Aberrant Glycosphingolipid Pattern in Human Cholangiocarcinoma Stem-Like Subsets

09-P56. Aberrant Glycosphingolipid Pattern in Human Cholangiocarcinoma Stem-Like Subsets A. Mannini1, C. Raggi1, M. Correnti2, E. Rovida3, M. Aureli4, E.V. Carsana4, B. Piombanti1, M. Pastore1, J. Andersen5, C. Coulouarn6, F. Marra1 1University of Florence, Experimental and Clinical Medicine, Florence, Italy, 2University of Milan, Biomedical Sciences for Health, Milan, Italy, 3University of Florence, Experimental and Clinical Biomedical Sciences, Florence, Italy, 4University of Milan, Medical Biotechnology and Translational Medicine, Milan, Italy, 5University of Copenhagen, Biotech Research and Innovation Centre, Copenhagen, Denmark, 6University of Rennes, Inserm, Inra, Institut NUMECAN (Nutrition Metabolisms and Cancer), Rennes, France. Introduction: It is well known that a crucial therapeutic target of cancer is represented by cancer stem cells (CSCs). A great deal of studies show that some glycosphingolipids, a peculiar class of plasma membrane lipids, are prevalently expressed in CSCs and could be considered as markers of cancer staminality (i.e., Burkitt lymphoma, pancreatic carcinoma, and other epithelial cancers). In particular, gangliosides (GS), sialic acid-containing GSL, have been investigated for their role in the malignant phenotype of several cancers (i.e., breast, melanoma, glioblastoma, ovary), and in tumor stem-like cells. However, there are no data regarding GSL composition in human cholangiocarcinoma (CCA). Thus, our study aims to provide GSL and GS profiling of both the stemlike subset and its parental cells in human CCA. Methods: Stem-like subset was enriched by sphere culture (SPH) in established human intrahepatic CCA cells (HUCCT1, CCLP1). CCA GS patterns were determined by chromatographic analytical procedures. Identification of GSL and GS molecular species and assessment of GS turnover were evaluated by feeding cells with 3H-sphingosine. GS role in modulation of stem features was investigated using D-threo-1phenyl-2- almitoylamino-3-N-morpholine-1-propanol (PPMP), a glucosylceramide synthase inhibitor. FACS-sorted GD2+ SPH cells were examined for stem-like gene expression compared to GD2- SPH. Results: In both CCA lines, stem-like subsets (SPHs) showed drastic changes in the number of specific sphingolipids (SLs) (Cer, Gb3, SM) compared to parental cells grown in monolayer conditions (MON). In both CCA lines, the number of total GSs was markedly different between MON and their SPH. In contrast to MON, CCA-SPH showed increased content of GM3, reduction of GM2, and appearance of GD2. This was corroborated by high levels of GM3 synthase as well as GD3 and GM2/GD2 synthase expression in CCA-SPH. GS biosynthesis enzymes, such as GlcCer-, LacCer-, and GM3 synthases, were strongly expressed in CCA-SPH compared to MON. Notably, sphere forming ability and expression of CSCrelated genes were affected by PPMP. Likewise, GD2+ SPH cells were enriched with CSC-markers (CD133, EpCAM, CD44) at protein and gene level in addition to several genes involved in pluripotency, self-renewal, and EM transition compared to GD2- SPH. Notably, expression of GM2/GD2 synthases was significantly expressed in tumor samples compared to paired nontumoral liver tissue of CCA patients (n = 104) and greatly correlated with presence of satellite nodules, lymphnode invasion, and recurrence. Conclusions: We show for the first time that the CCA stem-like properties may be associated with GSL synthetic pathway and pattern. GSL synthases could represent potential markers for CCA.