K. Moro1, J. Tsuchida1, A. Ohtani1, M. Endo1, T. Niwano1, K. Tatsuda1, C. Toshikawa1, M. Hasegawa1, M. Ikarashi1, M. Nakajima1, Y. Koyama1, J. Sakata1, T. Kobayashi1, H. Kameyama1, K. Takabe2, T. Wakai1, M. Nagahashi1 1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata, NIIGATA, Japan 2Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA
Introduction: Sphingolipids, including sphingosine-1-phosphate (S1P) and ceramide, have
emerged as key regulatory molecules that control various aspects of cell growth and survival in cancer. Despite their critical roles, the levels of sphingolipids have never been measured in patients due to lack of methods to precisely quantify them until recently. We have recently published high levels of S1P not only in breast tumor, but also in tumor microenvironment, such as tumor interstitial fluid (IF), and reported that S1P plays pivotal roles in breast cancer progression. On the other hand, the levels of ceramide, a bioactive metabolite of S1P, in breast cancer patients have not yet well investigated to date. The aim of this study is to determine the levels of ceramide in the tumor and its microenvironment by mass spectrometry utilizing surgical specimens obtained from breast cancer patients.
Methods: Surgical specimens were obtained from 45 breast cancer patients who underwent mastectomy in Niigata University Medical and Dental Hospital. Tissue samples from 1) breast cancer (tumor), 2) peri-tumor normal breast defined as tissue within 1 cm from the gross edge of cancer and 3) normal breast distant from the cancer were collected from the surgical specimens. IF from the tumor, peri-tumor and normal tissue was also collected by a centrifugation method. Sphingolipids, including ceramides (C14:0, C16:0, C18:1, C18:0, C20:0, C22:0, C24:1, C24:0, C26:0) and their metabolites of monohexosylceramides and sphingomyelin, in the tissue samples and the IF were determined by mass spectrometry. Results were analyzed for statistical significance with the Kruskal-Wallis test.
Results:Levels of all species of the ceramides, except for C26:0-ceramide, were significantly higher in tumor tissue than those in peri-tumor and those in normal breast tissue. Similarly, all species of the monohexosylceramides, except for C26:0-monohexosylceramide, were significantly higher in tumor tissue than those in peri-tumor and those in normal breast tissue. All of the sphingomyelin were also significantly higher in tumor tissue than those in peri-tumor and those in normal breast tissue. Importantly, ceramide levels; C14:0, C16:0, C18:1, C18:0, C22:0, C24:1 and the total ceramides, in IF from tumor tissue were significantly higher than those in IF from peri-tumor and from normal breast tissue.
Conclusion:This is the first report that not only the levels of ceramides in tumor tissue, but also those in the IF from tumor tissue shows higher than those in the tissue and IF from peri-tumor and normal breast tissue. At this point, it is unclear whether ceramides in IF is from dead cancer cells, stromal cells, inflammatory cells or adipocytes. Given the fact that ceramides are known to play major role in cancer cell survival, further studies are warranted to elucidate the mechanism.