ABSTRACT: Glycosphingolipids (GSL), a class of plasma membrane lipids, have been proposed as markers of cancer stem cells (CSC). In particular, gangliosides (GS), sialic acid-containing GSL, have been investigated for their role in the malignant phenotype of several cancers and in tumor-stem like cells, but there are no data about human cholangiocarcinoma (CCA). Our study aims to provide a GSL and GS profiling of both stem-like subsets and their parental cells in human CCA. Experimentals. Intrahepatic CCA cells (HUCCT1, CCLP1) were used. Stem-like subset was enriched by sphere culture (SPH) and compared to parental cells grown as monolayer (MON). CCA GS patterns were determined by chromatographic analytical procedures, and their molecular species identification was evaluated by feeding cells with 3H-sphingosine. GS role in modulation of stem features was investigated using D- threo-1phenyl-2-palmitoylamino-3-N-morpholine-1-propanol (PPMP, glucosylceramide synthase inhibitor), and CCA GD3S-transfected cells. FACS-sorted GD2+ SPH cells were examined for stem-like gene expression compared to GD2- SPH. GS biosynthesis enzymes were analyzed by RT-qPCR at different times of spherogenesis. Results: In both CCA lines, compared to MON, SPH showed drastic changes in specific sphingolipids (Cer, Gb3, SM), and in the amount of total GS. In contrast to MON, CCA-SPH shows increase content of GM3, reduction of GM2; among complex GS, strongly increase of GD1a and appearance of GD2, a finding also corroborated by high levels of GM3 synthase as well as GD3- and GM2/GD2 synthases expression in CCA-SPH. Notably, sphere-forming ability and expression of CSC-related genes were affected by PPMP. Importantly it has emerged that the cancer stem features related on GD2 availability are not due to the ganglioside GD2 synthase enzyme, but depend on the enzyme GD3S, the synthase that provides the precursor (GD3) of ganglioside GD2. Thus we have stably transfected both CCLP1 and HUCCT1 cells with the GD3S gene. GD3S-transfected MON (MON GD3S+) cells showed enhanced sphere-forming ability in vitro, superior invasive properties, as well as higher drug resistance when treated with cisplatin and oxaliplatin compare to the transfected control. To better clarify the molecular features and the altered pathways in MON GD3S+ CCA cells, a global transcriptomic analysis was performed. Likewise, GD2+ SPH cells were enriched with CSC-markers at protein and gene levels in addition to several genes involved in pluripotency, self-renewal, and EMT, compare to GD2- SPH. Notably, expression of GM2/GD2 synthases was significantly expressed in tumor samples compared to paired non-tumoral liver tissue of CCA patients (n=104) and greatly correlated with presence of satellite nodules, lymph node 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 and GS synthases could represent potential markers for CCA.
