M. Nagahashi1, A. Yamada2, T. Aoyagi2, J. Allegood3, T. Wakai1, S. Spiegel3, K. Takabe4 1Niigata University Graduate School Of Medical And Dental Sciences,Division Of Digestive And General Surgery,Niigata, NIIGATA, Japan 2Virginia Commonwealth University School Of Medicine,Division Of Surgical Oncology And The Massey Cancer Center,Richmond, VA, USA 3Virginia Commonwealth University School Of Medicine,Department Of Biochemistry And Molecular Biology And The Massey Cancer Center,Richmond, VA, USA 4Roswell Park Cancer Institute,Breast Surgery, Department Of Surgical Oncology,Buffalo, NY, USA
Introduction:
Sphingosine-1-phosphate (S1P) is a pleiotropic bioactive lipid mediator that regulates many physiological and pathological processes. S1P produced by sphingosine kinases (SphK1 and SphK2) is secreted from cells and signals in autocrine and/or paracrine manners by binding to its specific cell surface receptors. We have recently reported that S1P is strongly associated with lymphatic network development (FASEB J 2013) and lymphatic metastasis in cancer patients (Cancer Research 2012, JSR 2016). Further, we have shown that S1P links inflammation and cancer in colitis-associated colon cancer (Cancer Cell 2013). It has been suggested that S1P gradient with high concentrations in the blood and lymphatic fluid and low concentrations in the peripheral tissue plays important roles in immune cell trafficking and potentially cancer progression. Although S1P levels in blood have been published to be associated with lymphatic metastasis by our group and others, only a few reports have assessed its levels in lymphatic fluid due to lack of established method in experimental setting. Here, we report simple technique for collection of lymphatic fluid to measure sphingolipids in murine models.
Methods:
The lymphatic fluid was collected directly with a catheter needle (classical method) or was absorbed onto filter paper after incision of cisterna chyli (new method). Whole blood, serum, lymphatic fluid and mesenteric lymph nodes were corrected from WT and SphK2 knockout mice to determine levels of sphingolipids including S1P by mass spectrometry.
Results:
S1P levels were measured in the lymphatic fluid collected either by classical and new methods. The volume of the lymphatic fluid collected by the new method was at least three times greater than those collected by the old one. S1P levels in lymphatic fluid corrected by both classical and new methods showed consistent results with minimal variation. In 8 weeks old mice, S1P concentrations in lymphatic fluid were in the range of 100 to 400 nM, compared to more than 1500 nM in whole blood and more than 600 nM in serum, and less than 50 nM in the mesenteric lymph node tissue. SphK2 knockout mice showed higher levels of S1P in whole blood and serum than WT mice, partially due to overexpression of SphK1 in the blood endothelial cells. Interestingly, S1P levels in lymphatic fluid from SphK2 knockout mice were also significantly higher than those in the WT mice, suggesting an important role of SphK2 and/or SphK1 in the lymphatic endothelial cells to provide S1P into the lymphatic fluid.
Conclusion:
We determined the levels of S1P in lymphatic fluid, which is lower than blood and higher than lymph nodes. In agreement with the previous theory, our results confirm the “S1P gradient” among blood, lymphatic fluid and the peripheral lymphatic tissues. Convenient methods for collection and measurement of sphingolipids in lymphatic fluid are expected to provide new insights on functions of bioactive sphingolipids.