Michihiko Waki

Human Breast Cancer Tissues Contain Abundant Phosphatidylcholine(36∶1) with High Stearoyl-CoA Desaturase-1 Expression

Breast cancer is the leading cause of cancer and mortality in women worldwide. Recent studies have argued that there is a close relationship between lipid synthesis and cancer progression because some enzymes related to lipid synthesis are overexpressed in breast cancer tissues. However, lipid distribution in breast cancer tissues has not been investigated. We aimed to visualize phosphatidylcholines (PCs) and lysoPCs (LPCs) in human breast cancer tissues by performing matrix assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS), which is a novel technique that enables the visualization of molecules comprehensively. Twenty-nine breast tissue samples were obtained during surgery and subjected to MALDI-IMS analysis. We evaluated the heterogeneity of the distribution of PCs and LPCs on the tissues. Three species [PC(32∶1), PC(34∶1), and PC(36∶1)] of PCs with 1 mono-unsaturated fatty acid chain and 1 saturated fatty acid chain (MUFA-PCs) and one [PC(34∶0)] of PCs with 2 saturated fatty acid chains (SFA-PC) were relatively localized in cancerous areas rather than the rest of the sections (named reference area). In addition, the LPCs did not show any biased distribution. The relative amounts of PC(36∶1) compared to PC(36∶0) and that of PC(36∶1) to LPC(18∶0) were significantly higher in the cancerous areas. The protein expression of stearoyl-CoA desaturase-1 (SCD1), which is a synthetic enzyme of MUFA, showed accumulation in the cancerous areas as observed by the results of immunohistochemical staining. The ratios were further analyzed considering the differences in expressions of the estrogen receptor (ER), human epidermal growth factor receptor 2 (HER2), and Ki67. The ratios of the signal intensity of PC(36∶1) to that of PC(36∶0) was higher in the lesions with positive ER expression. The contribution of SCD1 and other enzymes to the formation of the observed phospholipid composition is discussed.

Accumulated phosphatidylcholine (16:0/16:1) in human colorectal cancer; possible involvement of LPCAT4

The identification of cancer biomarkers is critical for target‐linked cancer therapy. The overall level of phosphatidylcholine (PC) is elevated in colorectal cancer (CRC). To investigate which species of PC is overexpressed in colorectal cancer, an imaging mass spectrometry was performed using a panel of non‐neoplastic mucosal and CRC tissues. In the present study, we identified a novel biomarker, PC(16:0/16:1), in CRC using imaging mass spectrometry. Specifically, elevated levels of PC(16:0/16:1) expression were observed in the more advanced stage of CRC. Our data further showed that PC(16:0/16:1) was specifically localized in the cancer region when examined using imaging mass spectrometry. Notably, because the ratio of PC(16:0/16:1) to lyso‐PC(16:0) was higher in CRC, we postulated that lyso‐PC acyltransferase (LPCAT) activity is elevated in CRC. In an in vitro analysis, we showed that LPCAT4 is involved in the deregulation of PC(16:0/16:1) in CRC. In an immunohistochemical analysis, LPCAT4 was shown to be overexpressed in CRC. These data indicate the potential usefulness of PC(16:0/16:1) for the clinical diagnosis of CRC and implicate LPCAT4 in the elevated expression of PC(16:0/16:1) in CRC.

TSLP Directly Interacts with Skin-Homing Th2 Cells Highly Expressing its Receptor to Enhance IL-4 Production in Atopic Dermatitis

Thymic stromal lymphopoietin (TSLP) is overtly expressed on skin lesions of atopic dermatitis (AD), and the initiative role of TSLP-activated DCs in AD has gained much attention in the past few years, while its actions on other immune cells such as T cells have been given less notice. We aimed to clarify whether TSLP receptor (TSLPR) is expressed on certain populations of T cells and whether TSLP possesses the capability to directly interact with T cells from AD patients. Peripheral lymphocytes from 51 AD patients are analyzed by flow cytometry, and ex vivo experiments using peripheral blood and lesional skin-derived T cells were conducted. TSLPR expression was defined to CD4+ T cells, and CD4+CCR4+CXCR3-CCR7-CCR10+CLA+ T cells in AD patients exhibited enhanced TSLPR expression. The frequency of TSLPR+CD4+ T cells correlated with disease activity. CD4+ T cells from AD patients directly interacted with TSLP to produce a higher amount of IL-4 than those from normal subjects, and this action was attenuated with anti-TSLPR antibody. The importance of IL-4 in the induction of TSLPR expression was found in AD T cells. Our findings indicate that T cells from AD patients possess strong potential to directly interact with TSLP to promote Th2 response.

Investigation by imaging mass spectrometry of biomarker candidates for aging in the hair cortex.

Background Human hair is one of the essential components that define appearance and is a useful source of samples for non-invasive biomonitoring. We describe a novel application of imaging mass spectrometry (IMS) of hair biomolecules for advanced molecular characterization and a better understanding of hair aging. As a cosmetic and biomedical application, molecules whose levels in hair altered with aging were comprehensively investigated. Methods Human hair was collected from 15 young (20±5 years old) and 15 older (50±5 years old) volunteers. Matrix-free laser desorption/ionization IMS was used to visualize molecular distribution in the hair sections. Hair-specific ions displaying a significant difference in the intensities between the 2 age groups were extracted as candidate markers for aging. Tissue localization of the molecules and alterations in their levels in the cortex and medulla in the young and old groups were determined. Results Among the 31 molecules detected specifically in hair sections, 2—one at m/z 153.00, tentatively assigned to be dihydrouracil, and the other at m/z 207.04, identified to be 3,4-dihydroxymandelic acid (DHMA)—exhibited a higher signal intensity in the young group than in the old, and 1 molecule at m/z 164.00, presumed to be O-phosphoethanolamine, displayed a higher intensity in the old group. Among the 3, putative O-phosphoethanolamine showed a cortex-specific distribution. The 3 molecules in cortex presented the same pattern of alteration in signal intensity with aging, whereas those in medulla did not exhibit significant alteration. Conclusion Three molecules whose levels in hair altered with age were extracted. While they are all possible markers for aging, putative dihydrouracil and DHMA, are also suspected to play a role in maintaining hair properties and could be targets for cosmetic supplementation. Mapping of ion localization in hair by IMS is a powerful method to extract biomolecules in specified regions and determine their tissue distribution.

Single-cell time-of-flight secondary ion mass spectrometry reveals that human breast cancer stem cells have significantly lower content of palmitoleic acid compared to their counterpart non-stem cancer cells

Lipids comprise the primary component of cell membranes. Imaging mass spectrometry is increasingly being used to visualize membranous lipids in clinical specimens, and it has revealed that abnormal lipid metabolism is related to the development of diseases. To characterize cell populations which are rare and sparsely localized in tissues, we conducted time-of-flight secondary ion mass spectrometry (TOF-SIMS) analyses of individual cells sorted by fluorescence activated cell sorting (FACS) and applied the method to analyze breast cancer stem cells (CSCs). TOF-SIMS analyses visualized phosphoric acids and four fatty acid (FA) species in the sorted CD45(-)/CD44(+)/CD24(-) CSCs, and these ions are suspected to have originated from membranous phospholipids as they were uniformly detected from the locus where the cells attached. Integrated ion intensity of palmitoleic acids [FA(16:1)] normalized by phosphoric acid signals were decreased significantly in CSCs as compared to that of CD45(-)/CD44(-)/CD24(+) non-stem cancer cells (NSCCs). This finding was supported by liquid chromatography coupled electrospray ionization-tandem mass spectrometry analysis, which revealed phosphatidylcholine (PC)(16:0/16:1) to be less abundant and PC(16:0/16:0) to be more abundant in CSCs as compared to NSCCs. Therefore, our novel method successfully provided lipid composition analysis of individual cells classified by the expression of a complex combination of cell-surface markers. The lipid compositions of CSCs originating from the heterogeneous cellular populations of clinical specimens were successfully characterized by this method.

Nanoparticle-Assisted Laser Desorption/Ionization for Metabolite Imaging

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) has enabled the spatial analysis of various molecules, including peptides, nucleic acids, lipids, and drug molecules. To expand the capabilities of MALDI-IMS, we have established an imaging technique using metal nanoparticles (NPs) to visualize metabolites, termed nanoparticle-assisted laser desorption/ionization imaging mass spectrometry (nano-PALDI-IMS). By utilizing Ag-, Fe-, Au-, and TiO2-derived NPs, we have succeeded in visualizing various metabolites, including fatty acid and glycosphingolipids, with higher sensitivity and spatial resolution than conventional techniques. Herein, we describe the practical experimental procedures and methods associated with nano-PALDI-IMS for the visualization of these molecules.

Palmitic acid, verified by lipid profiling using secondary ion mass spectrometry, demonstrates anti-multiple myeloma activity

Recent studies indicate that lipid metabolic changes affect the survival of multiple myeloma (MM) cells. Time-of-flight secondary ion mass spectrometry (TOF-SIMS), an imaging mass spectrometry technique, is used to visualize the subcellular distribution of biomolecules including lipids. We therefore applied this method to human clinical specimens to analyze the membrane fatty acid composition and determine candidate molecules for MM therapies. We isolated MM cells and normal plasma cells (PCs) from bone marrow aspirates of MM patients and healthy volunteers, respectively, and these separated cells were analyzed by TOF-SIMS. Multiple ions including fatty acids were detected and their ion counts were estimated. In MM cells, the mean intensity of palmitic acid was significantly lower than the mean intensity in PCs. In a cell death assay, palmitic acid reduced U266 cell viability dose-dependently at doses between 50 and 1000 μM. The percentage of apoptotic cells increased from 24h after palmitic acid administration. In contrast, palmitic acid had no effect on the viability of normal peripheral blood mononuclear cells (PBMCs). The results of this study indicated that palmitic acid is a potential candidate for novel therapeutic agents that specifically attack MM cells.

Accumulation of arachidonic acid-containing phosphatidylinositol at the outer edge of colorectal cancer

Accumulating evidence indicates that cancer cells show specific alterations in phospholipid metabolism that contribute to tumour progression in several types of cancer, including colorectal cancer. Questions still remain as to what lipids characterize the outer edge of cancer tissues and whether those cancer outer edge-specific lipid compositions emerge autonomously in cancer cells. Cancer tissue-originated spheroids (CTOSs) that are composed of pure primary cancer cells have been developed. In this study, we aimed to seek out the cancer cell-autonomous acquisition of cancer outer edge-characterizing lipids in colorectal cancer by analysing phospholipids in CTOSs derived from colorectal cancer patients with matrix-assisted laser desorption/ionization (MALDI)-imaging mass spectrometry (IMS). A signal at m/z 885.5 in negative ion mode was detected specifically at the surface regions. The signal was identified as an arachidonic acid (AA)-containing phosphatidylinositol (PI), PI(18:0/20:4), by tandem mass spectrometry analysis. Quantitative analysis revealed that the amount of PI(18:0/20:4) in the surface region of CTOSs was two-fold higher than that in the medial region. Finally, PI(18:0/20:4) was enriched at the cancer cells/stromal interface in colorectal cancer patients. These data imply a possible importance of AA-containing PI for colorectal cancer progression, and suggest cells expressing AA-containing PI as potential targets for anti-cancer therapy.

Direct profiling of the phospholipid composition of adult Caenorhabditis elegans using whole-body imaging mass spectrometry

A protocol for the direct analysis of the phospholipid composition in the whole body of adult soil nematode, Caenorhabditis elegans (C. elegans), was developed, which combined freeze-cracking of the exoskeletal cuticle and matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS). Biomolecules in the m/z range from 700 to 900 were more effectively detected in the freeze-cracked than from simple frozen adult nematode bodies. Different distribution of biomolecules was observed in a nematode body when the matrix was applied with a sublimation deposition method. The whole-body IMS technique was applied on genetically deficient mutant C. elegans to combine whole-body lipidomics and genetics, by comparing the fatty acid compositions, especially of the phosphatidylcholine (PC) species, between the wild-type and fat-1 mutants, which lack the gene encoding an n-3 fatty acid desaturase. A significant reduction of PC(20:5/20:5) and PC(20:4/20:5) and a marked increase of PC(20:4/20:4), PC(20:3/20:4), and PC(20:3/20:3) were detected in the fat-1 mutants in positive ion mode. In addition, phospholipid compositions other than PCs were analyzed in negative ion mode. A loss of a possible phosphatidylinositol (PI) with 18:0/20:5 and a compensative accumulation of putative PI(18:0/20:4) were detected in the fat-1 mutants. In conclusion, the whole-body MALDI-IMS technique is useful for the profiling of multiple biomolecules in C. elegans in both intra- and inter-individual levels.

Acquired cystic disease‐associated renal cell carcinoma with a focal sarcomatoid component: Report of a case showing more pronounced polysomy of chromosomes 3 and 16 in the sarcomatoid component

Acquired cystic disease (ACD)‐associated renal cell carcinoma (RCC) has recently been established. Herein we report the sixth case of ACD‐associated RCC with a sarcomatoid change. The patient was a 77‐year‐old man who regularly underwent hemodialysis for 14 years due to chronic renal failure resulting from IgA nephropathy. On computed tomography, a large right RCC was observed with contrast enhancement in the arterial phase. A nodular protrusion into the perirenal fat was detected. Right nephrectomy was performed under laparoscopy. Surgically resected specimens revealed a tan‐to‐yellow tumor (95 × 75 × 55 mm) with a whitish nodule (20 × 15 × 15 mm) invading into the perirenal fat. Histopathologically, the large carcinoma component of the tumor displayed a cribriform or microcystic growth pattern with deposition of oxalate crystals. The whitish nodule corresponded to the sarcomatoid component, and the spindled and pleomorphic tumor cells showed diffuse positivity of p53 on immunohistochemistry. Fluorescence in situ hybridization revealed trisomy of chromosomes 3 and 16 in the carcinoma component, as was expected from the literature. In addition, increased polysomy of these chromosomes was also observed in the sarcomatoid component. This finding may be related to the development of the sarcomatoid component along with the TP53 mutation.