Teresa Delgado-Goñi

1H-MRSI pattern perturbation in a mouse glioma: the effects of acute hyperglycemia and moderate hypothermia.

MR spectroscopic Imaging (MRSI), with PRESS localization, is used here to monitor the effects of acute hyperglycemia in the spectral pattern of 11 mice bearing GL261 gliomas at normothermia (36.5–37.5°C) and at hypothermia (28.5–29.5°C). These in vivo studies were complemented by ex vivo high resolution magic angle spinning (HR‐MAS) analysis of GL261 tumor samples from 6 animals sacrificed by focused microwave irradiation, and blood glucose measurements in 12 control mice. Apparent glucose levels, monitored by in vivo MRSI in brain tumors during acute hyperglycemia, rose to an average of 1.6‐fold during hypothermia (p < 0.05), while no significant changes were detected at normothermia, or in control experiments performed at euglycemia, or in normal/peritumoral brain regions. Ex vivo analysis of glioma‐bearing mouse brains at hypothermia revealed higher glucose increases in distinct regions during the acute hyperglycemic challenge (up to 6.6‐fold at the tumor center), in agreement with maximal in vivo blood glucose changes (5‐fold). Phantom studies on taurine plus glucose containing solutions explained the differences between in vivo and ex vivo measurements. Our results also indicate brain tumor heterogeneity in the four animal tumors investigated in response to a defined metabolic challenge. Copyright

Improving the classification of brain tumors in mice with perturbation enhanced (PE)-MRSI

Classifiers based on statistical pattern recognition analysis of MRSI data are becoming important tools for the non-invasive diagnosis of human brain tumors. Here we investigate the potential interest of perturbation-enhanced MRSI (PE-MRSI), in this case acute hyperglycemia, for improving the discrimination between mouse brain MRS patterns of glioblastoma multiforme (GBM), oligodendroglioma (ODG), and non-tumor brain parenchyma (NT). Six GBM-bearing mice and three ODG-bearing mice were scanned at 7 Tesla by PRESS-MRSI with 12 and 136 ms echo-time, during euglycemia (Eug) and also during induced acute hyperglycemia (Hyp), generating altogether four datasets per animal (echo time + glycemic condition): 12Eug, 136Eug, 12Hyp, and 136Hyp. For classifier development all spectral vectors (spv) selected from the MRSI matrix were unit length normalized (UL2) and used either as a training set (76 GBM spv, four mice; 70 ODG spv, two mice; 54 NT spv) or as an independent testing set (61 GBM spv, two mice; 31 ODG, one mouse; 23 NT spv). All Fishers LDA classifiers obtained were evaluated as far as their descriptive performance-correctly classified cases of the training set (bootstrapping)-and predictive accuracy-balanced error rate of independent testing set classification. MRSI-based classifiers at 12Hyp were consistently more efficient in separating GBM, ODG, and NT regions, with overall accuracies always >80% and up to 95-96%; remaining classifiers were within the 48-85% range. This was also confirmed by user-independent selection of training and testing sets, using leave-one-out (LOO). This highlights the potential interest of perturbation-enhanced MRSI protocols for improving the non-invasive characterization of preclinical brain tumors.

Molecular imaging coupled to pattern recognition distinguishes response to temozolomide in preclinical glioblastoma

Non‐invasive monitoring of response to treatment of glioblastoma (GB) is nowadays carried out using MRI. MRS and MR spectroscopic imaging (MRSI) constitute promising tools for this undertaking.

The BRAF Inhibitor Vemurafenib Activates Mitochondrial Metabolism and Inhibits Hyperpolarized Pyruvate-Lactate Exchange in BRAF-Mutant Human Melanoma Cells.

Understanding the impact of BRAF signaling inhibition in human melanoma on key disease mechanisms is important for developing biomarkers of therapeutic response and combination strategies to improve long-term disease control. This work investigates the downstream metabolic consequences of BRAF inhibition with vemurafenib, the molecular and biochemical processes that underpin them, their significance for antineoplastic activity, and potential as noninvasive imaging response biomarkers. 1H NMR spectroscopy showed that vemurafenib decreases the glycolytic activity of BRAF-mutant (WM266.4 and SKMEL28) but not BRAFWT (CHL-1 and D04) human melanoma cells. In WM266.4 cells, this was associated with increased acetate, glycine, and myo-inositol levels and decreased fatty acyl signals, while the bioenergetic status was maintained. 13C NMR metabolic flux analysis of treated WM266.4 cells revealed inhibition of de novo lactate synthesis and glucose utilization, associated with increased oxidative and anaplerotic pyruvate carboxylase mitochondrial metabolism and decreased lipid synthesis. This metabolic shift was associated with depletion of hexokinase 2, acyl-CoA dehydrogenase 9, 3-phosphoglycerate dehydrogenase, and monocarboxylate transporters (MCT) 1 and 4 in BRAF-mutant but not BRAFWT cells and, interestingly, decreased BRAF-mutant cell dependency on glucose and glutamine for growth. Further, the reduction in MCT1 expression observed led to inhibition of hyperpolarized 13C-pyruvate–lactate exchange, a parameter that is translatable to in vivo imaging studies, in live WM266.4 cells. In conclusion, our data provide new insights into the molecular and metabolic consequences of BRAF inhibition in BRAF-driven human melanoma cells that may have potential for combinatorial therapeutic targeting as well as noninvasive imaging of response. Mol Cancer Ther; 15(12); 2987–99. ©2016 AACR.

A new ex vivo method to evaluate the performance of candidate MRI contrast agents: a proof-of-concept study

BackgroundMagnetic resonance imaging (MRI) plays an important role in tumor detection/diagnosis. The use of exogenous contrast agents (CAs) helps to improve the discrimination between lesion and neighbouring tissue, but most of the currently available CAs are non-specific. Assessing the performance of new, selective CAs requires exhaustive assays and large amounts of material. Accordingly, in a preliminary screening of new CAs, it is important to choose candidate compounds with good potential for in vivo efficiency. This screening method should reproduce as close as possible the in vivo environment. In this sense, a fast and reliable method to select the best candidate CAs for in vivo studies would minimize time and investment cost, and would benefit the development of better CAs.ResultsThe post-mortem ex vivo relative contrast enhancement (RCE) was evaluated as a method to screen different types of CAs, including paramagnetic and superparamagnetic agents. In detail, sugar/gadolinium-loaded gold nanoparticles (Gd-GNPs) and iron nanoparticles (SPIONs) were tested. Our results indicate that the post-mortem ex vivo RCE of evaluated CAs, did not correlate well with their respective in vitro relaxivities. The results obtained with different Gd-GNPs suggest that the linker length of the sugar conjugate could modulate the interactions with cellular receptors and therefore the relaxivity value. A paramagnetic CA (GNP (E_2)), which performed best among a series of Gd-GNPs, was evaluated both ex vivo and in vivo. The ex vivo RCE was slightly worst than gadoterate meglumine (201.9 ± 9.3% versus 237 ± 14%, respectively), while the in vivo RCE, measured at the time-to-maximum enhancement for both compounds, pointed to GNP E_2 being a better CA in vivo than gadoterate meglumine. This is suggested to be related to the nanoparticule characteristics of the evaluated GNP.ConclusionWe have developed a simple, cost-effective relatively high-throughput method for selecting CAs for in vivo experiments. This method requires approximately 800 times less quantity of material than the amount used for in vivo administrations.

Dimethyl sulfoxide (DMSO) as a potential contrast agent for brain tumors

Dimethyl sulfoxide (DMSO) is commonly used in preclinical studies of animal models of high‐grade glioma as a solvent for chemotherapeutic agents. A strong DMSO signal was detected by single‐voxel MRS in the brain of three C57BL/6 control mice during a pilot study of DMSO tolerance after intragastric administration. This led us to investigate the accumulation and wash‐out kinetics of DMSO in both normal brain parenchyma (n = 3 control mice) by single‐voxel MRS, and in 12 GL261 glioblastomas (GBMs) by single‐voxel MRS (n = 3) and MRSI (n = 9). DMSO accumulated differently in each tissue type, reaching its highest concentration in tumors: 6.18 ± 0.85 µmol/g water, 1.5‐fold higher than in control mouse brain (p < 0.05). A faster wash‐out was detected in normal brain parenchyma with respect to GBM tissue: half‐lives of 2.06 ± 0.58 and 4.57 ± 1.15 h, respectively. MRSI maps of time‐course DMSO changes revealed clear hotspots of differential spatial accumulation in GL261 tumors. Additional MRSI studies with four mice bearing oligodendrogliomas (ODs) revealed similar results as in GBM tumors. The lack of T1 contrast enhancement post‐gadolinium (gadopentetate dimeglumine, Gd‐DTPA) in control mouse brain and mice with ODs suggested that DMSO was fully able to cross the intact blood–brain barrier in both normal brain parenchyma and in low‐grade tumors. Our results indicate a potential role for DMSO as a contrast agent for brain tumor detection, even in those tumors ‘invisible’ to standard gadolinium‐enhanced MRI, and possibly for monitoring heterogeneities associated with progression or with therapeutic response. Copyright

Detecting human melanoma cell re-differentiation following BRAF or heat shock protein 90 inhibition using photoacoustic and magnetic resonance imaging

Targeted therapies specific to the BRAF-MEK-ERK signaling pathway have shown great promise in the treatment of malignant melanoma in the last few years, with these drugs now commonly used in clinic. Melanoma cells treated using these agents are known to exhibit increased levels of melanin pigment and tyrosinase activity. In this study we assessed the potential of non-invasive imaging approaches (photoacoustic imaging (PAI) and magnetic resonance imaging (MRI)) to detect melanin induction in SKMEL28 human melanoma cells, following inhibition of Hsp90 and BRAF signaling using 17-AAG and vemurafenib, respectively. We confirmed, using western blot and spectrophotometry, that Hsp90 or BRAF inhibitor-induced melanoma cell differentiation resulted in an upregulation of tyrosinase and melanin expression levels, in comparison to control cells. This post-treatment increase in cellular pigmentation induced a significant increase in PAI signals that are spectrally identifiable and shortening of the MRI relaxation times T1 and

Abstract 1130: Unveiling the metabolic response of BRAF mutant melanoma cells to BRAF inhibition

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