pressure gradient (HVPG) and indocyanine green (ICG) clearance TLBF were measured within 7 days of PCMRI. Bland-Altman (BA) analysis of agreement, coefficients of variation, and Pearson correlation coefficients were calculated for comparisons with direct inflow PCMRI, HVPG, and ICG clearance. RESULTS: The mean difference between caval subtraction TLBF and direct inflow PCMRI was 6.3 ± 4.2 mL/min/100 g (BA 95% limits of agreement, ±28.7 mL/min/100 g). Significant positive correlations were observed between HVPG and caval subtraction HA fraction (r = 0.780, P = 0.014), but not for HA flow (r = 0.625, P = 0.053), PV flow (r = 0.244, P = 0.469), or caval subtraction TLBF (r = 0.473, P = 0.141). Caval subtraction and ICG TLBF agreement was modest (mean difference, -32.6 ± 16.6 mL/min/100 g; BA 95% limits of agreement, ±79.7 mL/min/100 g), but coefficients of variation were not different (65.7% vs 48.1%, P = 0.28). CONCLUSIONS: In this proof-of-principle study, caval subtraction PCMRI measurements are consistent with direct inflow PCMRI, correlate with portal hypertension severity, and demonstrate modest agreement with invasive TLBF measurements. Larger studies investigating the clinical role of TLBF and HA flow measurement in patients with liver disease are justified.

Alzheimer’s disease can involve brain copper dyshomeostasis. We aimed to determine the effect of AD-like pathology on 64Cu trafficking in mice, using positron emission tomography (PET imaging), during 24 hours after intravenous administration of ionic 64Cu (Cu(II) acetate) and 64Cu-GTSM (GTSMH2 = glyoxalbis(thiosemicarbazone)). Copper trafficking was evaluated in 6-8-month-old and 13-15 month-old TASTPM transgenic and wild-type mice, by imaging 0-30 min and 24-25 h after intravenous administration of 64Cu tracer. Regional 64Cu distribution in brains was compared by ex vivo autoradiography to that of amyloid-β plaque. 64Cu-acetate showed uptake in, and excretion through, liver and kidneys. There was minimal uptake in other tissues by 30 minutes, and little further change after 24 h. Radioactivity within brain was focussed in and around the ventricles and was significantly greater in younger mice. 64CuGTSM was taken up in all tissues by 30 min, remaining high in brain but clearing substantially from other tissues by 24 h. Distribution in brain was not localised to specific regions. TASTPM mice showed no major changes in global or regional 64Cu brain uptake compared to wildtype after administration of 64Cu acetate (unlike 64Cu-GTSM) but efflux of 64Cu from brain by 24 h was slightly greater in 6-8 month-old TASTPM mice than in wildtype controls. Changes in copper trafficking associated with Alzheimer’s-like pathology after administration of ionic 64Cu are minor compared to those observed after administration of 64Cu-GTSM. PET imaging with 64Cu could help understand changes in brain copper dynamics in AD and underpin new clinical diagnostic imaging methods.

The aim of this study was to use the combined carbogen-ultrasmall superparamagnetic iron oxide (CUSPIO) magnetic resonance imaging (MRI) method, which uses spatial correlations in independent susceptibility imaging biomarkers, to investigate and compare the impact of tumor size and anatomical site on vascular structure and function in vivo. Mice bearing either subcutaneous or orthotopic PC3 LN3 prostate tumors were imaged at 7 T, using a multi-gradient echo sequence to quantify R2∗, before and during carbogen (95% O2/5% CO2) breathing, and subsequently following intravenous administration of USPIO particles. Carbogen and USPIO-induced changes in R2∗ were used to inform on hemodynamic vasculature and fractional blood volume (%), respectively. The CUSPIO imaging data were also segmented to identify and assess five categories of R2∗ response. Small and large subcutaneous and orthotopic tumor cohorts all exhibited significantly (P

The recently described combined carbogen USPIO (CUSPIO) magnetic resonance imaging (MRI) method uses spatial correlations in independent imaging biomarkers to assess specific components of tumor vascular structure and function. Our study aimed to evaluate CUSPIO biomarkers for the assessment of tumor response to antiangiogenic therapy. CUSPIO imaging was performed in subcutaneous rat C6 gliomas before and 2 days after treatment with the potent VEGF-signaling inhibitor cediranib (n = 12), or vehicle (n = 12). Histological validation of Hoechst 33342 uptake (perfusion), smooth muscle actin staining (maturation), pimonidazole adduct formation (hypoxia) and necrosis were sought. Following treatment, there was a significant decrease in fractional blood volume (-43%, p < 0.01) and a significant increase in hemodynamic vascular functionality (treatment alteredδ R 2* carbogen from 1.2 to -0.2 s -1, p < 0.05). CUSPIO imaging revealed an overall significant decrease in plasma perfusion (-27%, p < 0.05) following cediranib treatment, that was associated with selective effects on immature blood vessels. The CUSPIO responses were associated with a significant 15% reduction in Hoechst 33342 uptake (p < 0.05), but no significant difference in vascular maturation or necrosis. Additionally, treatment with cediranib resulted in a significant 40% increase in tumor hypoxia (p < 0.05). The CUSPIO imaging method provides novel and more specific biomarkers of tumor vessel maturity and vascular hemodynamics, and their response to VEGF-signaling inhibition, compared to current MR imaging biomarkers utilized in the clinic. Such biomarkers may prove effective in longitudinally monitoring tumor vascular remodeling and/or evasive resistance in response to antiangiogenic therapy. © 2011 UICC.

Vessel size index (Rv, μm) has been proposed as a quantitative magnetic resonance imaging (MRI) derived imaging biomarker in oncology, for the non-invasive assessment of tumour blood vessel architecture and vascular targeted therapies. Appropriate pre-clinical evaluation of Rv in animal tumour models will improve the interpretation and guide the introduction of the biomarker into clinical studies. The objective of this study was to compare Rv measured in vivo with vessel size measurements from high-resolution X-ray computed tomography (μCT) of vascular corrosion casts measured post mortem from the same tumours, with and without vascular targeted therapy. MRI measurements were first acquired from subcutaneous SW1222 colorectal xenografts in mice following treatment with 0 (n = 6), 30 (n = 6) or 200 mg/kg (n = 3) of the vascular disrupting agent ZD6126. The mice were then immediately infused with a low viscosity resin and, following polymerisation and maceration of surrounding tissues, the resulting tumour vascular casts were dissected and subsequently imaged using an optimised μCT imaging approach. Vessel diameters were not measurable by μCT in the 200 mg/kg group as the high dose of ZD6126 precluded delivery of the resin to the tumour vascular bed. The mean Rv for the three treatment groups was 24, 23 and 23.5 μm respectively; the corresponding μCT measurements from corrosion casts from the 0 and 30 mg/kg cohorts were 25 and 28 μm. The strong association between the in vivo MRI and post mortem μCT values supports the use of Rv as an imaging biomarker in clinical trials of investigational vascular targeted therapies., Highlights ► Non-invasive quantitation of vessel calibre in tumour xenografts in vivo ► Assessment of tumour vessel calibre response to a vascular disrupting agent ► Generation of vascular corrosion casts from the same tumours imaged by MRI ► Quantitation of vessel calibre from corrosion casts by microCT ► Excellent agreement between the in vivo MRI and post mortem microCT vessel calibres

Dimethylarginine dimethylaminohydrolase (DDAH) metabolizes the endogenous inhibitor of nitric oxide synthesis, asymmetric dimethylarginine (ADMA). Constitutive over-expression of DDAH1, the isoform primarily associated with neuronal nitric oxide synthase (nNOS) results in increased tumour growth and vascularization, and elevated VEGF secretion. To address whether DDAH1-mediated tumour growth is reliant upon the enzymatic activity of DDAH1, cell lines expressing an active site mutant of DDAH1 incapable of metabolizing ADMA were created. Xenografts derived from these cell lines grew significantly faster than those derived from control cells, yet not as fast as those over-expressing wild-type DDAH1. VEGF expression in DDAH1 mutant-expressing tumours did not differ from control tumours but was significantly lower than that of wild-type DDAH1-over-expressing tumours. Fluorescence microscopy for CD31 and pimonidazole adduct formation demonstrated that DDAH1 mutant-expressing tumours had a lower endothelial content and demonstrated less hypoxia, respectively, than wild-type DDAH1-expressing tumours. However, there was no difference in uptake of the perfusion marker Hoechst 33342. Non-invasive multiparametric quantitative MRI, including the measurement of native T(1) and T(2) relaxation times and apparent water diffusion coefficient, was indicative of higher cellularity in DDAH1-expressing xenografts, which was confirmed by histological quantification of necrosis. C6 xenografts expressing active site mutant DDAH1 displayed an intermediate phenotype between tumours over-expressing wild-type DDAH1 and control tumours. These data suggest that enhanced VEGF expression downstream of DDAH1 was dependent upon ADMA metabolism, but that the DDAH1-mediated increase in tumour growth was only partially dependent upon its enzymatic activity, and therefore must involve an as-yet unidentified mechanism. DDAH1 is an important mediator of tumour progression, but appears to have addition roles independent of its metabolism of ADMA, which need to be considered in therapeutic strategies targeted against the NO/DDAH pathway in cancer.

Background Research using orthotopic and transgenic models of cancer requires imaging methods to non-invasively quantify tumour burden. As the choice of appropriate imaging modality is wide-ranging, this study aimed to compare low-field (1T) magnetic resonance imaging (MRI), a novel and relatively low-cost system, against established preclinical techniques: bioluminescence imaging (BLI), ultrasound imaging (US), and high-field (9.4T) MRI. Methods A model of colorectal metastasis to the liver was established in eight mice, which were imaged with each modality over four weeks post-implantation. Tumour burden was assessed from manually segmented regions. Results All four imaging systems provided sufficient contrast to detect tumours in all of the mice after two weeks. No significant difference was detected between tumour doubling times estimated by low-field MRI, ultrasound imaging or high-field MRI. A strong correlation was measured between high-field MRI estimates of tumour burden and all the other modalities (p < 0.001, Pearson). Conclusion These results suggest that both low-field MRI and ultrasound imaging are accurate modalities for characterising the growth of preclinical tumour models.

To establish the experimental factors that dominate the uncertainty of hemodynamic parameters in commonly used pharmacokinetic models.By fitting simulation results from a multiregion tissue exchange model (Multiple path, Multiple tracer, Indicator Dilution, 4 region), the precision and accuracy of hemodynamic parameters in dynamic contrast-enhanced MRI with four tracer kinetic models is investigated. The impact of various injection rates as well as imprecise knowledge of the arterial input functions is examined.Fast injections are beneficial for K(trans) precision within the extended Tofts model and within the two-compartment exchange model but do not affect the other models under investigation. Biases from errors in the arterial input functions are mostly consistent in size and direction for the simple and the extended Tofts model, while they are hardly predictable for the other models. Errors in the hematocrit introduce the greatest loss in parameter accuracy, amounting to an average K(trans) bias of 40% for a 30% overestimation throughout all models.This simulation study allows the detailed inspection of the isolated impact from various experimental conditions on parameter uncertainty. Because parameter uncertainty comparable to human studies was found, this study represents a validation of preclinical dynamic contrast-enhanced MRI for modeling human tumor physiology.

There is a need for biomarkers that are useful for noninvasive imaging of tumor pathophysiology and drug efficacy. Through its use of endogenous water, diffusion-weighted MRI (DW-MRI) can be used to probe local tissue architecture and structure. However, most DW-MRI studies of cancer tissues have relied on simplistic mathematical models, such as apparent diffusion coefficient (ADC) or intravoxel incoherent motion (IVIM) models, which produce equivocal results on the relation of the model parameter estimate with the underlying tissue microstructure. Here, we present a novel technique called VERDICT (Vascular, Extracellular and Restricted Diffusion for Cytometry in Tumors) to quantify and map histologic features of tumors in vivo. VERDICT couples DW-MRI to a mathematical model of tumor tissue to access features such as cell size, vascular volume fraction, intra- and extracellular volume fractions, and pseudo-diffusivity associated with blood flow. To illustrate VERDICT, we used two tumor xenograft models of colorectal cancer with different cellular and vascular phenotypes. Our experiments visualized known differences in the tissue microstructure of each model and the significant decrease in cell volume resulting from administration of the cytotoxic drug gemcitabine, reflecting the apoptotic volume decrease. In contrast, the standard ADC and IVIM models failed to detect either of these differences. Our results illustrate the superior features of VERDICT for cancer imaging, establishing it as a noninvasive method to monitor and stratify treatment responses.

BACKGROUND: Non-invasive imaging biomarkers underpin the development of molecularly targeted anti-cancer drugs. This study evaluates tumour apparent diffusion coefficient (ADC), measured by diffusion-weighted magnetic resonance imaging (DW-MRI), as a biomarker of response to the MEK1/2 inhibitor selumetinib (AZD6244, ARRY-142886) in human tumour xenografts. METHODS: Nude mice bearing human BRAF(V600D) WM266.4 melanoma or BRAF(V600E) Colo205 colon carcinoma xenografts were treated for 4 days with vehicle or selumetinib. DW-MRI was performed before and 2 h after the last dose and excised tumours analysed for levels of phospho-ERK1/2, cleaved caspase 3 (CC3) and necrosis. RESULTS: Selumetinib treatment induced tumour stasis and reduced ERK1/2 phosphorylation in both WM266.4 and Colo205 tumour xenografts. Relative to day 0, mean tumour ADC was unchanged in the control groups but was significantly increased by up to 1.6-fold in selumetinib-treated WM266.4 and Colo205 tumours. Histological analysis revealed a significant increase in necrosis in selumetinib-treated WM266.4 and Colo205 xenografts and CC3 staining in selumetinib-treated Colo205 tumours relative to controls. CONCLUSION: Changes in ADC following treatment with the MEK1/2 inhibitor selumetinib in responsive human tumour xenografts were concomitant with induction of tumour cell death. ADC may provide a useful non-invasive pharmacodynamic biomarker for early clinical assessment of response to selumetinib and other MEK-ERK1/2 signalling-targeted therapies.

Background: Solid tumours can undergo cycles of hypoxia, followed by reoxygenation, which can have significant implications for the success of anticancer therapies. A need therefore exists to develop methods to aid its detection and to further characterise its biological basis. We present here a novel method for decomposing systemic and tumour-specific contributions to fluctuations in tumour deoxyhaemoglobin concentration, based on magnetic resonance imaging measurements. Methods: Fluctuations in deoxyhaemoglobin concentration in two tumour xenograft models of colorectal carcinoma were decomposed into distinct contributions using independent component analysis. These components were then correlated with systemic pulse oximetry measurements to assess the influence of systemic variations in blood oxygenation in tumours, compared with those that arise within the tumour itself (tumour-specific). Immunohistochemical staining was used to assess the physiological basis of each source of fluctuation. Results: Systemic fluctuations in blood oxygenation were found to contribute to cycling hypoxia in tumours, but tumour-specific fluctuations were also evident. Moreover, the size of the tumours was found to influence the degree of systemic, but not tumour-specific, oscillations. The degree of vessel maturation was related to the amplitude of tumour-specific, but not systemic, oscillations. Conclusions: Our results provide further insights into the complexity of spontaneous fluctuations in tumour oxygenation and its relationship with tumour pathophysiology. These observations could be used to develop improved drug delivery strategies.

To evaluate and histologically qualify carbogen-induced ΔR2 as a noninvasive magnetic resonance imaging biomarker of improved tumor oxygenation using a double 2-nitroimidazole hypoxia marker approach.Multigradient echo images were acquired from mice bearing GH3 prolactinomas, preadministered with the hypoxia marker CCI-103F, to quantify tumor R2 during air breathing. With the mouse remaining positioned within the magnet bore, the gas supply was switched to carbogen (95% O2, 5% CO2), during which a second hypoxia marker, pimonidazole, was administered via an intraperitoneal line, and an additional set of identical multigradient echo images acquired to quantify any changes in tumor R2. Hypoxic fraction was quantified histologically using immunofluorescence detection of CCI-103F and pimonidazole adduct formation from the same whole tumor section. Carbogen-induced changes in tumor pO2 were further validated using the Oxylite fiberoptic probe.Carbogen challenge significantly reduced mean tumor R2 from 116 ± 13 s(-1) to 97 ± 9 s(-1) (P.05). This was associated with a significantly lower pimonidazole adduct area (2.3 ± 1%), compared with CCI-103F (6.3 ± 2%) (P.05). A significant correlation was observed between ΔR2 and Δhypoxic fraction (r=0.55, P.01). Mean tumor pO2 during carbogen breathing significantly increased from 6.3 ± 2.2 mm Hg to 36.0 ± 7.5 mm Hg (P.01).The combined use of intrinsic susceptibility magnetic resonance imaging with a double hypoxia marker approach corroborates carbogen-induced ΔR2 as a noninvasive imaging biomarker of increased tumor oxygenation.

To investigate the combined use of hyperoxia-inducedΔR(2) * and ΔR(1) as a noninvasive imaging biomarker of tumor hypoxia.MRI was performed on rat GH3 prolactinomas (n = 6) and human PC3 prostate xenografts (n = 6) propagated in nude mice. multiple gradient echo and inversion recovery truefisp images were acquired from identical transverse slices to quantify tumor R(2) * and R(1)before and during carbogen (95% O2 /5% CO2 ) challenge, and correlates of ΔR(2) * and ΔR(1) assessed.Mean baseline R(2) * and R(1) were 119 ± 7 s(-1) and 0.6 ± 0.03 s(-1) for GH3 prolactinomas and 77 ± 12 s(-1) and 0.7 ± 0.02 s(-1) for PC3 xenografts, respectively. During carbogen breathing, mean ΔR(2) * and ΔR(1) were -20 ± 8 s(-1) and 0.08 ± 0.03 s(-1) for GH3 and -0.5 ± 1 s(-1) and 0.2 ± 0.08 s(-1) for the PC3 tumors, respectively. A pronounced relationship betweenΔR(2) * and ΔR(1) was revealed.Considering the blood oxygen-hemoglobin dissociation curve, fast R2 * suggested that GH3 prolactinomas were more hypoxic at baseline, and their carbogen response dominated by increased hemoglobin oxygenation, evidenced by highly negative ΔR(2) *. PC3 tumors were less hypoxic at baseline, and their response to carbogen dominated by increased dissolved oxygen, evidenced by highly positive ΔR(1) . Because the two biomarkers are sensitive to different oxygenation ranges, the combination of ΔR(2) * and ΔR(1) may better characterize tumor hypoxia than each alone.

Worldwide efforts to understand developmental processes demand new high-resolution 3D imaging methods to detect the consequences of gene function in embryo development and diseases. Encouragingly, recent studies have shown that MRI contrast agents can highlight specific tissue structures in ex vivo adult mouse brains. MR imaging of mouse embryos is currently limited by a lack of tissue staining capabilities that would provide the flexibility and specificity offered by histological stains conventionally used for mouse embryo phenotyping.The MRI staining properties of two readily available contrast agents, Mn-DPDP and Gd-DTPA, were investigated in mid-gestation mouse embryos.Brain tissue substructures not normally visible using MRI were detected. Mn-DPDP and Gd-DTPA provided spatially distinct tissue staining patterns. An initial assessment indicated that these agents utilized independent contrast enhancement mechanisms. Mn-DPDP was identified as a potential MRI contrast agent for enhancement of mouse embryonic cellular density and enabled identification of regions containing populations of neural stem and progenitor cells within the intact embryo brain.Different contrast agents may be used to provide tissue-specific contrast enhancement, suggesting that a host of specialized MRI stains may be available for probing the developing mouse brain and investigating developmental and disease mechanisms.

BACKGROUND: Non-invasive quantitative imaging biomarkers are essential for the evaluation of novel targeted therapeutics. Before deployment in clinical trials, such imaging biomarkers require qualification, typically through pre-clinical identification of imaging-pathology correlates. METHODS: First, in investigating imaging biomarkers of invasion, the response of orthotopic murine PC3 prostate xenografts to the Src inhibitor saracatinib was assessed using susceptibility contrast MRI. Second, the longitudinal response of chemically induced rat mammary adenocarcinomas to the VEGFR2 inhibitor vandetanib was monitored by intrinsic susceptibility MRI, to identify the time window of transient vascular normalisation. RESULTS: No significant differences in fractional blood volume (%), vessel calibre (μm), native T(1) (ms) or apparent water diffusion coefficient were determined, despite reduced expression of activated Fak and paxillin in the saracatinib cohort. Treatment with vandetanib elicited a 60% antitumour response (P

Background: Hypoxia-inducible factor-1 (HIF-1) mediates the transcriptional response to hypoxic stress, promoting tumour progression and survival. This study investigated the acute effects of the small-molecule HIF-pathway inhibitor NSC-134754. Methods: Human PC-3LN5 prostate cancer cells were treated with NSC-134754 for 24 h in hypoxia. Orthotopic prostate tumour-bearing mice were treated with a single dose of NSC-134754 for 6, 24 or 48 h. Treatment response was measured using magnetic resonance spectroscopy and imaging. Ex-vivo histological validation of imaging findings was also sought. Results: In vitro, NSC-134754 significantly reduced lactate production and glucose uptake (P

Chemical exchange saturation transfer (CEST) imaging has been used experimentally in a large range of applications. However, full quantification of CEST effects in vivo using standard imaging sequences is time consuming as a large number of saturation frequency offsets, each followed by an imaging readout, are required to define a z spectrum. Furthermore, outside the brain, the presence of fat can confound the interpretation of z spectra. A novel acquisition and post-processing technique is presented in this study, named exchange-modulated point-resolved spectroscopy (EXPRESS), which aims to address these limitations and to enable spatially localised, high signal-to-noise measurements of CEST effects in vivo. Using amide proton exchange (APT) measurements in tumours, it is demonstrated that the acquisition of two-dimensional EXPRESS spectra composed of chemical shift and saturation frequency offset dimensions allows the correction of CEST data containing both fat and water signals, which is a common confounding property of tissues found outside the brain.

Susceptibility contrast magnetic resonance imaging (MRI), utilising ultrasmall superparamagnetic iron oxide (USPIO) particles, was evaluated for the quantitation of vessel size index (Rv, mu m), a weighted average measure of tumour blood vessel calibre, and fractional tumour blood volume (fBV, %), in orthotopically propagated murine PC3 prostate tumour xenografts. Tumour vascular architecture was assessed in vivo by MRI prior to and 24 hr after treatment with 200 mg/kg of the vascular disrupting agent ZD6126. A Bayesian hierarchical model (BHM) was used to reduce the uncertainty associated with quantitation of Rv and fBV. Quantitative histological analyses of the uptake of Hoechst 33342 for perfused vasculature, and haematoxylin and eosin staining for necrosis, were also performed to qualify the MRI data. A relatively large median Rv of 40.3 38.mu m (90% confidence interval (CI90) = 37.4, 44.0 mu m) and a high fBV of 5.4% (CI90 = 5.3, 5.5%) were determined in control tumours, which agreed with histologically determined vessel size index. Treatment with ZD6126 significantly (p < 0.01) reduced tumour Rv (34.2 mu m, CI90 = 31.2, 38.0 mu m) and fBV (3.9%, CI90 = 3.8, 4.1%), which were validated against histologically determined significant reductions in perfusion and vessel size, and increased necrosis. Together these data (i) highlight the use of a BHM to optimise the inferential power available from susceptibility contrast MRI data, (ii) provide strong evaluation and qualification of Rv and fBV as non-invasive imaging biomarkers of tumour vascular morphology, (iii) reveal the presence of a different vascular phenotype and (iv) demonstrate that ZD6126 exhibits good anti-vascular activity against orthotopic prostate tumours.

Although the biasing of R(2)* estimates by assuming magnitude MR data to be normally distributed has been described, the effect on changes in R(2)* (DeltaR(2)*), such as induced by a paramagnetic contrast agent, has not been reported. In this study, two versions of a novel Bayesian maximum a posteriori approach for estimating DeltaR(2)* are described and evaluated: one that assumes normally distributed data and the other, Rice-distributed data. The approach enables the robust, voxelwise determination of the uncertainty in DeltaR(2)* estimates and provides a useful statistical framework for quantifying the probability that a pixel has been significantly enhanced. This technique was evaluated in vivo, using ultrasmall superparamagnetic iron oxide particles in orthotopic murine prostate tumors. It is shown that assuming magnitude data to be normally distributed causes DeltaR(2)* to be underestimated when signal-to-noise ratio is modest. However, the biasing effect is less than is found in R(2)* estimates, implying that the simplifying assumption of normally distributed noise is more justifiable when evaluating DeltaR(2)* compared with when evaluating precontrast R(2)* values.

A combined carbogen ultrasmall superparamagnetic iron oxide (USPIO) imaging protocol was developed and applied in vivo in two murine colorectal tumor xenograft models, HCT116 and SW1222, with established disparate vascular morphology, to investigate whether additional information could be extracted from the combination of two susceptibility MRI biomarkers. Tumors were imaged before and during carbogen breathing and subsequently following intravenous administration of USPIO particles. A novel segmentation method was applied to the image data, from which six categories of R(2)* response were identified, and compared with histological analysis of the vasculature. In particular, a strong association between a negative ΔR(2)*(carbogen) followed by positive ΔR(2)*(USPIO) with the uptake of the perfusion marker Hoechst 33342 was determined. Regions of tumor tissue where there was a significant ΔR(2)*(carbogen) but no significant ΔR(2)*(USPIO) were also identified, suggesting these regions became temporally isolated from the vascular supply during the experimental timecourse. These areas correlated with regions of tumor tissue where there was CD31 staining but no Hoechst 33342 uptake. Significantly, different combined carbogen USPIO responses were determined between the two tumor models. Combining ΔR(2)*(carbogen) and ΔR(2)*(USPIO) with a novel segmentation scheme can facilitate the interpretation of susceptibility contrast MRI data and enable a deeper interrogation of tumor vascular function and architecture.

The spatial distribution of apparent diffusion coefficient (ADC) estimates in tumors is typically heterogeneous, although this observed variability is composed of both true regional differences and random measurement uncertainty. In this study, an adaptive Bayesian adaptive smoothing (BAS) model for estimating ADC values is developed and applied to data acquired in two murine tumor models in vivo. BAS models have previously been shown to reduce parameter uncertainty through the use of a Markov random field. Here, diffusion data acquired with four averages was used as an empirical gold standard for evaluating the BAS model. ADC estimates using BAS displayed a significantly closer accordance with the gold standard data and, following analysis of uncertainty estimates, appeared to even outperform the gold standard. These observations were also reflected in simulations. These results have strong implications for clinical studies, as it suggests that the BAS postprocessing technique can be used to improve ADC estimates without the need to compromise on spatial resolution or signal-to-noise or for the adaptation of acquisition hardware. A novel measure of tumor ADC heterogeneity was also defined, which identified differences between tumors derived from different cell lines, which were reflected in histological variations within the tissue microenvironment.

Optical clearing, in combination with recently developed optical imaging techniques, enables visualization and acquisition of high-resolution, three-dimensional images of biological structures deep within the tissue. Many different approaches can be used to reduce light absorption and scattering within the tissue, but there is a paucity of research on the quantification of clearing efficacy. With the use of a custom-made spectroscopy system, we developed a way to quantify the quality of clearing in biological tissue and applied it to the mouse brain. Three clearing techniques were compared: BABB (1:2 mixture of benzyl alcohol and benzyl benzoate, also known as Murray’s clear), pBABB (peroxide BABB, a modification of BABB which includes the use of hydrogen peroxide), and passive CLARITY. We found that BABB and pBABB produced the highest degree of optical clearing. Furthermore, the approach allows regional measurement of light attenuation to be performed, and our results show that light is most attenuated in regions with high lipid content. We provide a way to choose between the multiple clearing protocols available, and it could prove useful for evaluating images that are acquired with cleared tissues.

Angiogenesis, the development of new blood vessels, is essential for tumour growth; this process is stimulated by the secretion of numerous growth factors including platelet derived growth factor (PDGF). PDGF signalling, through its receptor platelet derived growth factor receptor (PDGFR), is involved in vessel maturation, stimulation of angiogenesis and upregulation of other angiogenic factors, including vascular endothelial growth factor (VEGF). PDGFR is a promising target for anti-cancer therapy because it is expressed on both tumour cells and stromal cells associated with the vasculature. MLN0518 (tandutinib) is a potent inhibitor of type III receptor tyrosine kinases that demonstrates activity against PDGFRα/β, FLT3 and c-KIT. In this study a multi-parametric MRI and histopathological approach was used to interrogate changes in vascular haemodynamics, structural response and hypoxia in C6 glioma xenografts in response to treatment with MLN0518. The doubling time of tumours in mice treated with MLN0518 was significantly longer than tumours in vehicle treated mice. The perfused vessel area, number of alpha smooth muscle actin positive vessels and hypoxic area in MLN0518 treated tumours were also significantly lower after 10 days treatment. These changes were not accompanied by alterations in vessel calibre or fractional blood volume as assessed using susceptibility contrast MRI. Histological assessment of vessel size and total perfused area did not demonstrate any change with treatment. Intrinsic susceptibility MRI did not reveal any difference in baseline R2* or carbogen-induced change in R2*. Dynamic contrast-enhanced MRI revealed anti-vascular effects of MLN0518 following 3 days treatment. Hypoxia confers chemo- and radio-resistance, and alongside PDGF, is implicated in evasive resistance to agents targeted against VEGF signalling. PDGFR antagonists may improve potency and efficacy of other therapeutics in combination. This study highlights the challenges of identifying appropriate quantitative imaging response biomarkers in heterogeneous models, particularly considering the multifaceted roles of angiogenic growth factors.

To aid preclinical development of novel therapeutics for myeloma, an in vivo model which recapitulates the human condition is required. An important feature of such a model is the interaction of myeloma cells with the bone marrow microenvironment, as this interaction modulates tumour activity and protects against drug-induced apoptosis. Therefore NOD/SCIDγc(null) mice were injected intra-tibially with luciferase-tagged myeloma cells. Disease progression was monitored by weekly bioluminescent imaging (BLI) and measurement of paraprotein levels. Results were compared with magnetic resonance imaging (MRI) and histology. Assessment of model suitability for preclinical drug testing was investigated using bortezomib, melphalan and two novel agents. Cells engrafted at week 3, with a significant increase in BLI radiance occurring between weeks 5 and 7. This was accompanied by an increase in paraprotein secretion, MRI-derived tumour volume and CD138 positive cells within the bone marrow. Treatment with known anti-myeloma agents or novel agents significantly attenuated the increase in all disease markers. In addition, intra-tibial implantation of primary patient plasma cells resulted in development of myeloma within bone marrow. In conclusion, using both myeloma cell lines and primary patient cells, we have developed a model which recapitulates human myeloma by ensuring the key interaction of tumour cells with the microenvironment.

Objectives To use primed infusions of the magnetic resonance imaging (MRI) contrast agent Gd.DTPA (Magnevist), to achieve an equilibrium between blood and tissue (eqMRI). This may increase tumor Gd concentrations as a novel cancer imaging methodology for the enhancement of small tumor nodules within the low signal-to-noise background of the lung. Methods A primed infusion with a delay before equilibrium (eqMRI) of the Gd(III) chelator Gd.DTPA, via the intraperitoneal route, was used to evaluate gadolinium tumor enhancement as a function of a bolus injection, which is applied routinely in the clinic, compared to gadolinium maintained at equilibrium. A double gated (respiration and cardiac) spin-echo sequence at 9.4T was used to evaluate whole lungs pre contrast and then at 15 (representative of bolus enhancement), 25 and 35 minutes (representative of eqMRI). This was carried out in two lung metastasis models representative of high and low tumor cell seeding. Lungs containing discrete tumor nodes where inflation fixed and taken for haematoxylin and eosin staining as well as CD34 staining for correlation to MRI. Results We demonstrate that sustained Gd enhancement, afforded by Gd equilibrium, increases the detection of pulmonary metastases compared to bolus enhancement and those tumors which enhance at equilibrium are sub-millimetre in size (