Survival Pathways of HIF-Deficient Tumour Cells: TCA Inhibition, Peroxisomal Fatty Acid Oxidation Activation and an AMPK-PGC-1α Hypoxia Sensor.

The HIF-1 and HIF-2 (HIF1/2) hypoxia responses are frequently upregulated in cancers, and HIF1/2 inhibitors are being developed as anticancer drugs. How could cancers resist anti-HIF1/2 therapy? We studied metabolic and molecular adaptations of HIF-1ß-deficient Hepa-1c4, a hepatoma model lacking HIF1/2 signalling, which mimics a cancer treated by a totally effective anti-HIF1/2 agent. [1,2-13C2]-D-glucose metabolism was measured by SiDMAP metabolic profiling, gene expression by TaqMan, and metabolite concentrations by 1H MRS. HIF-1ß-deficient Hepa-1c4 responded to hypoxia by increasing glucose uptake and lactate production. They showed higher glutamate, pyruvate dehydrogenase, citrate shuttle, and malonyl-CoA fluxes than normal Hepa-1 cells, whereas pyruvate carboxylase, TCA, and anaplerotic fluxes decreased. Hypoxic HIF-1ß-deficient Hepa-1c4 cells increased expression of PGC-1α, phospho-p38 MAPK, and PPARα, suggesting AMPK pathway activation to survive hypoxia. They had higher intracellular acetate, and secreted more H2O2, suggesting increased peroxisomal fatty acid ß-oxidation. Simultaneously increased fatty acid synthesis and degradation would have "wasted" ATP in Hepa-1c4 cells, thus raising the [AMP]:[ATP] ratio, and further contributing to the upregulation of the AMPK pathway. Since these tumour cells can proliferate without the HIF-1/2 pathways, combinations of HIF1/2 inhibitors with PGC-1α or AMPK inhibitors should be explored.

Characterization and correction of center-frequency effects in X-nuclear eddy current compensations on a clinical MR system.

PURPOSE: The aim of the study was to investigate whether incorrectly compensated eddy currents are the source of persistent X-nuclear spectroscopy and imaging artifacts, as well as methods to correct this. METHODS: Pulse-acquire spectra were collected for 1 H and X-nuclei (23 Na or 31 P) using the minimum TR permitted on a 3T clinical MRI system. Data were collected in 3 orientations (axial, sagittal, and coronal) with the spoiler gradient at the end of the TR applied along the slice direction for each. Modifications to system calibration files to tailor eddy current compensation for each X-nucleus were developed and applied, and data were compared with and without these corrections for: slice-selective MRS (for 23 Na and 31 P), 2D spiral trajectories (for 13 C), and 3D cones trajectories (for 23 Na). RESULTS: Line-shape distortions characteristic of eddy currents were demonstrated for X-nuclei, which were not seen for 1 H. The severity of these correlated with the amplitude of the eddy current frequency compensation term applied by the system along the axis of the applied spoiler gradient. A proposed correction to eddy current compensation, taking account of the gyromagnetic ratio, was shown to dramatically reduce these distortions. The same correction was also shown to improve data quality of non-Cartesian imaging (2D spiral and 3D cones trajectories). CONCLUSION: A simple adaptation of the default compensation for eddy currents was shown to eliminate a range of artifacts detected on X-nuclear spectroscopy and imaging.

A novel Atg5-shRNA mouse model enables temporal control of Autophagy in vivo.

Macroautophagy/autophagy is an evolutionarily conserved catabolic pathway whose modulation has been linked to diverse disease states, including age-associated disorders. Conventional and conditional whole-body knockout mouse models of key autophagy genes display perinatal death and lethal neurotoxicity, respectively, limiting their applications for in vivo studies. Here, we have developed an inducible shRNA mouse model targeting Atg5, allowing us to dynamically inhibit autophagy in vivo, termed ATG5i mice. The lack of brain-associated shRNA expression in this model circumvents the lethal phenotypes associated with complete autophagy knockouts. We show that ATG5i mice recapitulate many of the previously described phenotypes of tissue-specific knockouts. While restoration of autophagy in the liver rescues hepatomegaly and other pathologies associated with autophagy deficiency, this coincides with the development of hepatic fibrosis. These results highlight the need to consider the potential side effects of systemic anti-autophagy therapies.

Quantitative and textural analysis of magnetization transfer and diffusion images in the early detection of brain metastases.

PURPOSE: The sensitivity of the magnetization transfer ratio (MTR) and apparent diffusion coefficient (ADC) for early detection of brain metastases was investigated in mice and humans. METHODS: Mice underwent MRI twice weekly for up to 31 d following intracardiac injection of the brain-homing breast cancer cell line MDA-MB231-BR. Patients with small cell lung cancer underwent quarterly MRI for 1 year. MTR and ADC were measured in regions of metastasis and matched contralateral tissue at the final time point and in registered regions at earlier time points. Texture analysis and linear discriminant analysis were performed to detect metastasis-containing slices. RESULTS: Compared with contralateral tissue, mouse metastases had significantly lower MTR and higher ADC at the final time point. Some lesions were visible at earlier time points on the MTR and ADC maps: 24% of these were not visible on corresponding T2 -weighted images. Texture analysis using the MTR maps showed 100% specificity and 98% sensitivity for metastasis at the final time point, with 77% sensitivity 2-4 d earlier and 46% 5-8 d earlier. Only 2 of 16 patients developed metastases, and their penultimate scans were normal. CONCLUSIONS: Some brain metastases may be detected earlier on MTR than conventional T2 ; however, the small gain is unlikely to justify "predictive" MRI. Magn Reson Med 77:1987-1995, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Apparent diffusion coefficient is highly reproducible on preclinical imaging systems: Evidence from a seven-center multivendor study.

PURPOSE: To evaluate between-site agreement of apparent diffusion coefficient (ADC) measurements in preclinical magnetic resonance imaging (MRI) systems. MATERIALS AND METHODS: A miniaturized thermally stable ice-water phantom was devised. ADC (mean and interquartile range) was measured over several days, on 4.7T, 7T, and 9.4T Bruker, Agilent, and Magnex small-animal MRI systems using a common protocol across seven sites. Day-to-day repeatability was expressed as percent variation of mean ADC between acquisitions. Cross-site reproducibility was expressed as 1.96 × standard deviation of percent deviation of ADC values. RESULTS: ADC measurements were equivalent across all seven sites with a cross-site ADC reproducibility of 6.3%. Mean day-to-day repeatability of ADC measurements was 2.3%, and no site was identified as presenting different measurements than others (analysis of variance [ANOVA] P = 0.02, post-hoc test n.s.). Between-slice ADC variability was negligible and similar between sites (P = 0.15). Mean within-region-of-interest ADC variability was 5.5%, with one site presenting a significantly greater variation than the others (P = 0.0013). CONCLUSION: Absolute ADC values in preclinical studies are comparable between sites and equipment, provided standardized protocols are employed.

Magnetic resonance spectroscopy of cancer metabolism and response to therapy.

Magnetic resonance spectroscopy allows noninvasive in vivo measurements of biochemical information from living systems, ranging from cultured cells through experimental animals to humans. Studies of biopsies or extracts offer deeper insights by detecting more metabolites and resolving metabolites that cannot be distinguished in vivo. The pharmacokinetics of certain drugs, especially fluorinated drugs, can be directly measured in vivo. This review briefly describes these methods and their applications to cancer metabolism, including glycolysis, hypoxia, bioenergetics, tumor pH, and tumor responses to radiotherapy and chemotherapy.

Can localised (19)F magnetic resonance spectroscopy pharmacokinetics of 5FU in colorectal metastases predict clinical response?

BACKGROUND: 5-Fluorouracil remains widely used in colorectal cancer treatment more than 40 years after its development. 19F magnetic resonance spectroscopy can be used in vivo to measure 5FU's half-life and metabolism to cytotoxic fluoronucleotides. Previous studies have shown better survival associated with longer 5FU tumour half-life. This work investigated 5FU pharmacokinetics in liver metastases of colorectal cancer. METHODS: A total of 32 subjects with colorectal cancer undergoing 5FU treatment, 15 of whom had liver metastases, were examined in a 1.5T MRI scanner, using a large coil positioned over the liver. Non-localised spectra were acquired in 1-min blocks for 32 min after injection of a 5FU bolus. The 5FU half-life was measured in each subject, and averaged spectra were examined for the presence of fluoronucleotides. Associations with progression-free survival were assessed. RESULTS: No association was observed between 5FU half-life, tumour burden and survival. Half-lives were all shorter than those associated with improved survival in the literature. Remarkably, in the group with liver metastases, high levels of fluoronucleotides were associated with poorer survival; this counterintuitive result may be due to the higher levels of fluoronucleotides (whose level is higher in tumour tissue than in normal liver) in patients with higher tumour burdens. CONCLUSIONS: It is recommended that future studies use chemical shift imaging at higher field strengths to better resolve tumour from normal liver. Non-localised spectroscopy retains prognostic potential by enabling straightforward detection of fluoronucleotides, which are present at very low concentrations distributed throughout the tissue.

Combined use of neuroradiology and 1H-MR spectroscopy may provide an intervention limiting diagnosis of glioblastoma multiforme.

PURPOSE: To evaluate the accuracy of (1)H-MR spectroscopy ((1)H-MRS) as an intervention limiting diagnostic tool for glioblastoma multiforme. GBM is the most common and aggressive primary brain tumor, with mean survival under a year. Oncological practice currently requires histopathological diagnosis before radiotherapy. MATERIALS AND METHODS: Eighty-nine patients had clinical computed tomography (CT) and MR imaging and 1.5T SV SE (1)H-MRS with PRESS localization for neuroradiological diagnosis and tumor classification with spectroscopic and automated pattern recognition analysis (TE 30 ms, TR 2000 ms, spectral width 2500 Hz and 2048 data points, 128-256 signal averages were acquired, depending on voxel size (8 cm(3) to 4 cm(3)). Eighteen patients from a cohort of 89 underwent stereotactic biopsy. RESULTS: The 18 stereotactic biopsies revealed 14 GBM, 2 grade II astrocytomas, 1 lymphoma, and 1 anaplastic astrocytoma. All 14 biopsied GBMs were diagnosed as GBM by a protocol combining an individual radiologist and an automated spectral pattern recognition program. CONCLUSION: In patients undergoing stereotactic biopsy combined neuroradiological and spectroscopic evaluation diagnoses GBM with accuracy that could replace the need for biopsy. We do not advocate the replacement of biopsy in all patients; instead our data suggest a specific intervention limiting role for the use of (1)H-MRS in brain tumor diagnosis.

Reduced N-acetylaspartate is consistent with axonal dysfunction in cerebral small vessel disease.

BACKGROUND: Cerebral small vessel disease (SVD) is an important cause of cognitive impairment, but the pathophysiological mechanisms remain unclear. We used (1)H MRS to investigate brain metabolic differences between patients with SVD and controls and correlated this with cognition. METHODS: 35 patients with SVD (lacunar stroke and radiological evidence of confluent leukoaraiosis) and 35 controls underwent multi-voxel spectroscopic imaging of white matter to obtain absolute metabolite concentrations of N-acetylaspartate (NAA), total creatines, total cholines, myo-inositol, and lactate. A range of cognitive tests was performed on patients with SVD, and composite scores were calculated. RESULTS: Scans of sufficient quality for data analysis were available in 29 cases and 35 controls. NAA was significantly reduced in patients compared with controls (lower by 7.27%, P = 0.004). However, when lesion load within each individual voxel (mean 22% in SVD vs 5% in controls, P < 0.001) was added as a covariate, these differences were no longer significant, suggesting that the metabolite differences arose primarily from differences in lesioned tissue. In patients with SVD, there was no correlation between cognitive scores and any brain metabolite. No lactate, an indicator of anaerobic metabolism, was detected. CONCLUSIONS: The most consistent change in SVD is a reduction in NAA, a marker of neuronal integrity. The lack of correlation with cognition does not support the use of MRS as a surrogate disease marker.

Vessel size index magnetic resonance imaging to monitor the effect of antivascular treatment in a rodent tumor model.

PURPOSE: Vascular disrupting agents are anticancer agents that typically produce a cytostatic tumor response. Vessel size index magnetic resonance imaging (MRI) allows for the estimation of the fractional blood volume (fBV) and blood vessel size (Rv). We assessed whether the vessel size index parameters provided imaging biomarkers for detecting early tumor response to a vascular disrupting agent. METHODS AND MATERIALS: GH3 prolactinomas were grown subcutaneously in 12 rats. Vessel size index MRI was performed with Sinerem, an ultrasmall superparamagnetic iron oxide intravascular contrast agent, to determine the tumor fBV and Rv. MRI was performed before and at 24 h after treatment with either the vascular disrupting agent, 5,6-dimethylxanthenone 4-acetic acid (DMXAA) (n = 6) or with the drug vehicle (n = 6). After treatment, the tumors were analyzed histologically and correlates with the MRI findings sought. RESULTS: Histogram analysis showed non-normal distributions of Rv and fBV. The 25th percentiles of the fBV and Rv were significantly reduced (p < 0.01) after treatment with DMXAA, with an increase in the regions of low-measured fBV. For the treated and control tumors, the fraction of tumor with an fBV of < or =1% correlated with the histologically determined percentage of necrosis (r = 0.77, p < 0.005). The fraction of tumor with an fBV of < or =1% in treated tumors was significantly increased compared with before treatment (p < 0.05) and with that in the controls (p < 0.05). CONCLUSION: The vessel size index results were consistent with the known action of DMXAA to cause vascular collapse, with histogram analysis of the fBV providing the most sensitive indicator of response. In particular, the parameter, the fraction of tumor with an fBV of < or =1% is a potential biomarker that correlates with the histopathologic measure of tumor necrosis.

Diffusion tensor imaging and MR spectroscopy in hypertension and presumed cerebral small vessel disease.

In patients with cerebral small vessel disease (SVD) diffusion tensor imaging (DTI) is sensitive to white matter damage and correlates better with cognitive function than conventional imaging. It has been proposed as a surrogate marker for treatment trials. However, the pathological changes underlying DTI are not known. The purpose of this study was to use magnetic resonance spectroscopy (MRS) to determine the pathological changes underlying DTI abnormalities in a range of patients from asymptomatic white matter hyperintensities to symptomatic cerebral SVD. 29 SVD patients, 63 hypertensive subjects, and 42 normotensive controls were recruited. The relationship between the DTI and MRS parameters in the centrum semiovale white matter was determined. There was a significant reduction in N-acetylaspartate (NAA; 2.067 +/- 0.042 vs 2.299 +/- 0.029 and 2.315 +/- 0.036, P = 9 x 10(-6)) and increase in mean diffusivity (mm2/s x 10(-3); 0.942 +/- 0.123 vs 0.822 +/- 0.064 and 0.792 +/- 0.057, P = 1 x 10(-8)) in symptomatic SVD patients compared with the other two groups. DTI parameters correlated with NAA in all three groups, in a graded manner depending on severity of disease (r -SVD -0.827, hypertensive subjects -0.457, controls -0.317). NAA is a marker of axonal loss/dysfunction. These findings are consistent with axonal loss/dysfunction being the principal process causing the DTI changes found in cerebral SVD and ageing.

Long and short echo time proton magnetic resonance spectroscopic imaging of the healthy aging brain.

PURPOSE: To investigate the relationship between subject age and white matter brain metabolite concentrations and R(2) relaxation rates in a cross-sectional study of human brain. MATERIALS AND METHODS: Long- and short-echo proton spectroscopic imaging were used to investigate concentrations and R2 relaxation rates of N-acetyl aspartate (NAA) + N-acetyl aspartyl glutamate (NAAG), choline (Cho), creatine (Cr), and myoinositol (mI) in the white matter of the centrum semiovale of 106 healthy volunteers aged 50-90 years; usable data were obtained from 79 subjects. A major aim was to identify which parameters were most sensitive to changes with age. Spectra were analyzed using the LCModel method. RESULTS: The apparent R2 of NAA and the LCModel concentration of Cr at short echo time were significantly correlated with age after multiplicity correction. Large lipid resonances were observed in the brain midline of some subjects, the incidence increasing significantly with age. We believe this to result from lipid deposits in the falx cerebri. CONCLUSION: Since only short-echo spectroscopy showed a robust relationship between Cr and subject age, and detects more metabolites than long echo time, we conclude that short-echo is superior to long-echo for future aging studies. Future studies could usefully determine whether the Cr-age relationship is due to changes in concentration, T1, or both.

Correlations between MRS and DTI in cerebral small vessel disease.

Cerebral small vessel disease results in lacunar infarcts and cognitive impairment. Diffusion tensor imaging (DTI) demonstrates a reduction in fractional anisotropy and increase in mean diffusivity, which correlates more strongly with cognition than conventional MRI. The underlying pathological basis for these DTI changes is not known. In this study magnetic resonance spectroscopy was used to determine the biochemical basis of these DTI alterations. Twenty-five patients with lacunar stroke and radiological leukoaraiosis were recruited. Chemical shift imaging (CSI) and DTI were performed on a 1.5 T MRI scanner. A region of interest was positioned in the white matter of the centrum semiovale. Multivoxel CSI data were processed and the metabolite ratios estimated. DTI parameters corresponding to the exact region of tissue excited by CSI were obtained. Mean spectroscopy data and DTI values for each subject were correlated. Univariate analysis revealed a positive correlation between N-acetyl aspartate-creatine (NAA/Cr) and fractional anisotropy (r = 0.52, p = 0.008), and a negative correlation with mean diffusivity (r = -0.51, p = 0.009). Results remained little changed after controlling for mean percentage lesion and mean percentage white matter per voxel (with fractional anisotropy r = 0.54, p = 0.008, and with mean diffusivity r = -0.52, p = 0.01). These findings are consistent with axonal loss or dysfunction, or both, accounting for at least part of the DTI abnormalities found in patients with small vessel disease. It provides evidence that DTI identifies axonal disruption in white matter tracts.

Rat tumor response to the vascular-disrupting agent 5,6-dimethylxanthenone-4-acetic acid as measured by dynamic contrast-enhanced magnetic resonance imaging, plasma 5-hydroxyindoleacetic acid levels, and tumor necrosis.

The dose-dependent effects of 5,6-dimethylxanthenone-4-acetic acid (DMXAA) on rat GH3 prolactinomas were investigated in vivo. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) was used to assess tumor blood flow/permeability pretreatment and 24 hours posttreatment with 0, 100, 200, or 350 mg/kg DMXAA. DCE-MRI data were analyzed using K(trans) and the integrated area under the gadolinium time curve (IAUGC) as response biomarkers. High-performance liquid chromatography (HPLC) was used to determine the plasma concentration of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) following treatment to provide an index of increased vessel permeability and vascular damage. Finally, tumor necrosis was assessed by grading hematoxylin and eosin-stained sections cut from the same tumors investigated by MRI. Both tumor K(trans) and IAUGC were significantly reduced 24 hours posttreatment with 350 mg/kg DMXAA only, with no evidence of dose response. HPLC demonstrated a significant increase in plasma 5-HIAA 24 hours posttreatment with 200 and 350 mg/kg DMXAA. Histologic analysis revealed some evidence of tumor necrosis following treatment with 100 or 200 mg/kg DMXAA, reaching significance with 350 mg/kg DMXAA. The absence of any reduction in K(trans) or IAUGC following treatment with 200 mg/kg, despite a significant increase in 5-HIAA, raises concerns about the utility of established DCE-MRI biomarkers to assess tumor response to DMXAA.

Acute tumor response to ZD6126 assessed by intrinsic susceptibility magnetic resonance imaging.

The effective magnetic resonance imaging (MRI) transverse relaxation rate R(2)* was investigated as an early acute marker of the response of rat GH3 prolactinomas to the vascular-targeting agent, ZD6126. Multigradient echo (MGRE) MRI was used to quantify R(2)*, which is sensitive to tissue deoxyhemoglobin levels. Tumor R(2)* was measured prior to, and either immediately for up to 35 minutes, or 24 hours following administration of 50 mg/kg ZD6126. Following MRI, tumor perfusion was assessed by Hoechst 33342 uptake. Tumor R(2)* significantly increased to 116 +/- 4% of baseline 35 minutes after challenge, consistent with an ischemic insult induced by vascular collapse. A strong positive correlation between baseline R(2)* and the subsequent increase in R(2)* measured 35 minutes after treatment was obtained, suggesting that the baseline R(2)* is prognostic for the subsequent tumor response to ZD6126. In contrast, a significant decrease in tumor R(2)* was found 24 hours after administration of ZD6126. Both the 35-minute and 24-hour R(2)* responses to ZD6126 were associated with a decrease in Hoechst 33342 uptake. Interpretation of the R(2)* response is complex, yet changes in tumor R(2)* may provide a convenient and early MRI biomarker for detecting the antitumor activity of vascular-targeting agents.

A method for interleaved acquisition of a vascular input function for dynamic contrast-enhanced MRI in experimental rat tumours.

Dynamic contrast-enhanced MRI is widely used for the evaluation of the response of experimental rodent tumours to antitumour therapy, particularly for the newly developing antiangiogenic and antivascular agents. However, standard models require a time-course for the plasma concentration of contrast agent (usually referred to as the arterial input function) to calculate the transfer constant K(trans) from the dynamic time-course data. Ideally, the plasma concentration time-course should be measured during each experiment to obtain the most accurate measure of K(trans). This is technically difficult in rodents, so assumed values are generally used. A method is presented here using interleaved acquisitions from a tail coil to obtain the plasma concentration simultaneously with DCE-MRI data obtained from a solenoid coil around the tumour. The SNR of the resulting vascular input function data is high compared with methods using a volume coil to acquire plasma concentrations from the aorta and vena cava.

Single dose of the antivascular agent, ZD6126 (N-acetylcolchinol-O-phosphate), reduces perfusion for at least 96 hours in the GH3 prolactinoma rat tumor model.

Tumor vasculature is an attractive therapeutic target as it differs structurally from normal vasculature, and the destruction of a single vessel can lead to the death of many tumor cells. The effects of antivascular drugs are frequently short term, with regrowth beginning less than 24 hours posttreatment. This study investigated the duration of the response to the vascular targeting agent, ZD6126, of the GH3 prolactinoma, in which efficacy and dose-response have previously been demonstrated. GH3 prolactinomas were grown in the flanks of eight Wistar Furth rats. All animals were treated with 50 mg/kg ZD6126. The tumors were examined with dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) 24 hours pretreatment and posttreatment, and at a single time between 48 and 96 hours posttreatment. No evidence of recovery of perfusion was observed even at the longest (96-hour) time point. Involvement of a statistician at the project planning stage and the use of DCE-MRI, which permits noninvasive quantitation of parameters related to blood flow in intact animals, allowed this highly significant result to be obtained using only eight rats.

Correction of spatial distortion in EPI due to inhomogeneous static magnetic fields using the reversed gradient method.

PURPOSE: To derive and implement a method for correcting spatial distortion caused by in vivo inhomogeneous static magnetic fields in echo-planar imaging (EPI). MATERIALS AND METHODS: The reversed gradient method, which was initially devised to correct distortion in images generated by spin-warp MRI, was adapted to correct distortion in EP images. This method provides point-by-point correction of distortion throughout the image. EP images, acquired with a 3 T MRI system, of a phantom and a volunteer's head were used to test the correction method. RESULTS: Good correction was observed in all cases. Spatial distortion in the uncorrected images ranged up to 4 pixels (12 mm) and was corrected successfully. CONCLUSION: The correction was improved by the application of a nonlinear interpolation scheme. The correction requires that two EP images be acquired at each slice position. This increases the acquisition time, but an improved signal-to-noise ratio (SNR) is seen in the corrected image. The local SNR gain decreases with increasing distortion. In many EPI acquisition schemes, multiple images are averaged at each slice position to increase the SNR; in such cases the reversed gradient correction method can be applied with no increase in acquisition duration.