The phenotypic landscape of a Tbc1d24 mutant mouse includes convulsive seizures resembling human early infantile epileptic encephalopathy.

Epilepsy, deafness, onychodystrophy, osteodystrophy and intellectual disability are associated with a spectrum of mutations of human TBC1D24. The mechanisms underlying TBC1D24-associated disorders and the functions of TBC1D24 are not well understood. Using CRISPR-Cas9 genome editing, we engineered a mouse with a premature translation stop codon equivalent to human S324Tfs*3, a recessive mutation of TBC1D24 associated with early infantile epileptic encephalopathy (EIEE). Homozygous S324Tfs*3 mice have normal auditory and vestibular functions but show an abrupt onset of spontaneous seizures at postnatal day 15 recapitulating human EIEE. The S324Tfs*3 variant is located in an alternatively spliced micro-exon encoding six perfectly conserved amino acids incorporated postnatally into TBC1D24 protein due to a micro-exon utilization switch. During embryonic and early postnatal development, S324Tfs*3 homozygotes produce predominantly the shorter wild-type TBC1D24 protein isoform that omits the micro-exon. S324Tfs*3 homozygotes show an abrupt onset of seizures at P15 that correlates with a developmental switch to utilization of the micro-exon. A mouse deficient for alternative splice factor SRRM3 impairs incorporation of the Tbc1d24 micro-exon. Wild-type Tbc1d24 mRNA is abundantly expressed in the hippocampus using RNAscope in situ hybridization. Immunogold electron microscopy using a TBC1D24-specific antibody revealed that TBC1D24 is associated with clathrin-coated vesicles and synapses of hippocampal neurons, suggesting a crucial role of TBC1D24 in vesicle trafficking important for neuronal signal transmission. This is the first characterization of a mouse model of human TBC1D24-associated EIEE that can now be used to screen for antiepileptogenic drugs ameliorating TBCID24 seizure disorders.

MRI demonstrates glutamine antagonist-mediated reversal of cerebral malaria pathology in mice.

The deadliest complication of Plasmodium falciparum infection is cerebral malaria (CM), with a case fatality rate of 15 to 25% in African children despite effective antimalarial chemotherapy. No adjunctive treatments are yet available for this devastating disease. We previously reported that the glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) rescued mice from experimental CM (ECM) when administered late in the infection, a time by which mice had already suffered blood-brain barrier (BBB) dysfunction, brain swelling, and hemorrhaging. Herein, we used longitudinal MR imaging to visualize brain pathology in ECM and the impact of a new DON prodrug, JHU-083, on disease progression in mice. We demonstrate in vivo the reversal of disease markers in symptomatic, infected mice following treatment, including the resolution of edema and BBB disruption, findings usually associated with a fatal outcome in children and adults with CM. Our results support the premise that JHU-083 is a potential adjunctive treatment that could rescue children and adults from fatal CM.

Preclinical evaluation of an 18F-trifluoroborate methionine derivative for glioma imaging.

PURPOSE: 11C-methionine (MET) is one of the most commonly used amino acid tracers for PET imaging of brain tumors. In this study, we report an 18F-labeled boron-derived methionine analogue, denoted as 18F-B-MET, as a potential substitute of 11C-MET for glioma PET imaging. METHODS: 19F-B-MET was synthesized from readily available chemicals according to our previous publication. For kit development, 19F-B-MET was aliquoted in quantities of 10 nmol for on-demand one-step labeling. The 18F-labeling was performed by 18F-19F isotope exchange, and quality control was performed by both HPLC and radio-TLC. Uptake of the tracer was determined in GL26, C6 and U87 tumor cells. PET imaging and the biodistribution assay were performed on mice bearing subcutaneous or orthotopic C6 and U87 tumor xenografts. RESULTS: Starting with 740-1110 MBq 18F-fluoride, >370 MBq of 18F-B-MET was obtained in 25 min (n = 5) with >99% purity and high specific activity (>37 GBq/µmol). 18F-B-MET demonstrated excellent in vitro stability with <1% decomposition after incubation with plasma for 2 h. In vitro cell uptake assay showed that 18F-B-MET accumulated in tumor cells in a time dependent manner and could be competitively inhibited by natural methionine and other L-type transporter transported amino acids. In vivo biodistribution and imaging studies showed high tumor accumulation (2.99 ± 0.23 %ID/g, n = 6) compared with low uptake of brain (0.262 ± 0.05 %ID/g, n = 6) at 60 min after injection in a subcutaneous C6 tumor model. Orthotropic C6 and U87 tumors were clearly visualized with high tumor to brain ratios at 60 min post-injection, corroborating with tumor L-type amino acid transporter 1 (LAT-1) expression levels. CONCLUSION: 18F-B-MET was radiolabeled with high yield in a one-step labeling process, showed excellent pharmacokinetic properties in vivo, with high tumor-to-brain contrast.

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.

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.

Simultaneous imaging of tumor oxygenation and microvascular permeability using Overhauser enhanced MRI.

Architectural and functional abnormalities of blood vessels are a common feature in tumors. A consequence of increased vascular permeability and concomitant aberrant blood flow is poor delivery of oxygen and drugs, which is associated with treatment resistance. In the present study, we describe a strategy to simultaneously visualize tissue oxygen concentration and microvascular permeability by using a hyperpolarized (1)H-MRI, known as Overhauser enhanced MRI (OMRI), and an oxygen-sensitive contrast agent OX63. Substantial MRI signal enhancement was induced by dynamic nuclear polarization (DNP). The DNP achieved up to a 7,000% increase in MRI signal at an OX63 concentration of 1.5 mM compared with that under thermal equilibrium state. The extent of hyperpolarization is influenced mainly by the local concentration of OX63 and inversely by the tissue oxygen level. By collecting dynamic OMRI images at different hyperpolarization levels, local oxygen concentration and microvascular permeability of OX63 can be simultaneously determined. Application of this modality to murine tumors revealed that tumor regions with high vascular permeability were spatio-temporally coincident with hypoxia. Quantitative analysis of image data from individual animals showed an inverse correlation between tumor vascular leakage and median oxygen concentration. Immunohistochemical analyses of tumor tissues obtained from the same animals after OMRI experiments demonstrated that lack of integrity in tumor blood vessels was associated with increased tumor microvascular permeability. This dual imaging technique may be useful for the longitudinal assessment of changes in tumor vascular function and oxygenation in response to chemotherapy, radiotherapy, or antiangiogenic treatment.

Tentorial venous anatomy of mice and humans.

We recently described a transtentorial venous system (TTVS), which to our knowledge was previously unknown, connecting venous drainage throughout the brain in humans. Prior to this finding, it was believed that the embryologic tentorial plexus regresses, resulting in a largely avascular tentorium. Our finding contradicted this understanding and necessitated further investigation into the development of the TTVS. Herein, we sought to investigate mice as a model to study the development of this system. First, using vascular casting and ex vivo micro-CT, we demonstrated that this TTVS is conserved in adult mice. Next, using high-resolution MRI, we identified the primitive tentorial venous plexus in the murine embryo at day 14.5. We also found that, at this embryologic stage, the tentorial plexus drains the choroid plexus. Finally, using vascular casting and micro-CT, we found that the TTVS is the dominant venous drainage in the early postnatal period (P8). Herein, we demonstrated that the TTVS is conserved between mice and humans, and we present a longitudinal study of its development. In addition, our findings establish mice as a translational model for further study of this system and its relationship to intracranial physiology.

Nitrite therapy after cardiac arrest reduces reactive oxygen species generation, improves cardiac and neurological function, and enhances survival via reversible inhibition of mitochondrial complex I.

BACKGROUND: Three-fourths of cardiac arrest survivors die before hospital discharge or suffer significant neurological injury. Except for therapeutic hypothermia and revascularization, no novel therapies have been developed that improve survival or cardiac and neurological function after resuscitation. Nitrite (NO(2)(-)) increases cellular resilience to focal ischemia/reperfusion injury in multiple organs. We hypothesized that nitrite therapy may improve outcomes after the unique global ischemia/reperfusion insult of cardiopulmonary arrest. METHODS AND RESULTS: We developed a mouse model of cardiac arrest characterized by 12 minutes of normothermic asystole and a high cardiopulmonary resuscitation rate. In this model, global ischemia and cardiopulmonary resuscitation were associated with blood and organ nitrite depletion, reversible myocardial dysfunction, impaired alveolar gas exchange, neurological injury, and an approximately 50% mortality. A single low dose of intravenous nitrite (50 nmol=1.85 micromol/kg=0.13 mg/kg) compared with blinded saline placebo given at cardiopulmonary resuscitation initiation with epinephrine improved cardiac function, survival, and neurological outcomes. From a mechanistic standpoint, nitrite treatment restored intracardiac nitrite and increased S-nitrosothiol levels, decreased pathological cardiac mitochondrial oxygen consumption resulting from reactive oxygen species formation, and prevented oxidative enzymatic injury via reversible specific inhibition of respiratory chain complex I. CONCLUSIONS: Nitrite therapy after resuscitation from 12 minutes of asystole rapidly and reversibly modulated mitochondrial reactive oxygen species generation during early reperfusion, limiting acute cardiac dysfunction, death, and neurological impairment in survivors.

Neuraxial dysraphism in EPAS1-associated syndrome due to improper mesenchymal transition.

OBJECTIVE: To investigate the effect of somatic, postzygotic, gain-of-function mutation of Endothelial Per-Arnt-Sim (PAS) domain protein 1 (EPAS1) encoding hypoxia-inducible factor-2α (HIF-2α) on posterior fossa development and spinal dysraphism in EPAS1 gain-of-function syndrome, which consists of multiple paragangliomas, somatostatinoma, and polycythemia. METHODS: Patients referred to our institution for evaluation of new, recurrent, and/or metastatic paragangliomas/pheochromocytoma were confirmed for EPAS1 gain-of-function syndrome by identification of the EPAS1 gain-of-function mutation in resected tumors and/or circulating leukocytes. The posterior fossa, its contents, and the spine were evaluated retrospectively on available MRI and CT images of the head and neck performed for tumor staging and restaging. The transgenic mouse model underwent Microfil vascular perfusion and subsequent intact ex vivo 14T MRI and micro-CT as well as gross dissection, histology, and immunohistochemistry to assess the role of EPAS1 in identified malformations. RESULTS: All 8 patients with EPAS1 gain-of-function syndrome demonstrated incidental posterior fossa malformations-one Dandy-Walker variant and 7 Chiari malformations without syringomyelia. These findings were not associated with a small posterior fossa; rather, the posterior fossa volume exceeded that of its neural contents. Seven of 8 patients demonstrated spinal dysraphism; 4 of 8 demonstrated abnormal vertebral segmentation. The mouse model similarly demonstrated features of neuraxial dysraphism, including cervical myelomeningocele and spinal dysraphism, and cerebellar tonsil displacement through the foramen magnum. Histology and immunohistochemistry demonstrated incomplete mesenchymal transition in the mutant but not the control mouse. CONCLUSIONS: This study characterized posterior fossa and spinal malformations seen in EPAS1 gain-of-function syndrome and suggests that gain-of-function mutation in HIF-2α results in improper mesenchymal transition.

A Multimodal Molecular Imaging Study Evaluates Pharmacological Alteration of the Tumor Microenvironment to Improve Radiation Response.

: Hypoxic zones in solid tumors contribute to radioresistance, and pharmacologic agents that increase tumor oxygenation prior to radiation, including antiangiogenic drugs, can enhance treatment response to radiotherapy. Although such strategies have been applied, imaging assessments of tumor oxygenation to identify an optimum time window for radiotherapy have not been fully explored. In this study, we investigated the effects of α-sulfoquinovosylacyl-1,3-propanediol (SQAP or CG-0321; a synthetic derivative of an antiangiogenic agent) on the tumor microenvironment in terms of oxygen partial pressure (pO2), oxyhemoglobin saturation (sO2), blood perfusion, and microvessel density using electron paramagnetic resonance imaging, photoacoustic imaging, dynamic contrast-enhanced MRI with Gd-DTPA injection, and T2*-weighted imaging with ultrasmall superparamagnetic iron oxide (USPIO) contrast. SCCVII and A549 tumors were grown by injecting tumor cells into the hind legs of mice. Five days of daily radiation (2 Gy) combined with intravenous injection of SQAP (2 mg/kg) 30 minutes prior to irradiation significantly delayed growth of tumor xenografts. Three days of daily treatment improved tumor oxygenation and decreased tumor microvascular density on T2*-weighted images with USPIO, suggesting vascular normalization. Acute effects of SQAP on tumor oxygenation were examined by pO2, sO2, and Gd-DTPA contrast-enhanced imaging. SQAP treatment improved perfusion and tumor pO2 (ΔpO2: 3.1 ± 1.0 mmHg) and was accompanied by decreased sO2 (20%-30% decrease) in SCCVII implants 20-30 minutes after SQAP administration. These results provide evidence that SQAP transiently enhanced tumor oxygenation by facilitating oxygen dissociation from oxyhemoglobin and improving tumor perfusion. Therefore, SQAP-mediated sensitization to radiation in vivo can be attributed to increased tumor oxygenation. SIGNIFICANCE: A multimodal molecular imaging study evaluates pharmacological alteration of the tumor microenvironment to improve radiation response.

On the detection of cerebral metabolic depression in experimental traumatic brain injury using Chemical Exchange Saturation Transfer (CEST)-weighted MRI.

Metabolic abnormalities are commonly observed in traumatic brain injury (TBI) patients exhibiting long-term neurological deficits. This study investigated the feasibility and reproducibility of using chemical exchange saturation transfer (CEST) MRI to detect cerebral metabolic depression in experimental TBI. Phantom and in vivo CEST experiments were conducted at 9.4 Tesla to optimize the selective saturation for enhancing the endogenous contrast-weighting of the proton exchanges over the range of glucose proton chemical shifts (glucoCEST) in the resting rat brain. The optimized glucoCEST-weighted imaging was performed on a closed-head model of diffuse TBI in rats with 2-deoxy-D-[14C]-glucose (2DG) autoradiography validation. The results demonstrated that saturation duration of 1‒2 seconds at pulse powers 1.5‒2µT resulted in an improved contrast-to-noise ratio between the gray and white matter comparable to 2DG autoradiographs. The intrasubject (n = 4) and intersubject (n = 3) coefficient of variations for repeated glucoCEST acquisitions (n = 4) ranged between 8‒16%. Optimization for the TBI study revealed that glucoCEST-weighted images with 1.5µT power and 1 s saturation duration revealed the greatest changes in contrast before and after TBI, and positively correlated with 2DG autoradiograph (r = 0.78, p < 0.01, n = 6) observations. These results demonstrate that glucoCEST-weighted imaging may be useful in detecting metabolic abnormalities following TBI.

Metabolic and Physiologic Imaging Biomarkers of the Tumor Microenvironment Predict Treatment Outcome with Radiation or a Hypoxia-Activated Prodrug in Mice.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by hypoxic niches that lead to treatment resistance. Therefore, studies of tumor oxygenation and metabolic profiling should contribute to improved treatment strategies. Here, we define two imaging biomarkers that predict differences in tumor response to therapy: (i) partial oxygen pressure (pO2), measured by EPR imaging; and (ii) [1-13C] pyruvate metabolism rate, measured by hyperpolarized 13C MRI. Three human PDAC xenografts with varying treatment sensitivity (Hs766t, MiaPaCa2, and Su.86.86) were grown in mice. The median pO2 of the mature Hs766t, MiaPaCa2, and Su.86.86 tumors was 9.1 ± 1.7, 11.1 ± 2.2, and 17.6 ± 2.6 mm Hg, and the rate of pyruvate-to-lactate conversion was 2.72 ± 0.48, 2.28 ± 0.26, and 1.98 ± 0.51 per minute, respectively (n = 6, each). These results are in agreement with steady-state data of matabolites quantified by mass spectroscopy and histologic analysis, indicating glycolytic and hypoxia profile in Hs766t, MiaPaca2, and Su.86.86 tumors. Fractionated radiotherapy (5 Gy × 5) resulted in a tumor growth delay of 16.7 ± 1.6 and 18.0 ± 2.7 days in MiaPaca2 and Su.86.86 tumors, respectively, compared with 6.3 ± 2.7 days in hypoxic Hs766t tumors. Treatment with gemcitabine, a first-line chemotherapeutic agent, or the hypoxia-activated prodrug TH-302 was more effective against Hs766t tumors (20.0 ± 3.5 and 25.0 ± 7.7 days increase in survival time, respectively) than MiaPaCa2 (2.7 ± 0.4 and 6.7 ± 0.7 days) and Su.86.86 (4.7 ± 0.6 and 0.7 ± 0.6 days) tumors. Collectively, these results demonstrate the ability of molecular imaging biomarkers to predict the response of PDAC to treatment with radiotherapy and TH-302.Significance: pO2 imaging data and clinically available metabolic imaging data provide useful insight into predicting the treatment efficacy of chemotherapy, radiation, and a hypoxia-activated prodrug as monotherapies and combination therapies in PDAC tumor xenograft models. Cancer Res; 78(14); 3783-92. ©2018 AACR.

Hyperpolarized [1-13C]-Pyruvate Magnetic Resonance Spectroscopic Imaging of Prostate Cancer In Vivo Predicts Efficacy of Targeting the Warburg Effect.

Purpose: To evaluate the potential of hyperpolarized [1-13C]-pyruvate magnetic resonance spectroscopic imaging (MRSI) of prostate cancer as a predictive biomarker for targeting the Warburg effect.Experimental Design: Two human prostate cancer cell lines (DU145 and PC3) were grown as xenografts. The conversion of pyruvate to lactate in xenografts was measured with hyperpolarized [1-13C]-pyruvate MRSI after systemic delivery of [1-13C] pyruvic acid. Steady-state metabolomic analysis of xenograft tumors was performed with mass spectrometry and steady-state lactate concentrations were measured with proton (1H) MRS. Perfusion and oxygenation of xenografts were measured with electron paramagnetic resonance (EPR) imaging with OX063. Tumor growth was assessed after lactate dehydrogenase (LDH) inhibition with FX-11 (42 µg/mouse/day for 5 days × 2 weekly cycles). Lactate production, pyruvate uptake, extracellular acidification rates, and oxygen consumption of the prostate cancer cell lines were analyzed in vitro LDH activity was assessed in tumor homogenates.Results: DU145 tumors demonstrated an enhanced conversion of pyruvate to lactate with hyperpolarized [1-13C]-pyruvate MRSI compared with PC3 and a corresponding greater sensitivity to LDH inhibition. No difference was observed between PC3 and DU145 xenografts in steady-state measures of pyruvate fermentation, oxygenation, or perfusion. The two cell lines exhibited similar sensitivity to FX-11 in vitro LDH activity correlated to FX-11 sensitivity.Conclusions: Hyperpolarized [1-13C]-pyruvate MRSI of prostate cancer predicts efficacy of targeting the Warburg effect. Clin Cancer Res; 24(13); 3137-48. ©2018 AACR.

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.

Epidermal growth factor receptor as a novel molecular target for aggressive papillary tumors in the middle ear and temporal bone.

Adenomatous tumors in the middle ear and temporal bone are rare but highly morbid because they are difficult to detect prior to the development of audiovestibular dysfunction. Complete resection is often disfiguring and difficult because of location and the late stage at diagnosis, so identification of molecular targets and effective therapies is needed. Here, we describe a new mouse model of aggressive papillary ear tumor that was serendipitously discovered during the generation of a mouse model for mutant EGFR-driven lung cancer. Although these mice did not develop lung tumors, 43% developed head tilt and circling behavior. Magnetic resonance imaging (MRI) scans showed bilateral ear tumors located in the tympanic cavity. These tumors expressed mutant EGFR as well as active downstream targets such as Akt, mTOR and ERK1/2. EGFR-directed therapies were highly effective in eradicating the tumors and correcting the vestibular defects, suggesting these tumors are addicted to EGFR. EGFR activation was also observed in human ear neoplasms, which provides clinical relevance for this mouse model and rationale to test EGFR-targeted therapies in these rare neoplasms.

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.

Pyruvate induces transient tumor hypoxia by enhancing mitochondrial oxygen consumption and potentiates the anti-tumor effect of a hypoxia-activated prodrug TH-302.

BACKGROUND: TH-302 is a hypoxia-activated prodrug (HAP) of bromo isophosphoramide mustard that is selectively activated within hypoxic regions in solid tumors. Our recent study showed that intravenously administered bolus pyruvate can transiently induce hypoxia in tumors. We investigated the mechanism underlying the induction of transient hypoxia and the combination use of pyruvate to potentiate the anti-tumor effect of TH-302. METHODOLOGY/RESULTS: The hypoxia-dependent cytotoxicity of TH-302 was evaluated by a viability assay in murine SCCVII and human HT29 cells. Modulation in cellular oxygen consumption and in vivo tumor oxygenation by the pyruvate treatment was monitored by extracellular flux analysis and electron paramagnetic resonance (EPR) oxygen imaging, respectively. The enhancement of the anti-tumor effect of TH-302 by pyruvate treatment was evaluated by monitoring the growth suppression of the tumor xenografts inoculated subcutaneously in mice. TH-302 preferentially inhibited the growth of both SCCVII and HT29 cells under hypoxic conditions (0.1% O2), with minimal effect under aerobic conditions (21% O2). Basal oxygen consumption rates increased after the pyruvate treatment in SCCVII cells in a concentration-dependent manner, suggesting that pyruvate enhances the mitochondrial respiration to consume excess cellular oxygen. In vivo EPR oxygen imaging showed that the intravenous administration of pyruvate globally induced the transient hypoxia 30 min after the injection in SCCVII and HT29 tumors at the size of 500-1500 mm(3). Pretreatment of SCCVII tumor bearing mice with pyruvate 30 min prior to TH-302 administration, initiated with small tumors (∼ 550 mm(3)), significantly delayed tumor growth. CONCLUSIONS/SIGNIFICANCE: Our in vitro and in vivo studies showed that pyruvate induces transient hypoxia by enhancing mitochondrial oxygen consumption in tumor cells. TH-302 therapy can be potentiated by pyruvate pretreatment if started at the appropriate tumor size and oxygen concentration.

Infusion-based manganese-enhanced MRI: a new imaging technique to visualize the mouse brain.

Manganese-enhanced magnetic resonance imaging is a technique that employs the divalent ion of the paramagnetic metal manganese (Mn(2+)) as an effective contrast agent to visualize, in vivo, the mammalian brain. As total achievable contrast is directly proportional to the net amount of Mn(2+) accumulated in the brain, there is a great interest in optimizing administration protocols to increase the effective delivery of Mn(2+) to the brain while avoiding the toxic effects of Mn(2+) overexposure. In this study, we investigated outcomes following continuous slow systemic infusion of manganese chloride (MnCl(2)) into the mouse via mini-osmotic pump administration. The effects of increasing fractionated rates of Mn(2+) infusion on signal enhancement in regions of the brain were analyzed in a three-treatment study. We acquired whole-brain 3-D T1-weighted images and performed region of interest quantitative analysis to compare mean normalized signal in Mn(2+) treatments spanning 3, 7, or 14 days of infusion (rates of 1, 0.5, and 0.25 µL/h, respectively). Evidence of Mn(2+) transport at the conclusion of each infusion treatment was observed throughout the brains of normally behaving mice. Regions of particular Mn(2+) accumulation include the olfactory bulbs, cortex, infralimbic cortex, habenula, thalamus, hippocampal formation, amygdala, hypothalamus, inferior colliculus, and cerebellum. Signals measured at the completion of each infusion treatment indicate comparable means for all examined fractionated rates of Mn(2+) infusion. In this current study, we achieved a significantly higher dose of Mn(2+) (180 mg/kg) than that employed in previous studies without any observable toxic effects on animal physiology or behavior.

Longitudinal imaging studies of tumor microenvironment in mice treated with the mTOR inhibitor rapamycin.

Rapamycin is an allosteric inhibitor of mammalian target of rapamycin, and inhibits tumor growth and angiogenesis. Recent studies suggested a possibility that rapamycin renormalizes aberrant tumor vasculature and improves tumor oxygenation. The longitudinal effects of rapamycin on angiogenesis and tumor oxygenation were evaluated in murine squamous cell carcinoma (SCCVII) by electron paramagnetic resonance imaging (EPRI) and magnetic resonance imaging (MRI) to identify an optimal time after rapamycin treatment for enhanced tumor radioresponse. Rapamycin treatment was initiated on SCCVII solid tumors 8 days after implantation (500-750 mm(3)) and measurements of tumor pO(2) and blood volume were conducted from day 8 to 14 by EPRI/MRI. Microvessel density was evaluated over the same time period by immunohistochemical analysis. Tumor blood volume as measured by MRI significantly decreased 2 days after rapamycin treatment. Tumor pO(2) levels modestly but significantly increased 2 days after rapamycin treatment; whereas, it decreased in non-treated control tumors. Furthermore, the fraction of hypoxic area (pixels with pO(2)<10 mm Hg) in the tumor region decreased 2 days after rapamycin treatments. Immunohistochemical analysis of tumor microvessel density and pericyte coverage revealed that microvessel density decreased 2 days after rapamycin treatment, but pericyte coverage did not change, similar to what was seen with anti-angiogenic agents such as sunitinib which cause vascular renormalization. Collectively, EPRI/MRI co-imaging can provide non-invasive evidence of rapamycin-induced vascular renormalization and resultant transient increase in tumor oxygenation. Improved oxygenation by rapamycin treatment provides a temporal window for anti-cancer therapies to realize enhanced response to radiotherapy.

Reporting of quantitative oxygen mapping in EPR imaging.

Oxygen maps derived from electron paramagnetic resonance spectral-spatial imaging (EPRI) are based upon the relaxivity of molecular oxygen with paramagnetic spin probes. This technique can be combined with MRI to facilitate mapping of pO(2) values in specific anatomic locations with high precision. The co-registration procedure, which matches the physical and digital dimensions of EPR and MR images, may present the pO(2) map at the higher MRI resolution, exaggerating the spatial resolution of oxygen, making it difficult to precisely distinguish hypoxic regions from normoxic regions. The latter distinction is critical in monitoring the treatment of cancer by radiation and chemotherapy, since it is well-established that hypoxic regions are three or four times more resistant to treatment compared to normoxic regions. The aim of this article is to describe pO(2) maps based on the intrinsic resolution of EPRI. A spectral parameter that affects the intrinsic spatial resolution of EPRI is the full width at half maximum (FWHM) height of the gradient-free EPR absorption line in frequency-encoded imaging. In single point imaging too, the transverse relaxation times (T(2)(∗)) limit the resolution since the signal decays by exp(-t(p)/T(2)(∗)) where the delay time after excitation pulse, t(p), is related to the resolution. Although the spin densities of two point objects may be resolved at this separation, it is inadequate to evaluate quantitative changes of pO(2) levels since the linewidths are proportionately affected by pO(2). A spatial separation of at least twice this resolution is necessary to correctly identify a change in pO(2) level. In addition, the pO(2) values are blurred by uncertainties arising from spectral dimensions. Blurring due to noise and low resolution modulates the pO(2) levels at the boundaries of hypoxic and normoxic regions resulting in higher apparent pO(2) levels in hypoxic regions. Therefore, specification of intrinsic resolution and pO(2) uncertainties are necessary to interpret digitally processed pO(2) illustrations.