The profile on intracellular gangliosides correlates with the malignant phenotype of cholangiocarcinoma cells and modulates cells adhesion

The profile on intracellular gangliosides correlates with the malignant phenotype of cholangiocarcinoma cells and modulates cells adhesion Chiara Raggi1, Antonella Mannini1, Margherita Correnti2, Elisabetta Rovida1, Jesper Andersen3, Cedric Coulouarn4, Fabio Marra1. 1University of Florence, Florence, Italy; 2Humanitas, Rozzano, Italy; 3Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark; 4Inserm, Inra, University Rennes, UMR 1241, Rennes, France Background and aims: Identification of cancer stem cell (CSC) has opened the way to the design of innovative diagnostic and therapeutic strategies in neoplastic diseases. Gangliosides (GS) a family of sialic acid-containing glycosphingolipids, have been associated with malignant phenotype of several cancers (i.e. breast, melanoma, glioblastoma, ovary). In particular, GD2, a complex GS, has been proposed as a novel CSC-marker. Nonetheless, limited information is available for many tumor types including human cholangiocarcinoma (CCA). This study aims to obtain a GS profiling of human CCA stem-like subset and their parental cells. Method: Stem-like compartment was enriched by sphere culture (SPH) in two lines of established human intrahepatic CCA cells (HUCCT1, CCLP1). CCA GS composition was performed by chromatographic analytical techniques and revealed by resorcinol-HCl reagent. GD2 detection as well as its co-expression with CD133 CSC-marker was carried out by FACS analysis. The role of the GM3 GS on cell adhesion was investigated using D-threo-1phenyl-2-palmitoylamino- 3-N-morpholine-1-propanol (PPMP), a GM3 synthase inhibitor. Results: In contrast to parental cells grown as adherent monolayers (MON), CCA-SPH showed strong expression of GD2. The time course of SPH-growth revealed a remarkable GD2 expression along with CD133 levels just few days after plating. Overall, CCA-SPH showed a GM1/GM2 reduction in parallel with an increase in GM. Notably, a cell-line specific GS profile was revealed. MON-CCA exhibited high levels of simple GS such as GM3 and GM2 in HUCCT1 and in CCLP1, respectively. Moreover, HUCCT1 showed a GM3-dependent adhesion capacityon fibronectin. Remarkably, since GM2 and GD2 biosynthesis is driven by the enzyme beta-1, 4 N-acetylgalactosaminyltransferase-1 (B4GALNT1) and GD3 by alpha-N-acetylneuraminide alpha-2, 8-sialyltransferase (ST8SIA1), mRNA expression of both enzymes were analyzed by transcriptomic data from surgically resected CCA samples. Both B4GALNT1 and ST8SIA1 were significantly increased in tumor samples compared to paired non-tumoral liver tissue. Strikingly, B4GALNT1 expression considerably correlated with recurrence, perineural invasion and presence of satellite nodules in CCA patients. Conclusion: We show for the first time that the GS composition is modulated in the CCA stem-like subset. GD2 should be explored as a candidate biomarker for CCA. GS composition may affect pivotal characteristics of CCA cells such as adhesion.