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.

The S enantiomer of 2-hydroxyglutarate increases central memory CD8 populations and improves CAR-T therapy outcome.

Cancer immunotherapy is advancing rapidly and gene-modified T cells expressing chimeric antigen receptors (CARs) show particular promise. A challenge of CAR-T cell therapy is that the ex vivo-generated CAR-T cells become exhausted during expansion in culture, and do not persist when transferred back to patients. It has become clear that naive and memory CD8 T cells perform better than the total CD8 T-cell populations in CAR-T immunotherapy because of better expansion, antitumor activity, and persistence, which are necessary features for therapeutic success and prevention of disease relapse. However, memory CAR-T cells are rarely used in the clinic due to generation challenges. We previously reported that mouse CD8 T cells cultured with the S enantiomer of the immunometabolite 2-hydroxyglutarate (S-2HG) exhibit enhanced antitumor activity. Here, we show that clinical-grade human donor CAR-T cells can be generated from naive precursors after culture with S-2HG. S-2HG-treated CAR-T cells establish long-term memory cells in vivo and show superior antitumor responses when compared with CAR-T cells generated with standard clinical protocols. This study provides the basis for a phase 1 clinical trial evaluating the activity of S-2HG-treated CD19-CAR-T cells in patients with B-cell malignancies.

Co-registration of optoacoustic tomography and magnetic resonance imaging data from murine tumour models.

As optoacoustic tomography (OT) emerges as a mainstream pre-clinical imaging modality, understanding the relationship between optoacoustic and other imaging biomarkers in the context of the underlying tissue biology becomes vitally important. Complementary insight into tumour vasculature and hypoxia can be gained using OT alongside magnetic resonance imaging (MRI)-based techniques. To evaluate the relationship between these metrics and the relative performance of the two modalities in assessment of tumour physiology, co-registration of their output imaging data is required. Unfortunately, this poses a significant challenge due to differences in animal positioning during imaging. Here, we present an integrated framework for registration of OT and MR image data in mice. Our framework combines a novel MR animal holder, to improve animal positioning during imaging, and a landmark-based software co-registration algorithm. We demonstrate that our protocol significantly improves registration of both body and tumour contours between these modalities, enabling more precise multi-modal tumour characterisation.

Carbonic anhydrase IX is a pH-stat that sets an acidic tumour extracellular pH in vivo.

BACKGROUND: Tumour carbonic anhydrase IX (CAIX), a hypoxia-inducible tumour-associated cell surface enzyme, is thought to acidify the tumour microenvironment by hydrating CO2 to form protons and bicarbonate, but there is no definitive evidence for this in solid tumours in vivo. METHODS: We used 1H magnetic resonance spectroscopic imaging (MRSI) of the extracellular pH probe imidazolyl succinic acid (ISUCA) to measure and spatially map extracellular pH in HCT116 tumours transfected to express CAIX and empty vector controls in SCID mice. We also measured intracellular pH in situ with 31P MRS and measured lactate in freeze-clamped tumours. RESULTS: CAIX-expressing tumours had 0.15 pH-unit lower median extracellular pH than control tumours (pH 6.71 tumour vs pH 6.86 control, P = 0.01). Importantly, CAIX expression imposed an upper limit for tumour extracellular pH at 6.93. Despite the increased lactate concentration in CAIX-expressing tumours, 31P MRS showed no difference in intracellular pH, suggesting that CAIX acidifies only the tumour extracellular space. CONCLUSIONS: CAIX acidifies the tumour microenvironment, and also provides an extracellular pH control mechanism. We propose that CAIX thus acts as an extracellular pH-stat, maintaining an acidic tumour extracellular pH that is tolerated by cancer cells and favours invasion and metastasis.

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.

Muscle metabolism and activation heterogeneity by combined 31P chemical shift and T2 imaging, and pulmonary O2 uptake during incremental knee-extensor exercise.

The integration of skeletal muscle substrate depletion, metabolite accumulation, and fatigue during large muscle-mass exercise is not well understood. Measurement of intramuscular energy store degradation and metabolite accumulation is confounded by muscle heterogeneity. Therefore, to characterize regional metabolic distribution in the locomotor muscles, we combined 31P magnetic resonance spectroscopy, chemical shift imaging, and T2-weighted imaging with pulmonary oxygen uptake during bilateral knee-extension exercise to intolerance. Six men completed incremental tests for the following: (1) unlocalized 31P magnetic resonance spectroscopy; and (2) spatial determination of 31P metabolism and activation. The relationship of pulmonary oxygen uptake to whole quadriceps phosphocreatine concentration ([PCr]) was inversely linear, and three of four knee-extensor muscles showed activation as assessed by change in T2. The largest changes in [PCr], [inorganic phosphate] ([Pi]) and pH occurred in rectus femoris, but no voxel (72 cm3) showed complete PCr depletion at exercise cessation. The most metabolically active voxel reached 11 ± 9 mM [PCr] (resting, 29 ± 1 mM), 23 ± 11 mM [Pi] (resting, 7 ± 1 mM), and a pH of 6.64 ± 0.29 (resting, 7.08 ± 0.03). However, the distribution of 31P metabolites and pH varied widely between voxels, and the intervoxel coefficient of variation increased between rest (∼10%) and exercise intolerance (∼30-60%). Therefore, the limit of tolerance was attained with wide heterogeneity in substrate depletion and fatigue-related metabolite accumulation, with extreme metabolic perturbation isolated to only a small volume of active muscle (<5%). Regional intramuscular disturbances are thus likely an important requisite for exercise intolerance. How these signals integrate to limit muscle power production, while regional "recruitable muscle" energy stores are presumably still available, remains uncertain.

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.

Inhibition of Hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDA) is among the most lethal human cancers in part because it is insensitive to many chemotherapeutic drugs. Studying a mouse model of PDA that is refractory to the clinically used drug gemcitabine, we found that the tumors in this model were poorly perfused and poorly vascularized, properties that are shared with human PDA. We tested whether the delivery and efficacy of gemcitabine in the mice could be improved by coadministration of IPI-926, a drug that depletes tumor-associated stromal tissue by inhibition of the Hedgehog cellular signaling pathway. The combination therapy produced a transient increase in intratumoral vascular density and intratumoral concentration of gemcitabine, leading to transient stabilization of disease. Thus, inefficient drug delivery may be an important contributor to chemoresistance in pancreatic cancer.

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.

Artefactual computed tomographic middle cerebral artery territory infarct.

We report an artefactual middle cerebral artery territory infarct on CT scan. This anomaly is suggested by the discrepancy between the clinical and radiological findings. Awareness of this possibility may avoid unnecessary further investigation or treatment.

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.