(C-114) Defining the contributions of N-linked, O-linked, and glycolipids to glycocalyx bulk and mechanically regulated migration in Glioblastoma Multiforme

Introduction:: Worldwide, one in five people develop cancer during their lifetime, and one of the most deadly forms of cancer is Glioblastoma multiform (GBM). GBM is one of the most aggressive cancer types that affect the brain and is characterized by a spindle shaped, mesenchymal morphology as compared to healthy, glioma cells. The glycocalyx is a sponge-like network of proteins, lipids, and glycans extending from the cell membrane which acts as a “buffer” to the mechanical and chemical cues of the surrounding environment and all signals must navigate this barrier to reach the cell. Any changes to the size or composition of the glycocalyx would in turn have drastic effects on how the cell interacts with its environment. In most cancer cells, specific glycocalyx signatures including increased thickness and more bulky side chains have been demonstrated to cause increased proliferation, migration, invasion, and immune evasion. So far, treatment options to prevent GBM metastasis or increase immune recognition have been ineffective, leaving modifying the glycocalyx as a promising therapeutic target. GBMs express a complex mixture of glycoproteins, glycolipids, and mucins so insight into the specific glycan structures mediating the mechanical forces and “bulkiness” of the glycocalyx is needed to effectively edit it. To this end, we hypothesize that editing one of the chain initiating enzymes of N-Linked glycans (MGAT1), O-linked glycans (COSMC), or glycolipids (UGCG) can edit the glycocalyx (Figure 1) in such a way that it disrupts the ability of GBMs to migrate, signal, and evade immune recognition.







Materials and Methods:: CRISPR plasmids with gRNA sequences against human COSMC, UGCG and MGAT1 in the pX330-U6-Chimeric_BB-CBh-hSpCas9 vector were available on Addgene (Plasmids# 80008, 80009, 80010). COSMC-KO (U87 COSMC-KO), MGAT1-KO (U87-MGAT1-KO) and UGCG-KO (U87-UGCG-KO) single KO cell lines were created by electroporating WT U87s with vectors containing target sequences against the respective genes in the BioRad Gene Pulser Xcell Systemwith 50/50 (v/v) mixture of fresh and U87 cell-culture conditioned EMEM (conditioned media). To characterize the KOs, the region surrounding the CRISPR target site will be PCR amplified from genomic DNA and specific deletions will be confirmed using the surveyor assay (IDT)^. Approximately 2–3 weeks after transfection, morphological changes were quantified by microscopy with firmly adherent, spindle like cells being more aggressive and mesenchymal GBMs and loosely attached, more rounded cells being pro-neural and less aggressive. Then we will fashion polyacrylamide gels at a range of stiffnesses (400-60,000 Pa) conjugated with Fibronectin and plate our cell lines. We will then monitor the migration profiles over time on our Nikon Tie2 inverted fluorescence microscope (Nikon) and calculate speed (μm/s), distance traveled (μm), and the motility coefficient (μm²/s) (MATLAB) to see if the knockouts migrate differently than the controls and if the stiffness effects the migration of the GBMs. Next we will check to see if the signaling (FAK) is altered in any of the GBM knockouts through use of FAK tyrosine specific activated antibodies (Cell Signaling Technologies) and western blotting specifically on the mutants which have altered migration profiles and response to mechanical cues.




Results, Conclusions, and Discussions:: To ask the question of whether a KO of any or combination of these three genes will affect cell division and proliferation in U87. To address this, we knocked out each glycosyltransferase in U87. We confirmed the efficiency of KO using PCR and surveyor assay and further plated each KO with the wild-type cell lines to check cell proliferation. Microscopic observation (Figure 2) showed a trend towards decline as expected in each of U87 COSMC-KO, MGAT1-KO and UGCG-KO. What is more surprising is that UGCG-KO showed a detached, less aggressive, pro-neural phenotype seen more often in normal glioma cells, an observation that is expected to correlate with the size reduction in the glycocalyx content and size.
Ongoing experiments are quantifying the changes in signalling and migration associated with the phenotypic changes seen in the U87-UGCG KOs with the hope of identifying glycolipids as key players in regulating the metastatic phenotype of GBMs. This could lead to new potential therapies that increase immune recognition and in turn patient prognosis in GBM.




Acknowledgements (Optional): : This work was funded partially by NIH GM143357 to ABJ.

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