Mass spectrometry images acylcarnitines, phosphatidylcholines, and sphingomyelin in MDA-MB-231 breast tumor models
2012; Elsevier BV; Volume: 54; Issue: 2 Linguagem: Inglês
10.1194/jlr.m027961
ISSN1539-7262
AutoresKamila Chughtai, Lu Jiang, Tiffany R. Greenwood, Kristine Glunde, Ron M. A. Heeren,
Tópico(s)Advanced Proteomics Techniques and Applications
ResumoThe lipid compositions of different breast tumor microenvironments are largely unknown due to limitations in lipid imaging techniques. Imaging lipid distributions would enhance our understanding of processes occurring inside growing tumors, such as cancer cell proliferation, invasion, and metastasis. Recent developments in MALDI mass spectrometry imaging (MSI) enable rapid and specific detection of lipids directly from thin tissue sections. In this study, we performed multimodal imaging of acylcarnitines, phosphatidylcholines (PC), a lysophosphatidylcholine (LPC), and a sphingomyelin (SM) from different microenvironments of breast tumor xenograft models, which carried tdTomato red fluorescent protein as a hypoxia-response element-driven reporter gene. The MSI molecular lipid images revealed spatially heterogeneous lipid distributions within tumor tissue. Four of the most-abundant lipid species, namely PC(16:0/16:0), PC(16:0/18:1), PC(18:1/18:1), and PC(18:0/18:1), were localized in viable tumor regions, whereas LPC(16:0/0:0) was detected in necrotic tumor regions. We identified a heterogeneous distribution of palmitoylcarnitine, stearoylcarnitine, PC(16:0/22:1), and SM(d18:1/16:0) sodium adduct, which colocalized primarily with hypoxic tumor regions. For the first time, we have applied a multimodal imaging approach that has combined optical imaging and MALDI-MSI with ion mobility separation to spatially localize and structurally identify acylcarnitines and a variety of lipid species present in breast tumor xenograft models The lipid compositions of different breast tumor microenvironments are largely unknown due to limitations in lipid imaging techniques. Imaging lipid distributions would enhance our understanding of processes occurring inside growing tumors, such as cancer cell proliferation, invasion, and metastasis. Recent developments in MALDI mass spectrometry imaging (MSI) enable rapid and specific detection of lipids directly from thin tissue sections. In this study, we performed multimodal imaging of acylcarnitines, phosphatidylcholines (PC), a lysophosphatidylcholine (LPC), and a sphingomyelin (SM) from different microenvironments of breast tumor xenograft models, which carried tdTomato red fluorescent protein as a hypoxia-response element-driven reporter gene. The MSI molecular lipid images revealed spatially heterogeneous lipid distributions within tumor tissue. Four of the most-abundant lipid species, namely PC(16:0/16:0), PC(16:0/18:1), PC(18:1/18:1), and PC(18:0/18:1), were localized in viable tumor regions, whereas LPC(16:0/0:0) was detected in necrotic tumor regions. We identified a heterogeneous distribution of palmitoylcarnitine, stearoylcarnitine, PC(16:0/22:1), and SM(d18:1/16:0) sodium adduct, which colocalized primarily with hypoxic tumor regions. For the first time, we have applied a multimodal imaging approach that has combined optical imaging and MALDI-MSI with ion mobility separation to spatially localize and structurally identify acylcarnitines and a variety of lipid species present in breast tumor xenograft models MALDI mass spectrometry imaging (MSI) can detect, localize, and identify multiple biologically relevant molecules directly from thin tissue sections without the necessity for any labeling (1Chughtai K. Heeren R.M. Mass spectrometric imaging for biomedical tissue analysis.Chem. Rev. 2010; 110: 3237-3277Crossref PubMed Scopus (493) Google Scholar). The sample preparation of thin tissue sections for MALDI-MSI requires the application of laser absorbing matrix crystals on the tissue surface. During an MSI experiment, the laser beam ablates the matrix coated tissue sample surface while the mass spectrometer acquires a collection of spectra that contain comprehensive information about the local biomolecular composition of the sample. Biomolecules present in the tissue are desorbed and separated by a mass spectrometer according to their m/z ratios. Each m/z value present in this spectral collection can be converted to an image by using dedicated imaging software. The chemical structure of each ion detected from the tissue surface can be identified after its isolation and fragmentation inside a mass spectrometer. An additional advantage of MSI is its compatibility with other imaging techniques such as optical bright field and fluorescence microscopy. Such a multimodal approach is particularly useful for analyzing the molecular composition of complex, heterogeneous tumor tissue, which comprises several distinct tumor microenvironments. One example of such heterogeneity is tissue hypoxia, a state of low oxygen tension (pO2 values ≤2.5 mmHg) observed in many solid tumors (2Vaupel P. Mayer A. Briest S. Hockel M. Oxygenation gain factor: a novel parameter characterizing the association between hemoglobin level and the oxygenation status of breast cancers.Cancer Res. 2003; 63: 7634-7637PubMed Google Scholar). Hypoxia increases overall tumor aggressiveness, induces radio and drug resistance, triggers resistance to apoptosis, and enhances cellular migration and invasiveness, eventually leading to cancer metastasis (3Hoogsteen, I. J., Marres, H. A., van den Hoogen, F. J., Rijken, P. F., Lok, J., Bussink, J., Kaanders, J. H., . Expression of EGFR under tumor hypoxia: identification of a subpopulation of tumor cells responsible for aggressiveness and treatment resistance. Int. J. Radiat. Oncol. Biol. Phys., Epub ahead of print. March 13, 2012; doi: 10.1016/j.ijrobp.2012.01.002.Google Scholar). At the molecular level, hypoxia triggers many known and unknown metabolome, lipidome, and proteome changes that modulate tumor vascular expansion and provide an overall advantage for malignant growth, causing rapid disease progression (4Vaupel P. Mayer A. Hypoxia in cancer: significance and impact on clinical outcome.Cancer Metastasis Rev. 2007; 26: 225-239Crossref PubMed Scopus (1680) Google Scholar). Severe, prolonged hypoxia can lead to cell death and tissue necrosis (4Vaupel P. Mayer A. Hypoxia in cancer: significance and impact on clinical outcome.Cancer Metastasis Rev. 2007; 26: 225-239Crossref PubMed Scopus (1680) Google Scholar). Tumor expansion results in the formation of metabolically diverse, viable and necrotic, normoxic and hypoxic, vascularized and avascular tissue regions, which, to date, have not been imaged in a single experiment or by a single imaging technique. We hypothesize that these different tumor tissue regions have different distributions of metabolites and lipids. MSI has the capability of parallel detection and visualization of biomolecules present in these specific tissue microenvironments, which allow for a more-comprehensive analysis of tumor biology. Unlike cancer cell cultures, human tumor xenograft models in mice provide a three-dimensional, native-like environment for studying different aspects of tumor growth (5Morton C.L. Houghton P.J. Establishment of human tumor xenografts in immunodeficient mice.Nat. Protoc. 2007; 2: 247-250Crossref PubMed Scopus (361) Google Scholar). Additionally, tumor xenograft models allow for genetic modifications such as incorporation of genes encoding fluorescent proteins for visualizing and monitoring selected processes such as the influence of tissue hypoxia on tumor extracellular matrix (6Kakkad S.M. Solaiyappan M. O'Rourke B. Stasinopoulos I. Ackerstaff E. Raman V. Bhujwalla Z.M. Glunde K. Hypoxic tumor microenvironments reduce collagen I fiber density.Neoplasia. 2010; 12: 608-617Crossref PubMed Scopus (61) Google Scholar) or cancer metastasis (7Penet M.F. Pathak A.P. Raman V. Ballesteros P. Artemov D. Bhujwalla Z.M. Noninvasive multiparametric imaging of metastasis-permissive microenvironments in a human prostate cancer xenograft.Cancer Res. 2009; 69: 8822-8829Crossref PubMed Scopus (34) Google Scholar). MALDI-MSI is well-suited for ex vivo detection and localization of different lipid categories and classes from samples of various origins and different sizes. MALDI-MSI has recently been applied in lipidome studies of human skin (8Hart P.J. Francese S. Claude E. Woodroofe M.N. Clench M.R. MALDI-MS imaging of lipids in ex vivo human skin.Anal. Bioanal. Chem. 2011; 401: 115-125Crossref PubMed Scopus (78) Google Scholar), veins (9Tanaka H. Zaima N. Yamamoto N. Sagara D. Suzuki M. Nishiyama M. Mano Y. Sano M. Hayasaka T. Goto-Inoue N. et al.Imaging mass spectrometry reveals unique lipid distribution in primary varicose veins.Eur. J. Vasc. Endovasc. Surg. 2010; 40: 657-663Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), and ovary (10Meriaux C. Franck J. Wisztorski M. Salzet M. Fournier I. Liquid ionic matrixes for MALDI mass spectrometry imaging of lipids.J. Proteomics. 2010; 73: 1204-1218Crossref PubMed Scopus (84) Google Scholar); mouse embryo implantation sites (11Burnum K.E. Cornett D.S. Puolitaival S.M. Milne S.B. Myers D.S. Tranguch S. Brown H.A. Dey S.K. Caprioli R.M. Spatial and temporal alterations of phospholipids determined by mass spectrometry during mouse embryo implantation.J. Lipid Res. 2009; 50: 2290-2298Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), tongue (12Enomoto H. Sugiura Y. Setou M. Zaima N. Visualization of phosphatidylcholine, lysophosphatidylcholine and sphingomyelin in mouse tongue body by matrix-assisted laser desorption/ionization imaging mass spectrometry.Anal. Bioanal. Chem. 2011; 400: 1913-1921Crossref PubMed Scopus (29) Google Scholar), and leg muscle (13Touboul D. Piednoel H. Voisin V. De La Porte S. Brunelle A. Halgand F. Laprevote O. Changes of phospholipid composition within the dystrophic muscle by matrix-assisted laser desorption/ionization mass spectrometry and mass spectrometry imaging.Eur. J. Mass Spectrom. (Chichester, Eng.). 2004; 10: 657-664Crossref PubMed Scopus (82) Google Scholar, 14Chughtai S. Chughtai K. Cillero-Pastor B. Kiss A. Agrawal P. MacAleese L. Heeren R.M.A. A multimodal mass spectrometry imaging approach for the study of musculoskeletal tissues.Int. J. Mass Spectrom. 2012; 325–327: 150-160Crossref Scopus (25) Google Scholar); and rat spinal cord (15Landgraf R.R. Prieto Conaway M.C. Garrett T.J. Stacpoole P.W. Yost R.A. Imaging of lipids in spinal cord using intermediate pressure matrix-assisted laser desorption-linear ion trap/Orbitrap MS.Anal. Chem. 2009; 81: 8488-8495Crossref PubMed Scopus (83) Google Scholar), cardiac tissue (16Menger R.F. Stutts W.L. Anbukumar D.S. Bowden J.A. Ford D.A. Yost R.A. MALDI mass spectrometric imaging of cardiac tissue following myocardial infarction in a rat coronary artery ligation model.Anal. Chem. 2012; 84: 1117-1125Crossref PubMed Scopus (44) Google Scholar), and liver (17Astigarraga E. Barreda-Gomez G. Lombardero L. Fresnedo O. Castano F. Giralt M.T. Ochoa B. Rodriguez-Puertas R. Fernandez J.A. Profiling and imaging of lipids on brain and liver tissue by matrix-assisted laser desorption/ ionization mass spectrometry using 2-mercaptobenzothiazole as a matrix.Anal. Chem. 2008; 80: 9105-9114Crossref PubMed Scopus (111) Google Scholar). Mammalian brain tissue, due to its high lipid content, has also been extensively studied by MSI. Ion images of lipid distributions have been obtained for rat (17Astigarraga E. Barreda-Gomez G. Lombardero L. Fresnedo O. Castano F. Giralt M.T. Ochoa B. Rodriguez-Puertas R. Fernandez J.A. Profiling and imaging of lipids on brain and liver tissue by matrix-assisted laser desorption/ ionization mass spectrometry using 2-mercaptobenzothiazole as a matrix.Anal. Chem. 2008; 80: 9105-9114Crossref PubMed Scopus (111) Google Scholar, 18Cha S. Yeung E.S. Colloidal graphite-assisted laser desorption/ionization mass spectrometry and MSn of small molecules. 1. Imaging of cerebrosides directly from rat brain tissue.Anal. Chem. 2007; 79: 2373-2385Crossref PubMed Scopus (157) Google Scholar, 19Koizumi S. Yamamoto S. Hayasaka T. Konishi Y. Yamaguchi-Okada M. Goto-Inoue N. Sugiura Y. Setou M. Namba H. Imaging mass spectrometry revealed the production of lyso-phosphatidylcholine in the injured ischemic rat brain.Neuroscience. 2010; 168: 219-225Crossref PubMed Scopus (112) Google Scholar, 20Puolitaival S.M. Burnum K.E. Cornett D.S. Caprioli R.M. Solvent-free matrix dry-coating for MALDI imaging of phospholipids.J. Am. Soc. Mass Spectrom. 2008; 19: 882-886Crossref PubMed Scopus (193) Google Scholar, 21Trim P.J. Atkinson S.J. Princivalle A.P. Marshall P.S. West A. Clench M.R. Matrix-assisted laser desorption/ionisation mass spectrometry imaging of lipids in rat brain tissue with integrated unsupervised and supervised multivariant statistical analysis.Rapid Commun. Mass Spectrom. 2008; 22: 1503-1509Crossref PubMed Scopus (71) Google Scholar, 22Wang H.Y. Jackson S.N. Post J. Woods A.S. A minimalist approach to MALDI imaging of glycerophospholipids and sphingolipids in rat brain sections.Int. J. Mass Spectrom. 2008; 278: 143-149Crossref PubMed Scopus (62) Google Scholar), mouse (23Chen Y. Allegood J. Liu Y. Wang E. Cachon-Gonzalez B. Cox T.M. Merrill Jr, A.H. Sullards M.C. Imaging MALDI mass spectrometry using an oscillating capillary nebulizer matrix coating system and its application to analysis of lipids in brain from a mouse model of Tay-Sachs/Sandhoff disease.Anal. Chem. 2008; 80: 2780-2788Crossref PubMed Scopus (130) Google Scholar, 24Murphy R.C. Hankin J.A. Barkley R.M. Imaging of lipid species by MALDI mass spectrometry.J. Lipid Res. 2009; 50: 317-322Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar), and human (25Veloso A. Astigarraga E. Barreda-Gomez G. Manuel I. Ferrer I. Giralt M.T. Ochoa B. Fresnedo O. Rodriguez-Puertas R. Fernandez J.A. Anatomical distribution of lipids in human brain cortex by imaging mass spectrometry.J. Am. Soc. Mass Spectrom. 2011; 22: 329-338Crossref PubMed Scopus (39) Google Scholar, 26Veloso A. Fernandez R. Astigarraga E. Barreda-Gomez G. Manuel I. Giralt M.T. Ferrer I. Ochoa B. Rodriguez-Puertas R. Fernandez J.A. Distribution of lipids in human brain.Anal. Bioanal. Chem. 2011; 401: 89-101Crossref PubMed Scopus (42) Google Scholar) brain sections. Lipid MSI has also been performed for various types of tumor tissue, such as human brain tumors (27Eberlin L.S. Norton I. Dill A.L. Golby A.J. Ligon K.L. Santagata S. Cooks R.G. Agar N.Y. Classifying human brain tumors by lipid imaging with mass spectrometry.Cancer Res. 2012; 72: 645-654Crossref PubMed Scopus (225) Google Scholar), seminoma (28Masterson T.A. Dill A.L. Eberlin L.S. Mattarozzi M. Cheng L. Beck S.D. Bianchi F. Cooks R.G. Distinctive glycerophospholipid profiles of human seminoma and adjacent normal tissues by desorption electrospray ionization imaging mass spectrometry.J. Am. Soc. Mass Spectrom. 2011; 22: 1326-1333Crossref PubMed Scopus (45) Google Scholar), ovarian cancer (29Gustafsson J.O. Oehler M.K. Ruszkiewicz A. McColl S.R. Hoffmann P. MALDI imaging mass spectrometry (MALDI-IMS)—application of spatial proteomics for ovarian cancer classification and diagnosis.Int. J. Mol. Sci. 2011; 12: 773-794Crossref PubMed Scopus (80) Google Scholar, 30Liu Y. Chen Y. Momin A. Shaner R. Wang E. Bowen N.J. Matyunina L.V. Walker L.D. McDonald J.F. Sullards M.C. et al.Elevation of sulfatides in ovarian cancer: an integrated transcriptomic and lipidomic analysis including tissue-imaging mass spectrometry.Mol. Cancer. 2010; 9: 186Crossref PubMed Scopus (103) Google Scholar), myxoid liposarcomas (31Willems S.M. van Remoortere A. van Zeijl R. Deelder A.M. McDonnell L.A. Hogendoorn P.C. Imaging mass spectrometry of myxoid sarcomas identifies proteins and lipids specific to tumour type and grade, and reveals biochemical intratumour heterogeneity.J. Pathol. 2010; 222: 400-409Crossref PubMed Scopus (90) Google Scholar), human astrocytoma (32Eberlin L.S. Dill A.L. Golby A.J. Ligon K.L. Wiseman J.M. Cooks R.G. Agar N.Y. Discrimination of human astrocytoma subtypes by lipid analysis using desorption electrospray ionization imaging mass spectrometry.Angew. Chem. Int. Ed. Engl. 2010; 49: 5953-5956Crossref PubMed Scopus (105) Google Scholar), human prostate cancer tissue (33Eberlin L.S. Dill A.L. Costa A.B. Ifa D.R. Cheng L. Masterson T. Koch M. Ratliff T.L. Cooks R.G. Cholesterol sulfate imaging in human prostate cancer tissue by desorption electrospray ionization mass spectrometry.Anal. Chem. 2010; 82: 3430-3434Crossref PubMed Scopus (147) Google Scholar), colon cancer liver metastasis (34Shimma S. Sugiura Y. Hayasaka T. Hoshikawa Y. Noda T. Setou M. MALDI-based imaging mass spectrometry revealed abnormal distribution of phospholipids in colon cancer liver metastasis.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2007; 855: 98-103Crossref PubMed Scopus (161) Google Scholar), and human liver adenocarcinoma (35Dill A.L. Ifa D.R. Manicke N.E. Ouyang Z. Cooks R.G. Mass spectrometric imaging of lipids using desorption electrospray ionization.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2009; 877: 2883-2889Crossref PubMed Scopus (111) Google Scholar). Lipid-rich tumors have been associated with increased tumor aggressiveness and metastasis (36Le T.T. Huff T.B. Cheng J.X. Coherent anti-Stokes Raman scattering imaging of lipids in cancer metastasis.BMC Cancer. 2009; 9: 42Crossref PubMed Scopus (146) Google Scholar, 37Ramos C.V. Taylor H.B. Lipid-rich carcinoma of the breast. A clinicopathologic analysis of 13 examples.Cancer. 1974; 33: 812-819Crossref PubMed Scopus (93) Google Scholar, 38Sijens P.E. Levendag P.C. Vecht C.J. van Dijk P. Oudkerk M. 1H MR spectroscopy detection of lipids and lactate in metastatic brain tumors.NMR Biomed. 1996; 9: 65-71Crossref PubMed Scopus (89) Google Scholar). To develop effective cancer treatment strategies, it is crucial not only to identify but also to localize the lipid molecules involved in cancer progression. To date, lipid distributions in breast tumors remain largely unknown due to limitations in lipid imaging methods, which often require incorporation of fluorescent tags and provide limited specificity (39Folick A. Min W. Wang M.C. Label-free imaging of lipid dynamics using coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy.Curr. Opin. Genet. Dev. 2011; 21: 585-590Crossref PubMed Scopus (68) Google Scholar, 40Le T.T. Yue S. Cheng J.X. Shedding new light on lipid biology with coherent anti-Stokes Raman scattering microscopy.J. Lipid Res. 2010; 51: 3091-3102Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 41Maier O. Oberle V. Hoekstra D. Fluorescent lipid probes: some properties and applications (a review).Chem. Phys. Lipids. 2002; 116: 3-18Crossref PubMed Scopus (155) Google Scholar). Here, we have adopted a novel multimodal imaging approach, which integrates microscopy and MSI, for a label-free, ex vivo visualization of different phospholipids directly from breast tumor xenograft tissue sections. We present spatial distributions of sphingomyelin (SM) and multiple phosphatidylcholine (PC) species, which were colocalized with normoxic, hypoxic, or necrotic tumor regions. MSI also localized the distribution of two acylcarnitine molecular ions, which were identified from tumor tissue by ion mobility separation (IMS) followed by tandem mass spectrometry. We have demonstrated that the incorporation of MSI into a multimodal imaging approach revealed the molecular complexity of tumor tissue at an unprecedented level. The matrix α-cyano-4-hydroxycinnamic acid (CHCA) was purchased from Sigma-Aldrich (Germany). Water, acetonitrile (ACN), and trifluoroacetic acid (TFA) were purchased from Biosolve (The Netherlands). Cresyl violet acetate, Ponceau S, and paraformaldehyde powder were purchased from Sigma-Aldrich. Gelatin Type A was purchased from Sigma. Mayer's hematoxylin was purchased from Sigma, and aqueous Eosin Y from EMD Chemicals, Inc. Cytoseal 60 Mounting Medium, Richard-Allan Scientific, was purchased from Thermo Scientific. The MDA-MB-231 human breast cancer cell line was purchased from the American Type Culture Collection (ATCC) and used within 6 months of obtaining from ATCC. This cell line was tested and authenticated by ATCC using two independent methods: the ATCC cytochrome C oxidase I PCR assay, and short tandem-repeat profiling using multiplex PCR. The MDA-MB-231 cell line was genetically modified to express tdTomato red fluorescent protein under the control of hypoxia response elements (MDA-MB-231-HRE-tdTomato) as previously described (42Krishnamachary, B., Penet, M. F., Nimmagadda, S., Mironchik, Y., Raman, V., Solaiyappan, M., Semenza, G. L., Pomper, M. G., Bhujwalla, Z. M., . Hypoxia regulates CD44 and its variant isoforms through HIF-1α in triple negative breast cancer. PLoS ONE., Epub August 28, 2012; doi: 10.1371/journal.pone.0044078.Google Scholar). Cells were injected into the upper thoracic mammary fat pad of athymic nude mice (2 × 106 cells/injection), and tumor growth was monitored using standard calipers. The research was conducted in conformity with the Public Health Service policy on Humane Care and Use of Laboratory Animals. All experimental animal protocols were approved by the Institutional Animal Care and Use Committee of the Johns Hopkins University School of Medicine. When tumors reached a volume of approximately 500 mm3, mice were euthanized and tumors were removed. Three individual tumors were embedded into 10% gelatin blocks prepared using 15 mm × 15 mm × 5 mm cryomolds (Sakura Finetek). Cresyl violet acetate and Ponceau S were prepared in 10% warm (37°C) gelatin at a concentration of 10 mg/ml, mixed in a 1:1 ratio, and injected into 10% gelatin blocks next to the tumor tissue. Each block was sectioned into serial 2 mm-thick fresh tumor sections using an acrylic adjustable tissue slicer (12 mm depth up to 25 mm width, Braintree Scientific, Inc.; Braintree, MA), and tissue slicer blades (Braintree Scientific, Inc.) as previously described (43Jiang L. Greenwood T.R. Artemov D. Raman V. Winnard Jr, P.T. Heeren R.M.A. Bhujwalla Z.M. Glunde K. Localized hypoxia results in spatially heterogeneous metabolic signatures in breast tumor models.Neoplasia. 2012.; 14: 732-741Crossref PubMed Scopus (32) Google Scholar, 44Jiang, L., Greenwood, T. R., Amstalden van Hove, E. R., Chughtai, K., Raman, V., Winnard, P. T., Jr, Heeren, R. M. A., Artemov, D., Glunde, K., . 2012. Combined MR, fluorescence and histology imaging strategy in a human breast tumor xenograft model. NMR Biomed., Epub ahead of print. September 4, 2012. doi: 10.1002/nbm.2846.Google Scholar). These serial fresh tumor xenograft sections were each placed on individual microscope slides (Fisherbrand catalog number 12-550-34, Fisher Scientific; Pittsburgh, PA), and stored in an ice box containing ice on the bottom, with the slides located on a perforated plate at approximately 1-cm above the ice to minimize tissue degradation (43Jiang L. Greenwood T.R. Artemov D. Raman V. Winnard Jr, P.T. Heeren R.M.A. Bhujwalla Z.M. Glunde K. Localized hypoxia results in spatially heterogeneous metabolic signatures in breast tumor models.Neoplasia. 2012.; 14: 732-741Crossref PubMed Scopus (32) Google Scholar, 44Jiang, L., Greenwood, T. R., Amstalden van Hove, E. R., Chughtai, K., Raman, V., Winnard, P. T., Jr, Heeren, R. M. A., Artemov, D., Glunde, K., . 2012. Combined MR, fluorescence and histology imaging strategy in a human breast tumor xenograft model. NMR Biomed., Epub ahead of print. September 4, 2012. doi: 10.1002/nbm.2846.Google Scholar). These fresh sections were imaged by bright-field and fluorescence microscopy with a 1× objective attached to a Nikon inverted microscope, equipped with a filter set for 528 to 553 nm excitation and 600 to 660 nm emission, and a Nikon Coolpix digital camera (Nikon Instruments, Inc.; Melville, NY). Bright-field imaging captured the position of the fiducial markers present inside the gelatin block, as well as the shape of the tumor tissue. The fluorescence from tdTomato expression in hypoxic regions of these tumor sections was detected by fluorescence microscopy. The GNU Image Manipulation Program (GIMP 2.6) was used for two-dimensional (2D) coregistration and overlay of bright-field and fluorescence images of 2 mm-thick tumor sections. All 2 mm-thick sections were snap-frozen immediately after microscopic imaging and stored at −80°C until further analysis. From each 2 mm-thick section, 10 µm-thick sections were cut at −16°C for MSI using a Microm HM550 cryo-microtome (Microm International GmbH; Walldorf, Germany) along with adjacent 10 µm-thick sections for histological staining. Tissue sections for MSI analysis were mounted onto 25 mm × 50 mm × 1.1 mm, Rs= 4–8 Ω Indium Tin Oxide-coated slides (Delta Technologies) and for histological staining onto Superfrost Slides (VWR International; Cat. 48311-600). Tissue sections were stained using a modified H and E staining protocol as previously described (43Jiang L. Greenwood T.R. Artemov D. Raman V. Winnard Jr, P.T. Heeren R.M.A. Bhujwalla Z.M. Glunde K. Localized hypoxia results in spatially heterogeneous metabolic signatures in breast tumor models.Neoplasia. 2012.; 14: 732-741Crossref PubMed Scopus (32) Google Scholar, 44Jiang, L., Greenwood, T. R., Amstalden van Hove, E. R., Chughtai, K., Raman, V., Winnard, P. T., Jr, Heeren, R. M. A., Artemov, D., Glunde, K., . 2012. Combined MR, fluorescence and histology imaging strategy in a human breast tumor xenograft model. NMR Biomed., Epub ahead of print. September 4, 2012. doi: 10.1002/nbm.2846.Google Scholar, 45Chughtai K. Jiang L. Greenwood T.R. Klinkert I. Amstalden van Hove E.R. Heeren R.M. Glunde K. Fiducial markers for combined 3-dimensional mass spectrometric and optical tissue imaging.Anal. Chem. 2012; 84: 1817-1823Crossref PubMed Scopus (40) Google Scholar). Briefly, 10 µm sections attached to Superfrost Slides (VWR International, Cat. 48311-600) were washed with PBS, fixed with freshly depolymerized 3% paraformaldehyde solution for 30 min, washed with distilled water (dH2O), and treated with Mayer's hematoxylin for 30 min at room temperature, followed by five washes with dH2O. Sections were immediately immersed in aqueous Eosin Y for 30 min, followed by five washes with dH2O, mounting with aqueous mounting medium, and attaching of a coverslip. Bright-field images of H and E-stained sections were acquired using 1× or 20× objectives attached to a Nikon microscope equipped with a Nikon Coolpix digital camera (Nikon Instruments, Inc.; Melville, NY). Six tissue sections obtained from three individual tumors were subjected to MSI analysis. Before MSI analysis, tissue sections were placed inside a vacuum desiccator for 30 min. CHCA matrix was prepared at a concentration of 10 mg/ml in 1:1 ACN:H2O/0.1% TFA and was applied by an ImagePrep (Bruker; Germany) application system. Tissue sections were analyzed on a MALDI-Q-TOF (Synapt HDMS; Waters, UK) instrument in IMS mode detecting positive ions. Images were acquired with 150 μm × 150 μm spatial resolution and a 100 μm laser beam spot size. For better detection of masses above m/z 700, the instrument was set to suppress all masses below m/z 700. For 2D MSI analysis and overlay of images, data were visualized using BioMap software (Novartis; Basel, Switzerland). Ion mobility data analysis and visualization were performed by using DriftScope software (version 2.1; Waters, UK) as well as high-definition imaging (HDI) software (version 31; Waters, UK). The LIPID Metabolites And Pathways Strategy database (LIPID MAPS, www.lipidmaps.org) was used to search for possible lipid structures (46Fahy E. Subramaniam S. Murphy R.C. Nishijima M. Raetz C.R. Shimizu T. Spener F. van Meer G. Wakelam M.J. Dennis E.A. Update of the LIPID MAPS comprehensive classification system for lipids.J. Lipid Res. 2009; 50: 9-14Abstract Full Text Full Text PDF PubMed Scopus (1054) Google Scholar, 47Fahy E. Sud M. Cotter D. Subramaniam S. LIPID MAPS online tools for lipid research.Nucleic Acids Res. 2007; 35: W606-612Crossref PubMed Scopus (577) Google Scholar). The identification of selected lipid species was performed on tissue in positive-ion mode using a MALDI-Q-TOF instrument (Synapt; Waters, UK) after completing MSI experiments. Lipid ion precursors were selected within a 2 Da selection window and fragmented using 20–30 V collision energy applied in the trap cell (in TOF mode) or 30–40 V applied in the transfer cell (in IMS mode). Spectra were analyzed using MassLynx software (Waters, UK). Ion mobility-separated ions were visualized by Driftscope software (Waters, UK), and selected drift time windows were exported to MassLynx for spectral analysis. FA chains of lipids of interest were identified from a combination of the mass of the lipid and the MS/MS fragmentation pattern obtained with a mass accuracy of 0.1 Da at m/z 1,000. The LIPID MAPS, Human Metabolome Database version 2.5 (www.hmdb.ca) and the METLIN Metabolite Database (metlin.scripps.edu) were used to search for possible lipid structures and MS/MS fragmentation spectra. We analyzed three MDA-MB-231-HRE-tdTomato breast tumor xenografts that were imaged by the multimodal approach described above. Necrotic tumor regions were outlined in H and E-stained 10 µm-thick sections. Hypoxic tumor regions were detected from the fluorescence images of tdTomato protein expression in 2 mm-thick sections. The remaining tumor regions were normoxic. Ion images of each individual m/z were overlaid with the corresponding optical images and coregistered by using the positions of fiducial markers and tumor boundaries as previously described (43Jiang L. Greenwood T.R. Artemov D. Raman V. Winnard Jr, P.T. Heeren R.M.A. Bhujwalla Z.M. Glunde K. Localized hypoxia results in spatially heterogeneous metabolic signatures in breast tumor models.Neoplasia. 2012.; 14: 732-741Crossref PubMed Scopus (32) Google Scholar, 44Jiang, L., Greenwood, T. R., Amstalden van Hove, E. R., Chughtai, K., Raman, V., Winnard, P. T., Jr, Heeren, R. M. A., Artemov, D., Glunde, K., . 2012. Com
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