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Jiang L.,Johns Hopkins University | Chughtai K.,FOM Institute for Atomic and Molecular Physics | Purvine S.O.,Pacific Northwest National Laboratory | Bhujwalla Z.M.,Johns Hopkins University | And 5 more authors.
Analytical Chemistry | Year: 2015

Hypoxic areas are a common feature of rapidly growing malignant tumors and their metastases and are typically spatially heterogeneous. Hypoxia has a strong impact on tumor cell biology and contributes to tumor progression in multiple ways. To date, only a few molecular key players in tumor hypoxia, such as hypoxia-inducible factor-1 (HIF-1), have been discovered. The distribution of biomolecules is frequently heterogeneous in the tumor volume and may be driven by hypoxia and HIF-1α. Understanding the spatially heterogeneous hypoxic response of tumors is critical. Mass spectrometric imaging (MSI) provides a unique way of imaging biomolecular distributions in tissue sections with high spectral and spatial resolution. In this paper, breast tumor xenografts grown from MDA-MB-231-HRE-tdTomato cells, with a red fluorescent tdTomato protein construct under the control of a hypoxia response element (HRE)-containing promoter driven by HIF-1α, were used to detect the spatial distribution of hypoxic regions. We elucidated the 3D spatial relationship between hypoxic regions and the localization of lipids and proteins by using principal component analysis-linear discriminant analysis (PCA-LDA) on 3D rendered MSI volume data from MDA-MB-231-HRE-tdTomato breast tumor xenografts. In this study, we identified hypoxia-regulated proteins active in several distinct pathways such as glucose metabolism, regulation of actin cytoskeleton, protein folding, translation/ribosome, splicesome, the PI3K-Akt signaling pathway, hemoglobin chaperone, protein processing in endoplasmic reticulum, detoxification of reactive oxygen species, aurora B signaling/apoptotic execution phase, the RAS signaling pathway, the FAS signaling pathway/caspase cascade in apoptosis, and telomere stress induced senescence. In parallel, we also identified colocalization of hypoxic regions and various lipid species such as PC(16:0/18:0), PC(16:0/18:1), PC(16:0/18:2), PC(16:1/18:4), PC(18:0/18:1), and PC(18:1/18:1), among others. Our findings shed light on the biomolecular composition of hypoxic tumor regions, which may be responsible for a given tumors resistance to radiation or chemotherapy. © 2015 American Chemical Society.

Ogrinc Potocnik N.,Maastricht Multimodal Molecular Imaging Institute | Porta T.,Maastricht Multimodal Molecular Imaging Institute | Becker M.,Bruker | Heeren R.M.A.,Maastricht Multimodal Molecular Imaging Institute | Ellis S.R.,Maastricht Multimodal Molecular Imaging Institute
Rapid Communications in Mass Spectrometry | Year: 2015

Rationale In mass spectrometry imaging (MSI) it is often desirable to analyse the same sample in both polarities to extract the most information. However, many matrices that produce high-quality spectra in matrix-assisted laser desorption/ionization (MALDI) are volatile, greatly limiting their use in long imaging experiments. We demonstrate that using a new high speed MALDI-MSI instrument, volatile matrices, including those that produce intense lipid signals in both positive and negative ion mode, can now be effectively used in MSI. Methods A prototype Bruker rapifleX MALDI Tissuetyper™ time-of-flight (TOF) instrument was used for high-speed imaging. This allows acquisition rates up to 50 pixels/s made possible by use of a 10 kHz laser and two rotating mirrors that allow the laser beam to be moved over, and synchronised with, the rapidly moving sample. MSI experiments were performed on mouse brain sections using non-vacuum stable dithranol and 2,6-dihydroxyacetophenone (DHA) matrices with pixel sizes ranging from 10 × 10 μm2 to 50 × 50 μm2. Results Both DHA and dithranol produced rich, complementary lipid spectra in both positive and negative ion modes. Due to the rapid acquisition speed of the instrument, both matrices could be effectively used for MSI despite their volatility. For example, an entire mouse brain could be imaged consecutively in both positive and negative ion mode with 50 × 50 μm2 pixels in ∼35 min. We demonstrate that these speeds make possible both faster and higher resolution imaging of biological tissues on practical timescales. Conclusions These high acquisition speeds now make possible whole new classes of matrices that are unstable under high vacuum for MALDI-MSI studies. This provides researchers with far greater range and flexibility in choosing the best matrix for the given sample and analytes that they wish to detect. In addition, such instruments allow MSI to be performed at higher resolution across larger areas on practical time scales. © 2015 John Wiley and Sons, Ltd.

Ellis S.R.,Maastricht Multimodal Molecular Imaging Institute | Cappell J.,Maastricht Multimodal Molecular Imaging Institute | Potocnik N.O.,Maastricht Multimodal Molecular Imaging Institute | Balluff B.,Maastricht Multimodal Molecular Imaging Institute | And 3 more authors.
Analyst | Year: 2016

The high ion signals produced by many lipids in mass spectrometry imaging (MSI) make them an ideal molecular class to study compositional changes throughout tissue sections and their relationship with disease. However, the large extent of structural diversity observed in the lipidome means optimal ion signal for different lipid classes is often obtained in opposite polarities. In this work we demonstrate how new high speed MALDI-MSI technologies combined with precise laser position control enables the acquisition of positive and negative ion mode lipid data from the same tissue section much faster than is possible with other MSI instruments. Critically, using this approach we explicitly demonstrate how such dual polarity acquisitions provide more information regarding molecular composition and spatial distributions throughout biological tissues. For example, in applying this approach to the zebra finch songbird brain we reveal the high abundance of DHA containing phospholipids (PC in positive mode and PE, PS in negative ion mode) in the nuclei that control song learning behaviour. To make the most of dual polarity data from single tissues we have also developed a pLSA-based multivariate analysis technique that includes both positive and negative ion data in the classification approach. In doing so the correlation amongst different lipid classes that ionise best in opposite polarities and contribute to certain spatial patterns within the tissue can be directly revealed. To demonstrate we apply this approach to studying the lipidomic changes throughout the tumor microenvironment within xenografts from a lung cancer model. © 2016 The Royal Society of Chemistry.

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