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The cell's endogenous antioxidant system is vital to maintain redox homeostasis. Despite its central role in normal and pathophysiology, no noninvasive tools exist to measure this system in patients. The cystine/glutamate antiporter system xc− maintains the balance between intracellular reactive oxygen species and antioxidant production through the provision of cystine, a key precursor in glutathione biosynthesis. Here, we show that tumor cell retention of a system xc−-specific PET radiotracer, (S)-4-(3-[18F]fluoropropyl)-L-glutamic acid ([18F]FSPG), decreases in proportion to levels of oxidative stress following treatment with a range of redox-active compounds. The decrease in [18F]FSPG retention correlated with a depletion of intracellular cystine resulting from increased de novo glutathione biosynthesis, shown through [U-13C6, U-15N2]cystine isotopic tracing. In vivo, treatment with the chemotherapeutic doxorubicin decreased [18F]FSPG tumor uptake in a mouse model of ovarian cancer, coinciding with markers of oxidative stress but preceding tumor shrinkage and decreased glucose utilization. Having already been used in pilot clinical trials, [18F]FSPG PET could be rapidly translated to the clinic as an early redox indicator of tumor response to treatment.Significance:[18F]FSPG PET imaging provides a sensitive noninvasive measure of tumor redox status and provides an early marker of tumor response to therapy.See related commentary by Lee et al., p. 701

Supplementary Methods and Figures 1 - 6. Fig. S1 shows effect of TBHP treatment on ROS and [18F]FSPG uptake in ovarian cancer cell lines; Fig. S2 illustrates the relationship between glutamate and cystine concentrations on [18F]FSPG uptake; Fig. S3 quantifies [U-13C6, U-15N2]cystine labeling of M+8 GSSG; Fig. S4 indicates extensive disulfide exchange between cystine and NAC; Fig. S5 depicts doxorubicin-mediated alterations in tumor cell redox state and [18F]FSPG; and Fig. S6 illustrates changes in [18F]FSPG and [18F]FDG tumor retention after doxil treatment of mice.

Purpose:Photoacoustic imaging (PAI) is a novel noninvasive and nonionizing imaging technique that allows longitudinal imaging of tumor vasculature in vivo and monitoring of response to therapy, especially for vascular targeted chemotherapy agents. In this study, we used a novel high-resolution all-optical PAI scanner to observe the pharmacodynamic response to the vascular-disrupting agent OXi4503.Experimental Design:Two models of colorectal carcinoma (SW1222 and LS174T) that possess differing pathophysiologic vascularization were established as subcutaneous tumors in mice. Monitoring of response was performed over a 16-day “regrowth” period following treatment at 40 mg/kg, and at day 2 for a “dose response” study at 40 mg/kg, 10 mg/kg, 1 mg/kg, and sham dose.Results:Qualitative and quantitative changes in PA signal are observed, with an initial decrease followed by a plateau and subsequent return of signal indicating regrowth. Both tumor types exhibited a decrease in signal; however, the more vascularized SW1222 tumors show greater response to treatment. Decreasing the dose of OXi4503 led to a decrease in PA signal intensity of 60%, 52%, and 20% in SW1222 tumors and 30%, 26%, and 4% for LS174T tumors.Conclusions:We have shown for the first time that PAI can observe the pharmacodynamic response of tumor vasculature to drug treatment both longitudinally and at different dose levels. Assessment of differing response to treatment based on vascular pathophysiologic differences among patients has the potential to provide personalized drug therapy; we have demonstrated that PAI, which is clinically translatable, could be a powerful tool for this purpose.

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Supplemental Movie 2: Animated EVAC streamlines (grey lines), showing the path taken by interstitial fluid, overlaid on vascular perfusion measurements (colour scale, measured in vivo using arterial spin labelling) and the location of blood vessels (yellow structures, measured ex vivo using micro-CT). The data were acquired in an LS174T colorectal carcinoma tumour xenograft.

Additional data describing the reproducibility of convectionMRI data, and a figure showing the convectionMRI acquisition sequence.

Movie S1

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.

Supplemental Movie 4: Animated EVAC streamlines (grey lines), showing the path taken by interstitial fluid, overlaid on vascular perfusion measurements (colour scale, measured in vivo using arterial spin labelling) and the location of blood vessels (yellow structures, measured ex vivo using micro-CT). The data were acquired in an SW1222 colorectal carcinoma tumour xenograft.

Purpose:Drug resistance is a major obstacle for the effective treatment of patients with high-grade serous ovarian cancer (HGSOC). Currently, there is no satisfactory way to identify patients with HGSOC that are refractive to the standard of care. Here, we propose the system xc− radiotracer (4S)-4-(3-[18F]fluoropropyl)-l-glutamate ([18F]FSPG) as a non-invasive method to measure upregulated antioxidant pathways present in drug-resistant HGSOC.Experimental Design:Using matched chemotherapy sensitive and resistant ovarian cancer cell lines, we assessed their antioxidant capacity and its relation to [18F]FSPG uptake, both in cells and in animal models of human ovarian cancer. We identified the mechanisms driving differential [18F]FSPG cell accumulation and evaluated [18F]FSPG tumor uptake as predictive marker of treatment response in drug-resistant tumors.Results:High intracellular glutathione (GSH) and low reactive oxygen species corresponded to decreased [18F]FSPG cell accumulation in drug-resistant versus drug-sensitive cells. Decreased [18F]FSPG uptake in drug-resistant cells was a consequence of changes in intracellular cystine, a key precursor in GSH biosynthesis. In vivo, [18F]FSPG uptake was decreased nearly 80% in chemotherapy-resistant A2780 tumors compared with parental drug-sensitive tumors, with nonresponding tumors displaying high levels of oxidized-to-reduced GSH. Treatment of drug-resistant A2780 tumors with doxorubicin resulted in no detectable change in tumor volume, GSH, or [18F]FSPG uptake.Conclusions:This study demonstrates the ability of [18F]FSPG to detect upregulated antioxidant pathways present in drug-resistant cancer. [18F]FSPG may therefore enable the identification of patients with HGSOC that are refractory to standard of care, allowing the transferal of drug-resistant patients to alternative therapies, thereby improving outcomes in this disease.

SW1222 subcutaneous tumor by PAT

Supplementary Figures 1 - 6. Fig. S1 shows the ability of PEO1, PEO4 and PEO6 cells to form colonies following cisplatin treatment; Fig. S2 demonstrates that [18F]FDG uptake in A2780 cells is unrelated to drug resistance; Fig. S3 examines the expression of key ABC transporters; Fig. S4 provides representative PET images of mice bearing A2780 WT and A2780 DoxR tumors; Fig. S5 quantifies the [18F]FSPG tumor-to-background ratio in WT and DoxR tumor-bearing mice; and Fig. S6 illustrates [18F]FSPG DoxR tumor retention after doxil treatment of mice.

Supplemental Movie 1: Schematic movie illustrating the physical principles underpinning the convection-MRI sequence

Supplemental Movie 5: Interstitial convection, measured using EVAC-MRI and animated using a particle simulation (left), compared with MRI signal enhancement with time, following injection with a contrast agent (Gd-DTPA), in an LS174T tumour.

High-resolution copy of Figure 6b. Fluid flow measured in vivo with convectionMRI is shown as grey streamlines and perfusion is shown as a colorscale. The location of blood vessels is represented by yellow volume renderings, acquired using ex vivo microvascular casting and imaged with micro-CT.

Supplemental Movie 3: Animated EVAC streamlines (grey lines), showing the path taken by interstitial fluid, overlaid on vascular perfusion measurements (colour scale, measured in vivo using arterial spin labelling) and the location of blood vessels (yellow structures, measured ex vivo using micro-CT). The data were acquired in an LS174T colorectal carcinoma tumour xenograft.

PDF file - 1763KB, This supplemental material includes S1), a figure with the fit of the VERDICT model to the diffusion data from all the samples, S2), a figure demonstrating the quality of the diffusion images and the signal attenuation from an example sample of each cell line, TS1) a table with the orientation angle estimates of the VERDICT model, S3) a figure to demonstrate that VERDICT performs better than a tensor model, a two-tensor model and an isotropic version of the VERDICT model. We also include details of all the histological procedures as well as extra information about the choice of the vascular compartment in the VERDICT model.

Mathematical oncology provides unique and invaluable insights into tumour growth on both the microscopic and macroscopic levels. This review presents state-of-the-art modelling techniques and focuses on their role in understanding glioblastoma, a malignant form of brain cancer. For each approach, we summarise the scope, drawbacks, and assets. We highlight the potential clinical applications of each modelling technique and discuss the connections between the mathematical models and the molecular and imaging data used to inform them. By doing so, we aim to prime cancer researchers with current and emerging computational tools for understanding tumour progression. Finally, by providing an in-depth picture of the different modelling techniques, we also aim to assist researchers who seek to build and develop their own models and the associated inference frameworks., 30 pages, 3 figures, 3 tables

The purpose of this study was to synthesize a fluorine-18 labeled, highly selective aldosterone synthase (hCYP11B2) inhibitor, [18F]AldoView, and to assess its potential for the detection of aldosterone-producing adenomas (APAs) with positron emission tomography in patients with primary hyperaldosteronism (PHA). Using dibenzothiophene sulfonium salt chemistry, [18F]AldoView was obtained in high radiochemical yield in one step from [18F]fluoride. In mice, the tracer showed a favorable pharmacokinetic profile, including rapid distribution and clearance. Imaging in the adrenal tissue from patients with PHA revealed diffuse binding patterns in the adrenal cortex, avid binding in some adenomas, and "hot spots" consistent with aldosterone-producing cell clusters. The binding pattern was in good visual agreement with the antibody staining of hCYP11B2 and distinguished areas with normal and excessive hCYP11B2 expression. Taken together, [18F]AldoView is a promising tracer for the detection of APAs in patients with PHA.

Chronic hypertension is a major risk factor for the development of neurodegenerative disease, yet the etiology of hypertension-driven neurodegeneration remains poorly understood. Forming a unique interface between the systemic circulation and the brain, the blood-cerebrospinal fluid barrier (BCSFB) at the choroid plexus (CP) has been proposed as a key site of vulnerability to hypertension that may initiate downstream neurodegenerative processes. However, our ability to understand BCSFB’s role in pathological processes has, to date, been restricted by a lack of non-invasive functional measurement techniques. In this work, we apply a novel Blood-Cerebrospinal Fluid Barrier Arterial Spin Labeling (BCSFB-ASL) Magnetic resonance imaging (MRI) approach with the aim of detecting possible derangement of BCSFB function in the Spontaneous Hypertensive Rat (SHR) model using a non-invasive, translational technique. SHRs displayed a 36% reduction in BCSFB-mediated labeled arterial water delivery into ventricular cerebrospinal fluid (CSF), relative to normotensive controls, indicative of down-regulated choroid plexus function. This was concomitant with additional changes in brain fluid biomarkers, namely ventriculomegaly and changes in CSF composition, as measured by T1 lengthening. However, cortical cerebral blood flow (CBF) measurements, an imaging biomarker of cerebrovascular health, revealed no measurable change between the groups. Here, we provide the first demonstration of BCSFB-ASL in the rat brain, enabling non-invasive assessment of BCSFB function in healthy and hypertensive rats. Our data highlights the potential for BCSFB-ASL to serve as a sensitive early biomarker for hypertension-driven neurodegeneration, in addition to investigating the mechanisms relating hypertension to neurodegenerative outcomes.

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.

Purpose A fundamental goal in the drive to understand and find better treatments for dementia is the identification of the factors that render the aging brain vulnerable to neurodegenerative disease. Recent evidence indicates the integrity of the blood–brain barrier (BBB) to be an important component of functional failure underlying age‐related cognitive decline. Practical and sensitive measurement is necessary, therefore, to support diagnostic and therapeutic strategies targeted at maintaining BBB integrity in aging patients. Here, we investigated changes in BBB permeability to endogenous blood water in the aging brain. Methods A multiple‐echo‐time arterial spin‐labeling MRI technique, implemented on a 9.4T Bruker imaging system, was applied to 7‐ and 27‐month‐old mice to measure changes in water permeability across the BBB with aging. Results We observed that BBB water permeability was 32% faster in aged mice. This occurred along with a 2.1‐fold increase in mRNA expression of aquaporin‐4 water channels and a 7.1‐fold decrease in mRNA expression of α‐syntrophin protein, which anchors aquaporin‐4 to the BBB. Conclusion Age‐related changes to water permeability across the BBB can be captured using noninvasive noncontrast MRI techniques., Click here for author‐reader discussions

Urinary tract cancer can be pure urothelial carcinoma, pure nonurothelial carcinoma or variant urothelial carcinoma (defined here as mixed urothelial carcinoma). Little is known regarding outcomes for patients with variant urothelial carcinoma receiving immune checkpoint inhibitors. We hypothesized that variant urothelial carcinoma does not compromise immune checkpoint inhibitor efficacy in patients with advanced urothelial carcinoma.We performed a retrospective cohort study across 18 institutions. Demographic, clinicopathological, treatment and outcomes data were collected for patients with advanced urothelial carcinoma who received immune checkpoint inhibitors. Patients were divided into pure vs variant urothelial carcinoma subgroups, with variant urothelial carcinoma further divided by type of variant (ie squamous, neuroendocrine etc). We compared overall response rate using univariate and multivariate logistic regression and progression-free survival and overall survival using Kaplan-Meier and univariate and multivariate Cox proportional hazards.Overall 519 patients were identified, with 395, 406 and 403 included in overall response rate, overall survival and progression-free survival analyses, respectively. Overall response rate to immune checkpoint inhibitors between patients with pure vs variant urothelial carcinoma was comparable (28% vs 29%, p=0.90) without significant differences for individual subtypes vs pure urothelial carcinoma. Median overall survival for patients with pure urothelial carcinoma was 11.0 months vs 10.1 months for variant urothelial carcinoma (p=0.60), but only 4.6 months for patients with neuroendocrine features (9 patients, HR 2.75, 95% CI 1.40-5.40 vs pure urothelial carcinoma, p=0.003). Median progression-free survival was 4.1 months for pure vs 5.2 months for variant urothelial carcinoma (p=0.43) and 3.7 months for neuroendocrine features (HR 1.87, 95% CI 0.92-3.79 vs pure urothelial carcinoma, p=0.09).Overall response rate to immune checkpoint inhibitors was comparable across histological types. However, overall survival was worse for patients with tumors containing neuroendocrine features. Variant urothelial carcinoma should not exclude patients from receiving immune checkpoint inhibitors.

Background MSCTRAIL is a cell-based therapy consisting of human allogeneic umbilical cord-derived MSCs genetically modified to express the anti-cancer protein TRAIL. Though cell-based therapies are typically designed with a target tissue in mind, delivery is rarely assessed due to a lack of translatable non-invasive imaging approaches. In this preclinical study, we demonstrate 89Zr-oxine labelling and PET-CT imaging as a potential clinical solution for non-invasively tracking MSCTRAIL biodistribution. Future implementation of this technique should improve our understanding of MSCTRAIL during its evaluation as a therapy for metastatic lung adenocarcinoma. Methods MSCTRAIL were radiolabelled with 89Zr-oxine and assayed for viability, phenotype, and therapeutic efficacy post-labelling. PET-CT imaging of 89Zr-oxine-labelled MSCTRAIL was performed in a mouse model of lung cancer following intravenous injection, and biodistribution was confirmed ex vivo. Results MSCTRAIL retained the therapeutic efficacy and MSC phenotype in vitro at labelling amounts up to and above those required for clinical imaging. The effect of 89Zr-oxine labelling on cell proliferation rate was amount- and time-dependent. PET-CT imaging showed delivery of MSCTRAIL to the lungs in a mouse model of lung cancer up to 1 week post-injection, validated by in vivo bioluminescence imaging, autoradiography, and fluorescence imaging on tissue sections. Conclusions 89Zr-oxine labelling and PET-CT imaging present a potential method of evaluating the biodistribution of new cell therapies in patients, including MSCTRAIL. This offers to improve understanding of cell therapies, including mechanism of action, migration dynamics, and inter-patient variability.

Cell-based therapies are promising treatments for various kidney diseases. However, the major hurdle in initiating therapeutic responses is the inefficiency of injection routes to deliver cells to the kidney parenchyma. Systemic injection, such as intravenous injection only delivers a small proportion of cells to the kidney. Whereas direct delivery, such as renal artery injection requires surgical procedures. A minimally invasive renal artery injection was therefore developed to enhance cell delivery to kidney. In this study, luciferase expressing human adipocyte derived stem cells (ADSC) were labelled with gold nanorods (GNR) and injected into the renal artery using ultrasound guidance. The ADSCs were tracked using bioluminescence and photoacoustic imaging serially over 7 days. Imaging confirmed that the majority of signal was within the kidney, indicative of successful injection and that the cells remained viable for 3 days. Histology showed co-localization of GNRs with ADSC staining throughout the kidney with no indication of injury caused by injection. These findings demonstrate that ultrasound-guided renal artery injection is feasible in mice and can successfully deliver a large proportion of cells which are retained within the kidney for 3 days. Therefore, the techniques developed here will be useful for optimising cell therapy in kidney diseases.

The blood–cerebrospinal fluid barrier (BCSFB) is a highly dynamic transport interface that serves brain homeostasis. To date, however, understanding of its role in brain development and pathology has been hindered by the absence of a non-invasive technique for functional assessment. Here we describe a method for non-invasive measurement of BSCFB function by using tracer-free MRI to quantify rates of water delivery from arterial blood to ventricular cerebrospinal fluid. Using this method, we record a 36% decrease in BCSFB function in aged mice, compared to a 13% decrease in parenchymal blood flow, itself a leading candidate biomarker of early neurodegenerative processes. We then apply the method to explore the relationship between BCSFB function and ventricular morphology. Finally, we provide proof of application to the human brain. Our findings position the BCSFB as a promising new diagnostic and therapeutic target, the function of which can now be safely quantified using non-invasive MRI., The blood–cerebrospinal fluid barrier (BCSFB) is an important interface for brain homeostasis. Here the authors describe a non-invasive MRI technique for the quantitative assessment of BCSFB function.

Neurovascular coupling is a fundamental brain mechanism that regulates local cerebral blood flow (CBF) in response to changes in neuronal activity. Functional imaging techniques are commonly used to record these changes in CBF as a proxy of neuronal activity to study the human brain. However, the mechanisms of neurovascular coupling remain incompletely understood. Here we show in experimental animal models (laboratory rats and mice) that the neuronal activity-dependent increases in local CBF in the somatosensory cortex are prevented by saturation of the CO2-sensitive vasodilatory brain mechanism with surplus of exogenous CO2 or disruption of brain CO2/HCO3− transport by genetic knockdown of electrogenic sodium-bicarbonate cotransporter 1 (NBCe1) expression in astrocytes. A systematic review of the literature data shows that CO2 and increased neuronal activity recruit the same vasodilatory signaling pathways. These results and analysis suggest that CO2 mediates signaling between neurons and the cerebral vasculature to regulate brain blood flow in accord with changes in the neuronal activity.

Magnetomechanical Stimulation Magnetomechanical stimulation enables remote and selective control of brain cell activity without any need for genetic modification. Antibody‐functionalized magnetic particles are targeted to the mechanosensitive astrocytes and a magnetic field is applied to activate signaling pathways that regulate a wide range of brain functions. More details can be found in article number 2104194 by Yichao Yu, Mark F. Lythgoe, and co‐workers. [Image: see text]

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.

Mechanosensitivity is a well-known feature of astrocytes, however, its underlying mechanisms and functional significance remain unclear. There is evidence that astrocytes are acutely sensitive to decreases in cerebral perfusion pressure and may function as intracranial baroreceptors, tuned to monitor brain blood flow. This study investigated the mechanosensory signaling in brainstem astrocytes, as these cells reside alongside the cardiovascular control circuits and mediate increases in blood pressure and heart rate induced by falls in brain perfusion. It was found that mechanical stimulation-evoked Ca2+responses in astrocytes of the rat brainstem were blocked by (1) antagonists of connexin channels, connexin 43 (Cx43) blocking peptide Gap26, or Cx43 gene knock-down; (2) antagonists of TRPV4 channels; (3) antagonist of P2Y1receptors for ATP; and (4) inhibitors of phospholipase C or IP3 receptors. Proximity ligation assay demonstrated interaction between TRPV4 and Cx43 channels in astrocytes. Dye loading experiments showed that mechanical stimulation increased open probability of carboxyfluorescein-permeable membrane channels. These data suggest that mechanosensory Ca2+responses in astrocytes are mediated by interaction between TRPV4 and Cx43 channels, leading to Cx43-mediated release of ATP which propagates/amplifies Ca2+signals via P2Y1receptors and Ca2+recruitment from the intracellular stores. In astrocyte-specific Cx43 knock-out mice the magnitude of heart rate responses to acute increases in intracranial pressure was not affected by Cx43 deficiency. However, these animals displayed lower heart rates at different levels of cerebral perfusion, supporting the hypothesis of connexin hemichannel-mediated release of signaling molecules by astrocytes having an excitatory action on the CNS sympathetic control circuits.SIGNIFICANCE STATEMENTThere is evidence suggesting that astrocytes may function as intracranial baroreceptors that play an important role in the control of systemic and cerebral circulation. To function as intracranial baroreceptors, astrocytes must possess a specialized membrane mechanism that makes them exquisitely sensitive to mechanical stimuli. This study shows that opening of connexin 43 (Cx43) hemichannels leading to the release of ATP is the key central event underlying mechanosensory Ca2+responses in astrocytes. This astroglial mechanism plays an important role in the autonomic control of heart rate. These data add to the growing body of evidence suggesting that astrocytes function as versatile surveyors of the CNS metabolic milieu, tuned to detect conditions of potential metabolic threat, such as hypoxia, hypercapnia, and reduced perfusion.

Purpose: Photoacoustic imaging (PAI) is a novel noninvasive and nonionizing imaging technique that allows longitudinal imaging of tumor vasculature in vivo and monitoring of response to therapy, especially for vascular targeted chemotherapy agents. In this study, we used a novel high-resolution all-optical PAI scanner to observe the pharmacodynamic response to the vascular-disrupting agent OXi4503. Experimental Design: Two models of colorectal carcinoma (SW1222 and LS174T) that possess differing pathophysiologic vascularization were established as subcutaneous tumors in mice. Monitoring of response was performed over a 16-day “regrowth” period following treatment at 40 mg/kg, and at day 2 for a “dose response” study at 40 mg/kg, 10 mg/kg, 1 mg/kg, and sham dose. Results: Qualitative and quantitative changes in PA signal are observed, with an initial decrease followed by a plateau and subsequent return of signal indicating regrowth. Both tumor types exhibited a decrease in signal; however, the more vascularized SW1222 tumors show greater response to treatment. Decreasing the dose of OXi4503 led to a decrease in PA signal intensity of 60%, 52%, and 20% in SW1222 tumors and 30%, 26%, and 4% for LS174T tumors. Conclusions: We have shown for the first time that PAI can observe the pharmacodynamic response of tumor vasculature to drug treatment both longitudinally and at different dose levels. Assessment of differing response to treatment based on vascular pathophysiologic differences among patients has the potential to provide personalized drug therapy; we have demonstrated that PAI, which is clinically translatable, could be a powerful tool for this purpose.

Introduction There is a growing appreciation for including a complex, vascularised stroma in three-dimensional (3D) tumour models to recapitulate the native tumour microenvironment in situ. Methods Using a compartmentalised, biomimetic, 3D cancer model, comprising a central cancer mass surrounded by a vascularised stroma, we have tested the invasive capability of colorectal cancer cells. Results We show histological analysis of dense collagen I/laminin scaffolds, forming necrotic cores with cellular debris. Furthermore, cancer cells within this 3D matrix form spheroids, which is corroborated with high EpCAM expression. We validate the invasive growth of cancer cells into the stroma through quantitative image analysis and upregulation of known invasive gene markers, including metastasis associated in colon cancer 1, matrix metalloproteinase 7 and heparinase. Tumouroids containing highly invasive HCT116 cancer masses form less complex and less branched vascular networks, recapitulating ‘leaky’ vasculature associated with highly metastatic cancers. Angiogenic factors regulating this were vascular endothelial growth factor A and hepatocyte growth factor active protein. Where vascular networks were formed with less invasive cancer masses (HT29), higher expression of vascular endothelial cadherin active protein resulted in more complex and branched networks. To eliminate the cell–cell interaction between the cancer mass and stroma, we developed a three-compartment model containing an acellular ring to test the chemoattractant pull from the cancer mass. This resulted in migration of endothelial networks through the acellular ring accompanied by alignment of vascular networks at the cancer/stroma boundary. Discussion This work interrogates to the gene and protein level how cancer cells influence the development of a complex stroma, which shows to be directly influenced by the invasive capability of the cancer.

Early measurements of tissue viability after myocardial infarction (MI) are essential for accurate diagnosis and treatment planning but are challenging to obtain. Here, manganese, a calcium analogue and clinically approved magnetic resonance imaging (MRI) contrast agent, is used as an imaging biomarker of myocardial viability in the first hours after experimental MI. Safe Mn2+ dosing is confirmed by measuring in vitro beating rates, calcium transients, and action potentials in cardiomyocytes, and in vivo heart rates and cardiac contractility in mice. Quantitative T1 mapping‐manganese‐enhanced MRI (MEMRI) reveals elevated and increasing Mn2+ uptake in viable myocardium remote from the infarct, suggesting MEMRI offers a quantitative biomarker of cardiac inotropy. MEMRI evaluation of infarct size at 1 h, 1 and 14 days after MI quantifies myocardial viability earlier than the current gold‐standard technique, late‐gadolinium‐enhanced MRI. These data, coupled with the re‐emergence of clinical Mn2+‐based contrast agents open the possibility of using MEMRI for direct evaluation of myocardial viability early after ischemic onset in patients., Early measurements of tissue viability after myocardial infarction (MI) are essential for accurate diagnosis and treatment planning. Here, the safety and efficacy of manganese‐enhanced magnetic resonance imaging (MEMRI) in vitro in cardiomyocytes and in vivo in mouse models of MI are evaluated. It is demonstrated that MEMRI offers a novel biomarker of myocardial viability in the first hours after MI.