| Warthin tumor (War-T), also known as papillary cystadenoma or lymphaticmonomorphic adenoma, is the second common benign tumor of salivary gland.It consists of two components, neoplastical epithelium and lymphoid stroma.War-T is popular in the people between60to70years old, and smoking isconsidered strongly related to War-T. Although big efforts were made by otherresearches for investigating the occurrence and development of War-T, Thepathogenesis of this lesion still remains unclear. Previous researches werefocused on genomics and proteomics. However, lipidomic as an emergingdiscipline, there is little study for the role of it in War-T. Especially, the thedistribution of lipids in War-T, there is no study for it.Lipids is the important part in human body, it is including fat, Water-solublevitamins, triglycerides, phospholipids and so on. It is not only involved in thestructure of biomembrane, cell proliferation, differentiation, metabolism andimmune regulation but also energy storage, signal transmission and some otherbiological activities. Phosphatidylcholines (PCs) are one kind of phospholipids. It is main component of phospholipid bilayer of biomembrane, and more presentin the outside of bilayer. The alteration of PCs in the membrane reflects the thesignlling and cells metabolism. Therefore, PCs play an important role in theformation, growth and prognosis of tumors. The characristics of PCs are decidedby the types of binding fatty acids. Therefore, comaparing the distribution ofPCs, especially the distribution of fatty acids in normal and pathological samplewill provide the role of PCs in tumors.Imaging Mass Spectrometry (IMS) is one of the newest in-situ analysistechniques. It can directly scan the biological samples and analyze theinformations about the "structure, spatial and time distribution of molecules" ina cell or tissue. This techniques is based on the mass spectra, it is not limited inanalyzing only one or a few specifical biomolecules. IMS can detect a variety ofbiomolecules in the same time without complex sample preparation and label.IMS is the only technique which makes the binding fatty acids of lipid visual. Itcan locate and observe the distribution of the PCs containing different kinds offatty acids in an intuitive way on the biological samples. In order to deeplyunderstand the characristics of War-T and provide information for futherresearches of pathogenesis of War-T, we applied IMS analysis for detecting theprofile of PCs in War-T and Non-T of human samples, and five War-T specificPCs were found. These five PCs were identified by MS/MS analysis and themeaning of these finding was also discussed in this manuscript.Methods:CLINICAL SAMPLES Eight human samples (five from men and three fromwomen) were analyzed. The ages of the patients ranged from30to78years(Table1). None of the patients had received medical treatment for War-T, and there was no recurrent patient.IMAGING MASS SPECTROMETRY (IMS) The tissue blocks were sliced to athickness of10μm at20°C with a cryostat (CM1950; Leica, Wetzler,Germany). The consecutive tissue sections were mounted on an indium-tin-oxide (ITO)-coated glass slide (Bruker Daltonics) and a MAS-coated glass slide(Matsunami, Osaka, Japan) for the IMS analysis and HE staining, respectively.DHB matrix solution was sprayed uniformly by a0.2-mm nozzle caliberairbrush (Procon Boy FWA Platinum; Mr. Hobby, Tokyo, Japan) MALDI-TOF-TOF-type instrument (ultraflex II TOF/TOF, Bruker Daltonics) was applied toanalyze the tissue sections. The setting parameters for the laser energy, detectorgain and random walk function were optimized for each measurement manuallyto obtain the highest sensitivity for the m/z range of460–1000for detectingphospholipids. All data were acquired and visualized by flexImaging2.1software (Bruker Daltonics). The War-T and Non-T regions of interest (ROIs)were determined by comparison with the HE staining results of the consecutivetissue section. After the exclusion of the matrix-derived peaks and isotopic peaksand the peaks were picked up within m/z460–1000according to their intensitieswith the SIMtool software from the War-T and Non-T ROIs, respectively. Thedistributions of these peaks were visualized on a tissue section. The signalintensities of these peaks in each ROI were statistically compared by Welch t-test. Differences with p <0.01were considered significant. All datasets wereanalyzed by this way.MS/MS ANALYSIS The specific peaks in the War-T and Non-T regions wereidentified by MS/MS analyses performed on consecutive tissue sections in thepositive-ion mode by QSTAR Elite (Applied Biosystems/MDS Sciex, FosterCity, CA). The PL species and its FA component were determined by ion- fragment pattern referring to the candidate molecule hits in an MS Search on theHuman Metabolome Database (http://www.hmdb.ca/spectra/ms/search)Results:(1) HE staining was performed to reveal the morphological characteristics of theWar-T and Non-T regions. According to the HE staining, the War-T and Non-Tregions can be distinguished by their histological features. In addition, theneoplastic epithelium and lymphoid stroma can be distinguished in the War-Tregion.(2) Comparison of the mass spectra from the War-T and Non-T regions of case1,a clear difference in signal intensity was observed in m/z740–940between theWar-T and Non-T regions.15peaks were picked up. In comparison with thesemass spectra, most of the peaks were higher in War-T than Non-T.(3) The ion images from these signals for case1showed four signals (m/z796.5,m/z895.5, m/z897.5and m/z923.5) located mainly in the Non-T region. Thedistributions of m/z782.5and m/z798.5appeared almost uniform. The othernine signals (m/z741.5, m/z772.5, m/z820.5, m/z822.5, m/z824.5, m/z826.5m/z844.5, m/z846.5and m/z848.5) were accumulated in the War-T region.(4) Statistical analysis of signal intensities from case1showed that10of the15peaks had a significant difference in signal intensity (p <0.01) between the War-T and Non-T regions. One signal, m/z796.5, was higher in Non-T. The othernine signals, m/z741.5, m/z772.5, m/z820.5, m/z822.5, m/z824.5, m/z826.5,m/z844.5, m/z846.5and m/z848.5, had higher signal intensity in War-T.(5) These nine signals in War-T were visualized for all cases, and the ion imagesshowed that the signals m/z822.5, m/z824.5and m/z826.5were located at theregion which seemed to be neoplastic epithelium in the War-T region. The signals at m/z820.5, m/z844.5, m/z846.5and m/z848.5were located at a regionwhich was considered to include neoplastic epithelium and lymphoid stroma.However, the signals at m/z741.5and m/z772.5appeared partially in the War-Tregion and seemed to be involved in the lymphoid stroma structure. Finally, afterwe applied the War-T signals to all samples, those at m/z772.5, m/z820.5, m/z822.5, m/z844.5and m/z846.5were found to be common to all War-T regions.(6) MS/MS analysis identified these five signals (m/z772.5, m/z820.5, m/z822.5, m/z844.5and m/z846.5) from tissue sections. According to the ionfragmentation and the candidate from The MS Search of the HumanMetabolome Database, m/z772.5were identified as [PC (diacyl-16:0/16:0)+K]+. The molecules at m/z820.5and m/z822.5were identified as [PC (diacyl-16:0/20:4)+K]+and [PC (diacyl-16:0/20:3)+K]+. The molecules at m/z844.5and m/z846.5were identified as [PC (diacyl-18:2/20:4)+K]+and [PC (diacyl-18:0/20:5)+K]+.(7) By comparing the hot spots in the ion image and the enlarged HE-stainingimages, we found that the strong signal of PC (diacyl-16:0/16:0) was from thefolliculus lymphaticus structure.Summary and Conclusions: An IMS analysis revealed that10signals havesignificant differences in signal intensities between the War-T and Non-Tregions from case1. There were nine signals specifically detected in the War-Tregion. After applied these War-T positive signals to all dataset, five War-Tspecific signals were confirmed. One signal, identified as were PC (diacyl-16:0/20:3), located mainly in neoplastic epithelium; three signals identified asPC (diacyl-16:0/20:4), PC (diacyl-18:2/20:4) and PC (diacyl-18:0/20:5), locatedmainly in neoplastic epithelium and lymphoid stroma; one signal was identified as PC (diacyl-16:0/16:0) and was specifically distributed in the folliculuslymphaticus of lymphoid stroma in War-T. |
PDF Full Text Request