Membrane glycome is impacted by the cell culturing mode of neuroblastoma cells with differing migration and invasion potential

Neuroblastoma is a highly metastatic childhood cancer for which studies indicate an association between protein glycosylation and tumor behavior. However, there is a lack of detailed glycome analysis on neuroblastoma cells that have varying metastatic potential. Furthermore, the impact of the cell culturing mode, i.e. 2-dimensional (2D) versus 3-dimensional (3D) spheroids, on the membrane protein glycome is unknown. To address these gaps in knowledge, we mapped membrane protein N- and O-glycosylation of neuroblastoma cells that have lower invasive and metastatic potential (Stathmin shRNA-expressing cells, StmnSeq2_SH, and StmnSeq3_SH) compared with control cells (control shRNA-expressing cells, Ctrl_SH). We showed that the neuroblastoma cells with different migratory and invasive potential underwent drastic changes in their membrane protein N-glycosylation exclusively when cultured in 3D spheroids. We also investigated the impact of 2D and 3D cell culture methods on cellular glycosylation using the neuroblastoma cells and found the cell N-glycome was markedly impacted by the culture method, with the 2D grown cells showing an abundance of oligomannosidic glycans, whereas 3D spheroids expressed more complex type glycans on their membrane proteins. In summary, this study provides the first comprehensive protein glycome profiling of neuroblastoma cells that have varying invasiveness and migratory potential and unravels the distinct membrane glycan features of cells that are grown under 2D versus 3D culture conditions.