Differential O- and Glycosphingolipid Glycosylation in Human Pancreatic Adenocarcinoma Cells With Opposite Morphology and Metastatic Behavior

Abstract

Changes in the glycosylation profile of cancer cells have been strongly associated with cancer progression. To increase our insights into the role of glycosylation in human pancreatic ductal adenocarcinoma (PDAC), we performed a study on O-glycans and glycosphingolipid (GSL) glycans of the PDAC cell lines Pa-Tu-8988T (PaTu-T) and Pa-Tu-8988S (PaTu-S). These cell lines are derived from the same patient, but show an almost opposite phenotype, morphology and capacity to metastasize, and may thus provide an attractive model to study the role of glycosylation in progression of PDAC. Gene-array analysis revealed that 24% of the glycosylation-related genes showed a ≥ 1.5-fold difference in expression level between the two cell lines. Subsequent validation of the data by porous graphitized carbon nano-liquid chromatography coupled to a tandem ion trap mass spectrometry and flow cytometry established major differences in O-glycans and GSL-glycans between the cell lines, including lower levels of T and sialylated Tn (sTn) antigens, neoexpression of globosides (Gb3 and Gb4), and higher levels of gangliosides in the mesenchymal-like PaTu-T cells compared to the epithelial-like PaTu-S. In addition, PaTu-S cells demonstrated a significantly higher binding of the immune-lectins macrophage galactose-type lectin and galectin-4 compared to PaTu-T. In summary, our data provide a comprehensive and differential glycan profile of two PDAC cell lines with disparate phenotypes and metastatic behavior. This will allow approaches to modulate and monitor the glycosylation of these PDAC cell lines, which opens up avenues to study the biology and metastatic behavior of PDAC.

Dissection of N-, O- and glycosphingolipid glycosylation changes in PaTu-S pancreatic adenocarcinoma cells upon TGF-β challenge

“…Importantly, the sialylated Tn antigen was notably decreased by TGF-β stimulation in Patu-S cells, which is believed to be carried by various glycoproteins and be associated with cancer progression, invasion and metastasis in some published studies (81,82). In line with this TGF-β-induced downregulation, we have also observed the loss of sialylated Tn antigen in the mesenchymal-like Pa-Tu-8988T (PaTu-T) cells (83), which was derived from the same was not certified by peer review) is the author/funder. All rights reserved.…”

“…The neo-expression of globosides with TGF-β treatment is an interesting observation, since it has been reported that Gb3 is associated with tumor metastasis and invasion in many cancers, including lung cancer (86), colon cancer (87,88), breast cancer (89), gastric adenocarcinomas (90), and pancreatic cancer (91). In our previous study, globoside Gb3 and Gb4 were specifically expressed in the mesenchymal-like PaTu-T cells compared to PaTu-S cells (83). Again, these findings indicate the globosides may contribute to the invasion and metastasis in PDAC.…”

“…The copyright holder for this preprint (which this version posted May 17, 2021. ; https://doi.org/10.1101/2021.05.14.444203 doi: bioRxiv preprint multiple signaling events relevant for T cell-mediated tumor immunity in triple-negative breast cancer cells (92). Our previous study of glycosylation in PaTu-S revealed that Patu-S cells bind to dendritic cells (DCs) by galectins and other lectins, which could directly influence immune responses toward cancer cells (83). Thus, the TGF-β-SOX4 signaling pathway-induced upregulation of N-glycosylation especially branching, sialylation and core fucosylation might impact on immune responses.…”

“…Extraction of GSLs and preparation of GSL-glycan alditols from PaTu-S cells were performed in triplicate as previously described (83). Shortly, 2 x 10 6 cells were harvested, was not certified by peer review) is the author/funder.…”

Deciphering the Importance of Glycosphingolipids on Cellular and Molecular Mechanisms Associated with Epithelial-to-Mesenchymal Transition in Cancer

“…Furthermore, it is known that alteration of glycosylation on lipids at the cell surface can affect cell adhesion, recognition, and signal transduction [ 27 , 28 ] through the regulation of different signaling pathways involved during EMT. The EMT-associated alteration of glycosphingolipids (GSLs) has been identified in several cancer types; e.g., reduced globosides and enhanced gangliosides in mesenchymal-like PaTu-T cells compared to epithelial-like PaTu-S cells have been detected using mass spectrometry-based glycomics and flow cytometry [ 29 ], the functional loss of globosides induces EMT in ovarian cancer using the CRISPR- Cas9 approach [ 30 ], or during transition of breast cancer stem cells [ 31 ]. In addition, the emerging role of GSLs in epithelial-to-mesenchymal transition (EMT) was previously mentioned as a process that enables metastatic cellular invasion in the context of cancer progression, highlighting the spontaneous and transforming growth factor β (TGFβ)-induced alterations of GSLs [ 32 ].…”