Yoshimi Ide

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.

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.

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.

Abstract P5-01-13: The spectroscopic feature of breast cancer

Objectives: To examine optical properties of breast cancer by time-resolved spectroscopy. Materials and Methods: We irradiated a pulsed laser of 760, 800, and 830 nm wave-length lights at multiple sites of both breasts including the site just above the cancer and detected the light transmitted through the breast with TRS-20SH (Hamamatsu Photonics K.K.). Absorption coefficient (μa), reduced scattering coefficient (μs’), total hemoglobin (tHb), and oxygen saturation (SO2) of the breast were calculated by photon diffusion equation. The clinical trial started in January 2007. A total of one hundred fifty-two breast cancer patients participated in the trial and written informed consent were obtained from all of the patients. Results: The absorption coefficient (μa) in 760, 800 and 830 nm wave-length of breast cancer tissue was significantly high compared with contra-lateral normal breast (760nm:cancer;0.078 normal;0.063 p There was no difference in reduced scattering coefficient (μs’) between breast cancer tissue and contra-lateral normal breast(760nm: cancer;9.58, normal;9.71, 800nm: cancer:9.23, normal;9.40. 830nm; cancer;9.07, normal;9.22). The tHb of breast cancer tissue was significantly high, compared with normal breast (cancer:32.3±14.6, normal breast;22.0±8.6, p=0.001). There was no difference in oxygen saturation (SO2) between breast cancer tissue and contra-lateral normal breast (cancer:73.2±4.3, normal breast;73.6±5.9, p=0.31). Conclusion: Absorption coefficient (μa) and tHb increased in breast cancer, whereas reduced scattering coefficient (μs’) and oxygen saturation did not. Citation Format: Hiroyuki Ogura, Nobuko Yoshizawa, Kenji Yoshimoto, Hatsuko Nasu, Yumiko Taki, Youko Hosokawa, Ryouichi Matsunuma, Yoshimi Ide, Etsuko Yamaki, Toshihiko Suzuki, Motoki Oda, Yukio Ueda, Yutaka Yamashita, Harumi Sakahara. The spectroscopic feature of breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P5-01-13.

Abstract P6-07-05: Single-cell TOF-SIMS reveals that human breast cancer stem cells have significantly lower content of palmitoleic acid compared to their counterpart non-stem cancer cells

Background Distinguishing individual cancers according to their biochemical heterogeneity have provided much useful information to clinical site. Recently, the cancer stem cell (CSC) theory has been accepted as a concept that explains the mechanism of cancer recurrence and resistance to treatment. To characterize such particular cell populations in heterogeneous tissues, we conducted combination of fluorescence activated cell sorting (FACS) and time-of-flight secondary-ion mass spectrometry (TOF-SIMS) and applied the method to analyses of breast CSCs. TOF-SIMS, which enables to visualize the composition of molecules with mass over 100 Da that were obtained in specimen, has been employed to analyze surface of industrial materials and biomaterials. This method is thus suitable for performing single cell analysis of membranous lipids. Methods Breast cancer specimens surgically resected from two patients were enzymatically dispersed into cells. They were labeled with fluorescence-conjugated antibodies of CD45, CD44, and CD24. The cells of CD45-CD44+ CD24- were sorted as CSCs with FACS as well as CD45-CD44- CD24+ cells as non-stem cancer cells (NSCCs). TOF-SIMS analysis and fatty acid analysis was performed according to our previous study published in Surface and Interface Analysis (1). The surface of the sorted cells was analyzed by a PHI TRIFT V (ULVAC-PHI Inc., Kanagawa, Japan) TOF-SIMS instrument. Primary ion beam is irradiated to the surface of the samples and, secondary ions derived from samples are calculated by time-of-flight with the information of the place where the molecular ions were ejected. Negative secondary ions were obtained with a mass range of m/z 0–1850. Mass spectra were analyzed by WinCadenceN software (ULVAC-PHI Inc.) to obtain ion counts and ion images. Integrated ion intensities of FA were normalized using phosphoric acid intensity. The Welch’s t-test was used to compare the normalized ion counts with P-value Results FACS analyses successfully collected CD45-/CD44+/CD24- CSCs and CD45-/CD44-/CD24+ non-stem cancer cells (NSCCs) in both two cases, which were corresponding to o.33% and 0.74% of all cells in case 1, and 0.14% and 1.14% in case 2. TOF-SIMS analyses visualized phosphoric acids and four fatty acid (FA) species in the sorted CSCs and NSCCs. These ions probably came from membranous phosphopolipids and they were uniformly detected from the locus where the cell attached. Integrated ion intensity of palmitoleic acids [FA(16:1)] of CSCs normalized by phosphoric acids signals were significantly decreased than that of CD45-/CD44-/CD24+ NSCCs as a counterpart. Therefore, our novel method successfully provided lipid composition analysis of individual cells classified with complicated combination of marker expressions in clinical specimens composed of heterogeneous cellular populations, and characterized lipid composition of CSCs. Reference 1. Nagata Y, Ishizaki I, Waki M, Ide Y, Hossen A, Ohnishi K, et al. Glutaraldehyde Fixation Method for Single-Cell Lipid Analysis by Time-of-Flight Secondary Ion-Mass Spectrometry. Surface and interface analysis : SIA. 2014:DOI: 10.1002/sia/5522. Citation Format: Yoshimi Ide, Michihiko Waki, Itsuko Ishizaki, Yasuyuki Nagata, Yumiko Taki, Yuko Hosokawa, Ryoichi Matsunuma, Hiroyuki Ogura, Norihiko Shiiya, Noriaki Sanada, Mitsutoshi Setou. Single-cell TOF-SIMS reveals that human breast cancer stem cells have significantly lower content of palmitoleic acid compared to their counterpart non-stem cancer cells [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P6-07-05.

Abstract P4-03-05: The Clinical Trial of New Optical Mammography

Objectives: Optical mammography (O-MMG), which utilizes near-infrared light for the detection of abnormal breast tissue, can be a potentially useful and non-invasive tool for the screening of breast cancer patients. Here we report on the preliminary results of our clinical trial using a time-resolved O-MMG system developed by Hamamatsu Photonics (Hamamatsu, Japan). In our past examination, negative results included cases with mismatch for breast cup because of small breast, lesion outside of cup because of big breast, and bad positioning. So, size and shape of a gantry was improved (3 cup sizes; S, M and L cup). We reported the initial experience of the improved O-MMG systems. Materials and Methods: In principle, the technique involves sequential irradiation of the breast tissue with a pulsed laser with a wavelength of 760, 800 and, 830nm, which is then detected by the variable capacity gantry at multiple sites after passing through the breast tissue. Measurement time has improved to about 7 minutes (a third of the conventional measurement time). The data are analyzed and tomographic images of the absorption coefficient of the breast are then reconstructed based on our tomographic reconstruction algorithm. Between October 2011 and May 2012, fifteen patients with breast cancer and one patient with fibroadenoma participated in the trial. Results: In patients with breast cancer, S cup was used for one patient, M cup for 12 patients and L cup for two patients. Five patients after neo-adjuvant chemotherapy were excluded from the analysis. The lesions were depicted as an area of high hemoglobin concentration in 7 of 10 patients with cancer. One lesion of fibroadenoma was also identified as an area of high hemoglobin concentration. Conclusion: Further examination with our new O-MMG systems will be of interest. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-03-05.

P4-05-05: Imaging Mass Spectrometry Based Lipid Metabolites Analysis for Breast Cancer.

Background: Activation of lipid metabolism is an early event of carcinogenesis and a central hallmark of many cancers including breast cancer. Recent findings argue that stearoyl CoA desaturase-1 (SCD1), a key regulator of the fatty acid (FA) composition and the endoplasmic reticulum resident enzyme that converts saturated FA (SFA) into monounsaturated FA (MUFA) is a novel regulator of carcinogenesis. The distinctive lipids composition of membrane in cancer cells and the biological functions of SCD1, however, still remain uncertain. Imaging mass spectrometry (IMS) is a mass spectrometry-based analyzing technique that enables visualization of the individual molecules without requiring antibodies. It allows comprehensive detection of a wide range of biomolecules, such as lipids. We attempted to visualize the localization of lipids in breast cancer by IMS for better understanding of cancer proliferation. Materials and methods: 13 specimens were obtained from the primary breast cancer patients. All were Japanese woman and aged 41–86 years (mean 61.5y.o.). Only one patient received preoperative systematic therapy. 6 were estrogen receptor (ER) and/or progesterone receptor (PgR) positive and human epidermal growth factor receptor 2 (HER2) negative, 2 were ER and/or PgR positive and HER2 positive, 2 were both ER and PgR negative and HER2 positive and 2 were triple negative. IMS: Samples were immediately chilled in liquid Hexan and stored at −80°. All specimens were sliced into 10 mm thin sections, mounted onto one indium-tin oxide-coated glass slides (Bruker Daltonics) and then sprayed by 2,5-Dihydroxybenzoic acid. Matrix assisted laser desorption ionization (MALDI) technique was used as a soft ionization method. We used time of flight (TOF)/TOF type instrument (Ultraflex, Bruker Daltonics) and all the spectrum were acquired automatically using Fleximaging software (Bruker Daltonics). Each spectral intensity at any mass-to-charge ratio (m/z) was measured at 16 regions of interest (ROI); 13 ROI were picked up from cancerous parts and 3 were from non-cancerous parts. Spectral intensities were compared and statistical analysis was performed by Mann Whitney test. The software was also used to create two-dimensional ion-density maps. Results: In the cancerous parts of all the 13 specimens, two distinct peaks of the molecular ions were detected at m/z 798.5 and 810.5, which were not found in the non-cancerous parts. Median intensity of the molecular ions at m/z 798.5 and 810.5 were 38.9 and 3.18 in the cancerous part, while they were 0.84 and 1.02 in the non-cancerous part (p=0.010 and 0.015, respectively). Tandem mass spectrometry analysis for these two molecules revealed that they were two kinds of phosphatidylcholine (PC), PC (16:0/18:1) and PC (18:0/18:1). Localization of the individual PC was visualized by means of IMS, which showed that in cancerous part accumulation of PCs containing MUFA was more pronounced than those containing SFA only. Conclusion: Two kinds of PC containing MUFA were found to highly accumulate in cancerous parts, which may suggest involvement of SCD1 in the membrane composition regulation and cancer proliferation. Further studies may thus be warranted to explore the relation between PC localization and the SCD1 expression. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-05-05.