Ibrutinib disrupts blood-tumor barrier integrity and prolongs survival in rodent glioma model.

In malignant glioma, cytotoxic drugs are often inhibited from accessing the tumor site due to the blood-tumor barrier (BTB). Ibrutinib, FDA-approved lymphoma agent, inhibits Bruton tyrosine kinase (BTK) and has previously been shown to independently impair aortic endothelial adhesion and increase rodent glioma model survival in combination with cytotoxic therapy. Yet additional research is required to understand ibrutinib's effect on BTB function. In this study, we detail baseline BTK expression in glioma cells and its surrounding vasculature, then measure endothelial junctional expression/function changes with varied ibrutinib doses in vitro. Rat glioma cells and rodent glioma models were treated with ibrutinib alone (1-10 µM and 25 mg/kg) and in combination with doxil (10-100 µM and 3 mg/kg) to assess additive effects on viability, drug concentrations, tumor volume, endothelial junctional expression and survival. We found that ibrutinib, in a dose-dependent manner, decreased brain endothelial cell-cell adhesion over 24 h, without affecting endothelial cell viability (p < 0.005). Expression of tight junction gene and protein expression was decreased maximally 4 h after administration, along with inhibition of efflux transporter, ABCB1, activity. We demonstrated an additive effect of ibrutinib with doxil on rat glioma cells, as seen by a significant reduction in cell viability (p < 0.001) and increased CNS doxil concentration in the brain (56 ng/mL doxil alone vs. 74.6 ng/mL combination, p < 0.05). Finally, Ibrutinib, combined with doxil, prolonged median survival in rodent glioma models (27 vs. 16 days, p < 0.0001) with brain imaging showing a - 53% versus - 75% volume change with doxil alone versus combination therapy (p < 0.05). These findings indicate ibrutinib's ability to increase brain endothelial permeability via junctional disruption and efflux inhibition, to increase BTB drug entry and prolong rodent glioma model survival. Our results motivate the need to identify other BTB modifiers, all with the intent of improving survival and reducing systemic toxicities.

A PARP inhibitor, rucaparib, improves cardiac dysfunction in ADP-ribose-acceptor hydrolase 3 ( Arh3 ) deficiency.

Aims: Patients with ADP-ribose-acceptor hydrolase 3 ( ARH3 ) deficiency exhibit stress-induced childhood-onset neurodegeneration with ataxia and seizures (CONDSIAS). ARH3 degrades protein-linked poly(ADP- ribose) (PAR) synthesized by poly(ADP-ribose)polymerase (PARP)-1 during oxidative stress, leading to cleavage of the ADP-ribose linked to protein. ARH3 deficiency leads to excess accumulation of PAR, resulting in PAR-dependent cell death or parthanatos. Approximately one-third of patients with homozygous mutant ARH3 die from cardiac arrest, which has been described as neurogenic, suggesting that ARH3 may play an important role in maintaining myocardial function. To address this question, cardiac function was monitored in Arh3 -knockout (KO) and - heterozygous (HT) mice. Methods and results: Arh3 -KO male mice displayed cardiac hypertrophy by histopathology and decreased cardiac contractility assessed by MRI. In addition, both genders of Arh3 -KO and -HT mice showed decreased cardiac contractility by dobutamine stress test assessed by echocardiography. A direct role of ARH3 on myocardial function was seen with a Langendorff-perfused isolated heart model . Arh3 -KO male mouse hearts showed decreased post-ischemic rate pressure products, increased size of ischemia-reperfusion (IR) infarcts, and elevated PAR levels. Consistently, in vivo IR injury showed enhanced infarct size in Arh3 -KO mice in both genders. In addition, Arh3 -HT male mice showed increased size of in vivo IR infarcts. Treatment with an FDA-approved PARP inhibitor, rucaparib, improved cardiac contractility during dobutamine-induced stress and exhibited reduced size of in vivo IR infarcts. To understand better the role of ARH3, CRISPR-Cas9 was used to generate different Arh3 genotypes of myoblasts and myotubes. Incubation with H2O2 decreased viability of Arh3 -KO and -HT myoblasts and myotubes, resulting in PAR-dependent cell death that was reduced by PARP inhibitors or by transfection with the Arh3 gene. Conclusion: ARH3 regulates PAR homeostasis in myocardium to preserve function and protect against oxidative stress; PARP inhibitors reduce the myocardial dysfunction seen with Arh3 mutations.

Mono-ADP-ribosyltransferase 1 ( Artc1 )-deficiency decreases tumorigenesis, increases inflammation, decreases cardiac contractility, and reduces survival.

Arginine-specific mono-ADP-ribosylation is a reversible post-translational modification; arginine-specific, cholera toxin-like mono-ADP-ribosyltransferases (ARTCs) transfer ADP-ribose from NAD + to arginine, followed by cleavage of ADP-ribose-(arginine)protein bond by ADP-ribosylarginine hydrolase 1 (ARH1), generating unmodified (arginine)protein. ARTC1 has been shown to enhance tumorigenicity as does Arh1 deficiency. In this study, Artc1 -KO and Artc1/Arh1 -double-KO mice showed decreased spontaneous tumorigenesis and increased age-dependent, multi-organ inflammation with upregulation of pro-inflammatory cytokine TNF- α . In a xenograft model using tumorigenic Arh1 -KO mouse embryonic fibroblasts (MEFs), tumorigenicity was decreased in Artc1 -KO and heterozygous recipient mice, with tumor infiltration by CD8 + T cells and macrophages, leading to necroptosis, suggesting that ARTC1 promotes the tumor microenvironment. Furthermore, Artc1/Arh1 -double-KO MEFs showed decreased tumorigenesis in nude mice, showing that tumor cells as well as tumor microenvironment require ARTC1. By echocardiography and MRI, Artc1 -KO and heterozygous mice showed male-specific, reduced myocardial contractility. Furthermore, Artc1 -KO male hearts exhibited enhanced susceptibility to myocardial ischemia-reperfusion-induced injury with increased receptor-interacting protein kinase 3 (RIP3) protein levels compared to WT mice, suggesting that ARTC1 suppresses necroptosis. Overall survival rate of Artc1 -KO was less than their Artc1 -WT counterparts, primarily due to enhanced immune response and inflammation. Thus, anti-ARTC1 agents may reduce tumorigenesis but may increase multi-organ inflammation and decrease cardiac contractility.

ADP-ribose-acceptor hydrolase 2 ( Arh2 ) deficiency results in cardiac dysfunction, tumorigenesis, inflammation, and decreased survival.

ADP-ribosylation is a reversible reaction with ADP-ribosyltransferases catalyzing the forward reaction and ADP-ribose-acceptor hydrolases (ARHs) hydrolyzing the ADP-ribose acceptor bond. ARH2 is a member of the 39-kDa ARH family (ARH1-3), which is expressed in heart and skeletal muscle. ARH2 failed to exhibit any in vitro enzymatic activity. To determine its possible in vivo activities, Arh2 -knockout (KO) and - heterozygous (Het) mice were generated using CRISPR-Cas9. Arh2 -KO mice exhibited decreased cardiac contractility by MRI, echocardiography and dobutamine stress with cardiomegaly and abnormal motor function. Arh2 -Het mice showed results similar to those seen in Arh2 -KO mice except for cardiomegaly. Arh2 -KO and -Het mice and mouse embryonic fibroblasts (MEFs) developed spontaneous tumors and subcutaneous tumors in nude mice. We identified 13 mutations in Arh2 -Het MEFs and heterozygous tumors, corresponding to human ARH2 mutations in cancers obtained from COSMIC. Of interest, the L116R mutation in Arh2 gene plays a critical role in aggressive tumorigenesis in nude mice, corresponding to human ARH2 mutations in stomach adenocarcinoma. Both genders of Arh2 -KO and -Het mice showed increased unexpectedly deaths and decreased survival rate during a 24-month observation, caused by tumor, inflammation, non-inflammation (e.g., cardiomegaly, dental dysplasia), and congenital diseases. Thus, Arh2 plays a pivotal role in cardiac function, tumorigenesis, inflammation, and overall survival.

Glb1 knockout mouse model shares natural history with type II GM1 gangliosidosis patients.

GM1 gangliosidosis is a rare lysosomal storage disorder affecting multiple organ systems, primarily the central nervous system, and is caused by functional deficiency of ß-galactosidase (GLB1). Using CRISPR/Cas9 genome editing, we generated a mouse model to evaluate characteristics of the disease in comparison to GM1 gangliosidosis patients. Our Glb1-/- mice contain small deletions in exons 2 and 6, producing a null allele. Longevity is approximately 50 weeks and studies demonstrated that female Glb1-/- mice die six weeks earlier than male Glb1-/- mice. Gait analyses showed progressive abnormalities including abnormal foot placement, decreased stride length and increased stance width, comparable with what is observed in type II GM1 gangliosidosis patients. Furthermore, Glb1-/- mice show loss of motor skills by 20 weeks assessed by adhesive dot, hanging wire, and inverted grid tests, and deterioration of motor coordination by 32 weeks of age when evaluated by rotarod testing. Brain MRI showed progressive cerebellar atrophy in Glb1-/- mice as seen in some patients. In addition, Glb1-/- mice also show significantly increased levels of a novel pentasaccharide biomarker in urine and plasma which we also observed in GM1 gangliosidosis patients. Glb1-/- mice also exhibit accumulation of glycosphingolipids in the brain with increases in GM1 and GA1 beginning by 8 weeks. Surprisingly, despite being a null variant, this Glb1-/- mouse most closely models the less severe type II disease and will guide the development of new therapies for patients with the disorder.

Leukocyte chemotactic receptor Fpr1 protects against aging-related posterior subcapsular cataract formation.

Cataracts are a common consequence of aging; however, pathogenesis remains poorly understood. Here, we observed that after 3 months of age mice lacking the G protein-coupled leukocyte chemotactic receptor Fpr1 (N-formyl peptide receptor 1) began to develop bilateral posterior subcapsular cataracts that progressed to lens rupture and severe degeneration, without evidence of either systemic or local ocular infection or inflammation. Consistent with this, Fpr1 was detected in both mouse and human lens in primary lens epithelial cells (LECs), the only cell type present in the lens; however, expression was confined to subcapsular LECs located along the anterior hemispheric surface. To maximize translucency, LECs at the equator proliferate and migrate posteriorly, then differentiate into lens fiber cells by nonclassical apoptotic signaling, which results in loss of nuclei and other organelles, including mitochondria which are a rich source of endogenous N-formyl peptides. In this regard, denucleation and posterior migration of LECs were abnormal in lenses from Fpr1-/- mice, and direct stimulation of LECs with the prototypic N-formyl peptide agonist fMLF promoted apoptosis. Thus, Fpr1 is repurposed beyond its immunoregulatory role in leukocytes to protect against cataract formation and lens degeneration during aging.

Amphotericin B Penetrates into the Central Nervous System Through Focal Disruption of the Blood Brain Barrier in Experimental Hematogenous Candida Meningoencephalitis.

Hematogenous Candida meningoencephalitis (HCME) is a life-threatening complication of neonates and immunocompromised children. Amphotericin B (AmB) shows poor permeability and low cerebrospinal fluid (CSF) concentrations, but is effective in treatment of HCME. In order to better understand the mechanism of CNS penetration of AmB, we hypothesized that AmB may achieve focally higher concentrations in infected CNS lesions. An in vitro BBB model was serially infected with C. albicans. Liposomal AmB (LAMB) or deoxycholate AmB (DAMB) at 5 µg/ml were then provided, vascular and CNS compartments were sampled 4h later. For in vivo correlation, rabbits with experimental HCME received a single dose of DAMB 1 mg/kg or LAMB 5 mg/kg, and were euthanized after 1, 3, 6 and 24h. Evans blue solution (2%) 2 ml/kg administered IV one hour prior to euthanasia stained infected regions of tissue but not histologically normal areas. AmB concentrations in stained and unstained tissue regions were measured using UPLC. For selected rabbits, MRI scans performed on days 1-7 postinoculation were acquired before and after IV bolus Gd-DTPA at 15min intervals through 2h post-injection. The greatest degree of penetration of DAMB and LAMB through the in vitro BBB occurred after 24h of exposure (P=0.0022). In vivo the concentrations of LAMB and DAMB in brain abscesses were 4.35±0.59 and 3.14±0.89-times higher vs. normal tissue (P≤0.019). MRI scans demonstrated that Gd-DTPA accumulated in infected areas with disrupted BBB. Localized BBB disruption in HCME allows high concentrations of AmB within infected tissues, despite the presence of low CSF concentrations.

In vivo kinetics and nonradioactive imaging of rapidly proliferating cells in graft-versus-host disease.

Hematopoietic stem cell transplantation (HSCT) offers a cure for cancers that are refractory to chemotherapy and radiation. Most HSCT recipients develop chronic graft-versus-host disease (cGVHD), a systemic alloimmune attack on host organs. Diagnosis is based on clinical signs and symptoms, as biopsies are risky. T cells are central to the biology of cGVHD. We found that a low Treg/CD4+ T effector memory (Tem) ratio in circulation, lymphoid, and target organs identified early and established mouse cGVHD. Using deuterated water labeling to measure multicompartment in vivo kinetics of these subsets, we show robust Tem and Treg proliferation in lymphoid and target organs, while Tregs undergo apoptosis in target organs. Since deuterium enrichment into DNA serves as a proxy for cell proliferation, we developed a whole-body clinically relevant deuterium MRI approach to nonradioactively detect cGVHD and potentially allow imaging of other diseases characterized by rapidly proliferating cells.

ISMRM Raw data format: A proposed standard for MRI raw datasets.

PURPOSE: This work proposes the ISMRM Raw Data format as a common MR raw data format, which promotes algorithm and data sharing. METHODS: A file format consisting of a flexible header and tagged frames of k-space data was designed. Application Programming Interfaces were implemented in C/C++, MATLAB, and Python. Converters for Bruker, General Electric, Philips, and Siemens proprietary file formats were implemented in C++. Raw data were collected using magnetic resonance imaging scanners from four vendors, converted to ISMRM Raw Data format, and reconstructed using software implemented in three programming languages (C++, MATLAB, Python). RESULTS: Images were obtained by reconstructing the raw data from all vendors. The source code, raw data, and images comprising this work are shared online, serving as an example of an image reconstruction project following a paradigm of reproducible research. CONCLUSION: The proposed raw data format solves a practical problem for the magnetic resonance imaging community. It may serve as a foundation for reproducible research and collaborations. The ISMRM Raw Data format is a completely open and community-driven format, and the scientific community is invited (including commercial vendors) to participate either as users or developers. Magn Reson Med 77:411-421, 2017. © 2016 Wiley Periodicals, Inc.

EGFR-targeted therapy results in dramatic early lung tumor regression accompanied by imaging response and immune infiltration in EGFR mutant transgenic mouse models.

Lung adenocarcinoma patients harboring kinase domain mutations in Epidermal growth factor receptor (EGFR) have significant clinical benefit from EGFR-targeted tyrosine kinase inhibitors (TKIs). Although a majority of patients experience clinical symptomatic benefit immediately, an objective response can only be demonstrated after 6-8 weeks of treatment. Evaluation of patient response by imaging shows that 30-40% of patients do not respond due to intrinsic resistance to these TKIs. We investigated immediate-early effects of EGFR-TKI treatment in mutant EGFR-driven transgenic mouse models by FDG-PET and MRI and correlated the effects on the tumor and the tumor microenvironment. Within 24 hours of erlotinib treatment we saw approximately 65% tumor regression in mice with TKI-sensitive EGFRL858R lung adenocarcinoma. However, mice with EGFRL858R/T790M-driven tumors did not respond to either erlotinib or afatinib monotherapy, but did show a significant tumor response to afatinib-cetuximab combination treatment. The imaging responses correlated with the inhibition of downstream EGFR signaling, increased apoptosis, and decreased proliferation in the tumor tissues. In EGFRL858R-driven tumors, we saw a significant increase in CD45+ leukocytes, NK cells, dendritic cells, macrophages and lymphocytes, particularly CD8+ T cells. In response to erlotinib, these dendritic cells and macrophages had significantly higher MHC class II expression, indicating increased antigen-presenting capabilities. Together, results of our study provide novel insight into the immediate-early therapeutic response to EGFR TKIs in vivo.

PP2A inhibition with LB100 enhances cisplatin cytotoxicity and overcomes cisplatin resistance in medulloblastoma cells.

The protein phosphatase 2A (PP2A) inhibitor, LB100, has been shown in pre-clinical studies to be an effective chemo- and radio-sensitizer for treatment of various cancers. We investigated effects associated with LB100 treatment alone and in combination with cisplatin for medulloblastoma (MB) in vitro and in vivo in an intracranial xenograft model. We demonstrated that LB100 had a potent effect on MB cells. By itself, LB100 inhibited proliferation and induced significant apoptosis in a range of pediatric MB cell lines. It also attenuated MB cell migration, a pre-requirement for invasion. When used in combination, LB100 enhanced cisplatin-mediated cytotoxic effects. Cell viability in the presence of 1 uM cisplatin alone was 61% (DAOY), 100% (D341), and 58% (D283), but decreased with the addition of 2 µM of LB100 to 26% (DAOY), 67% (D341), and 27% (D283), (p < 0.005). LB100 suppressed phosphorylation of the STAT3 protein and several STAT3 downstream targets. Also, LB100 directly increased cisplatin uptake and overcame cisplatin-resistance in vitro. Finally, LB100 exhibited potent in vivo anti-neoplastic activity in combination with cisplatin in an intracranial xenograft model.

Imaging Candida Infections in the Host.

Disseminated fungal infections caused by Candida species are associated with homing of the pathogen to specific organs in human and murine hosts. Kidneys are a primary target organ of Candida albicans, and invasion into the kidney medulla can lead to loss of renal function and death. Therefore, development of noninvasive methods to assess kidney infections could aid in the management of disseminated candidemia. We describe a magnetic resonance imaging method utilizing iron oxide-based contrast agents to noninvasively assess recruitment of phagocytes and kidney inflammation. C. albicans also colonizes the brain and can cause meningoencephalitis. We describe additional imaging methods to assess loss of the blood-brain barrier function that initiates brain infections.

13C-MR Spectroscopic Imaging with Hyperpolarized [1-13C]pyruvate Detects Early Response to Radiotherapy in SCC Tumors and HT-29 Tumors.

PURPOSE: X-ray irradiation of tumors causes diverse effects on the tumor microenvironment, including metabolism. Recent developments of hyperpolarized (13)C-MRI enabled detecting metabolic changes in tumors using a tracer [1-(13)C]pyruvate, which participates in important bioenergetic processes that are altered in cancers. Here, we investigated the effects of X-ray irradiation on pyruvate metabolism in squamous cell carcinoma (SCCVII) and colon cancer (HT-29) using hyperpolarized (13)C-MRI. EXPERIMENTAL DESIGN: SCCVII and HT-29 tumors were grown by injecting tumor cells into the hind legs of mice. [1-(13)C]pyruvate was hyperpolarized and injected intravenously into tumor-bearing mice, and (13)C-MR signals were acquired using a 4.7 T scanner. RESULTS: [1-(13)C]pyruvate and [1-(13)C]lactate were detected in the tumor-bearing legs immediately after hyperpolarized [1-(13)C]pyruvate administration. The [1-(13)C]lactate to [1-(13)C]pyruvate ratio (Lac/Pyr) increased as the tumors grew in nonirradiated SCCVII tumors. The increase in Lac/Pyr was suppressed modestly with a single 10 Gy of irradiation, but it significantly decreased by further irradiation (10 Gy × 3). Similar results were obtained in HT-29; Lac/Pyr significantly dropped with fractionated 30 Gy irradiation. Independent ex vivo measurements revealed that the lactate dehydrogenase (LDH) activity and protein level were significantly smaller in the irradiated SCCVII tumors compared with the nonirradiated tumors, indicating that a decrease in LDH activity was one of the main factors responsible for the decrease of Lac/Pyr observed on (13)C-MRI. CONCLUSIONS: Robust changes of Lac/Pyr observed in the HT-29 after the radiation suggested that lactate conversion from pyruvate monitored with hyperpolarized (13)C-MRI could be useful for the evaluation of early response to radiotherapy. See related commentary by Lai et al., p. 4996.

Cannabinoid receptor 1 promotes hepatocellular carcinoma initiation and progression through multiple mechanisms.

UNLABELLED: Hepatocellular carcinoma (HCC) has high mortality and no adequate treatment. Endocannabinoids interact with hepatic cannabinoid 1 receptors (CB1Rs) to promote hepatocyte proliferation in liver regeneration by inducing cell cycle proteins involved in mitotic progression, including Forkhead Box M1. Because this protein is highly expressed in HCC and contributes to its genesis and progression, we analyzed the involvement of the endocannabinoid/CB1R system in murine and human HCC. Postnatal diethylnitrosamine treatment induced HCC within 8 months in wild-type mice but fewer and smaller tumors in CB1R(-/-) mice or in wild-type mice treated with the peripheral CB1R antagonist JD5037, as monitored in vivo by serial magnetic resonance imaging. Genome-wide transcriptome analysis revealed CB1R-dependent, tumor-induced up-regulation of the hepatic expression of CB1R, its endogenous ligand anandamide, and a number of tumor-promoting genes, including the GRB2 interactome as well as Forkhead Box M1 and its downstream target, the tryptophan-catalyzing enzyme indoleamine 2,3-dioxygenase. Increased indoleamine 2,3-dioxygenase activity and consequent induction of immunosuppressive T-regulatory cells in tumor tissue promote immune tolerance. CONCLUSION: The endocannabinoid/CB1R system is up-regulated in chemically induced HCC, resulting in the induction of various tumor-promoting genes, including indoleamine 2,3-dioxygenase; and attenuation of these changes by blockade or genetic ablation of CB1R suppresses the growth of HCC and highlights the therapeutic potential of peripheral CB1R blockade.

Diffusion tensor and volumetric magnetic resonance measures as biomarkers of brain damage in a small animal model of HIV.

BACKGROUND: There are currently no widely accepted neuro-HIV small animal models. We wanted to validate the HIV-1 Transgenic rat (Tg) as an appropriate neuro-HIV model and then establish in vivo imaging biomarkers of neuropathology, within this model, using MR structural and diffusion tensor imaging (DTI). METHODS: Young and middle-aged Tg and control rats were imaged using MRI. A subset of middle-aged animals underwent longitudinal repeat imaging six months later. Total brain volume (TBV), ventricular volume (VV) and parenchymal volume (PV = TBV-VV) were measured. Fractional anisotropy (FA) and mean diffusivity (MD) values of the corpus callosum (CC) were calculated from DTI data. RESULTS: TBV and PV were smaller in Tg compared to control rats in young and middle-aged cohorts (p<0.0001). VV increased significantly (p = 0.005) over time in the longitudinal Tg cohort. There were lower FA (p<0.002) and higher MD (p<0.003) values in the CC of middle-aged Tg rats compared to age-matched controls. Longitudinally, MD significantly decreased over time in Tg rats (p<0.03) while it did not change significantly in the control cohort over the same period of time (p>0.05). CONCLUSIONS: We detected brain volume loss in the Tg rat, probably due to astrocytic dysfunction/loss, loss of structural/axonal matrix and striatal neuronal loss as suggested by immunofluorescence. Increased MD and decreased FA in the CC probably reflect microstructural differences between the Tg and Control rats which could include increased extracellular space between white matter tracts, demyelination and axonal degeneration, among other pathologies. We believe that the Tg rat is an adequate model of neuropathology in HIV and that volumetric MR and DTI measures can be potentially used as biomarkers of disease progression.

In vivo imaging of tumor physiological, metabolic, and redox changes in response to the anti-angiogenic agent sunitinib: longitudinal assessment to identify transient vascular renormalization.

AIMS: The tumor microenvironment is characterized by a highly reducing redox status, a low pH, and hypoxia. Anti-angiogenic therapies for solid tumors frequently function in two steps: the transient normalization of structurally and functionally aberrant tumor blood vessels with increased blood perfusion, followed by the pruning of tumor blood vessels and the resultant cessation of nutrients and oxygen delivery required for tumor growth. Conventional anatomic or vascular imaging is impractical or insufficient to distinguish between the two steps of tumor response to anti-angiogenic therapies. Here, we investigated whether the noninvasive imaging of the tumor redox state and energy metabolism could be used to characterize anti-angiogenic drug-induced transient vascular normalization. RESULTS: Daily treatment of squamous cell carcinoma (SCCVII) tumor-bearing mice with the multi-tyrosine kinase inhibitor sunitinib resulted in a rapid decrease in tumor microvessel density and the suppression of tumor growth. Tumor pO2 imaging by electron paramagnetic resonance imaging showed a transient increase in tumor oxygenation after 2-4 days of sunitinib treatment, implying improved tumor perfusion. During this window of vascular normalization, magnetic resonance imaging of the redox status using an exogenously administered nitroxide probe and hyperpolarized (13)C MRI of the metabolic flux of pyruvate/lactate couple revealed an oxidative shift in tumor redox status. INNOVATION: Redox-sensitive metabolic couples can serve as noninvasive surrogate markers to identify the vascular normalization window in tumors with imaging techniques. CONCLUSION: A multimodal imaging approach to characterize physiological, metabolic, and redox changes in tumors is useful to distinguish between the different stages of anti-angiogenic treatment.

MRI confirms loss of blood-brain barrier integrity in a mouse model of disseminated candidiasis.

Disseminated candidiasis primarily targets the kidneys and brain in mice and humans. Damage to these critical organs leads to the high mortality associated with such infections, and invasion across the blood-brain barrier can result in fungal meningoencephalitis. Candida albicans can penetrate a brain endothelial cell barrier in vitro through transcellular migration, but this mechanism has not been confirmed in vivo. MRI using the extracellular vascular contrast agent gadolinium diethylenetriaminepentaacetic acid demonstrated that integrity of the blood-brain barrier is lost during C. albicans invasion. Intravital two-photon laser scanning microscopy was used to provide the first real-time demonstration of C. albicans colonizing the living brain, where both yeast and filamentous forms of the pathogen were found. Furthermore, we adapted a previously described method utilizing MRI to monitor inflammatory cell recruitment into infected tissues in mice. Macrophages and other phagocytes were visualized in kidney and brain by the administration of ultrasmall iron oxide particles. In addition to obtaining new insights into the passage of C. albicans across the brain microvasculature, these imaging methods provide useful tools to study further the pathogenesis of C. albicans infections, to define the roles of Candida virulence genes in kidney versus brain infection and to assess new therapeutic measures for drug development.

EPR oxygen imaging and hyperpolarized 13C MRI of pyruvate metabolism as noninvasive biomarkers of tumor treatment response to a glycolysis inhibitor 3-bromopyruvate.

The hypoxic nature of tumors results in treatment resistance and poor prognosis. To spare limited oxygen for more crucial pathways, hypoxic cancerous cells suppress mitochondrial oxidative phosphorylation and promote glycolysis for energy production. Thereby, inhibition of glycolysis has the potential to overcome treatment resistance of hypoxic tumors. Here, EPR imaging was used to evaluate oxygen dependent efficacy on hypoxia-sensitive drug. The small molecule 3-bromopyruvate blocks glycolysis pathway by inhibiting hypoxia inducible enzymes and enhanced cytotoxicity of 3-bromopyruvate under hypoxic conditions has been reported in vitro. However, the efficacy of 3-bromopyruvate was substantially attenuated in hypoxic tumor regions (pO2<10 mmHg) in vivo using squamous cell carcinoma (SCCVII)-bearing mouse model. Metabolic MRI studies using hyperpolarized 13C-labeled pyruvate showed that monocarboxylate transporter-1 is the major transporter for pyruvate and the analog 3-bromopyruvate in SCCVII tumor. The discrepant results between in vitro and in vivo data were attributed to biphasic oxygen dependent expression of monocarboxylate transporter-1 in vivo. Expression of monocarboxylate transporter-1 was enhanced in moderately hypoxic (8-15 mmHg) tumor regions but down regulated in severely hypoxic (<5 mmHg) tumor regions. These results emphasize the importance of noninvasive imaging biomarkers to confirm the action of hypoxia-activated drugs.

Urea amidolyase (DUR1,2) contributes to virulence and kidney pathogenesis of Candida albicans.

The intracellular enzyme urea amidolyase (Dur1,2p) enables C. albicans to utilize urea as a sole nitrogen source. Because deletion of the DUR1,2 gene reduces survival of C. albicans co-cultured with a murine macrophage cell line, we investigated the role of Dur1,2p in pathogenesis using a mouse model of disseminated candidiasis. A dur1,2Δ/dur1,2Δ strain was significantly less virulent than the wild-type strain, showing significantly higher survival rate, better renal function, and decreased and less sustained fungal colonization in kidney and brain. Complementation of the mutant restored virulence. DUR1,2 deletion resulted in a milder host inflammatory reaction. Immunohistochemistry, flow cytometry, and magnetic resonance imaging showed decreased phagocytic infiltration into infected kidneys. Systemic cytokine levels of wild-type mice infected with the dur1,2 mutant showed a more balanced systemic pro-inflammatory cytokine response. Host gene expression and protein analysis in infected kidneys revealed parallel changes in the local immune response. Significant differences were observed in the kidney IL-1 inflammatory pathway, IL-15 signaling, MAP kinase signaling, and the alternative complement pathway. We conclude that Dur1,2p is important for kidney colonization during disseminated candidiasis and contributes to an unbalanced host inflammatory response and subsequent renal failure. Therefore, this Candida-specific enzyme may represent a useful drug target to protect the host from kidney damage associated with disseminated candidiasis.

Transient decrease in tumor oxygenation after intravenous administration of pyruvate.

MRI using hyperpolarized (13) C-labeled pyruvate is a promising tool to biochemically profile tumors and monitor their response to therapy. This technique requires injection of pyruvate into tumor-bearing animals. Pyruvate is an endogenous entity but the influence of exogenously injected bolus doses of pyruvate on tumor microenvironment is not well understood. In this study, the effect of injecting a bolus of pyruvate on tumor oxygen status was investigated. EPR oxygen imaging revealed that the partial pressure of oxygen (pO(2)) in squamous cell carcinoma implanted in mice decreased significantly 30 min after [1-(13) C]pyruvate injection, but recovered to preinjection levels after 5 h. Dynamic contrast-enhanced-MRI studies showed that, at the dose of pyruvate used, no changes in tumor perfusion were noticed. Immunohistochemical analysis of hypoxic marker pimonidazole independently verified that the squamous cell carcinoma tumor transiently became more hypoxic by pyruvate injection. Efficacy of radiotherapy was suppressed when X-irradiation was delivered during the period of pyruvate-induced transient hypoxia. These results suggest importance of taking into account the transient decrease in tumor pO(2) after pyruvate injection in hyperpolarized (13) C MRI, because tumor oxygen status is an important factor in determining outcomes of therapies.