Poster T8 - CRISPR editing of the Cancer Glycocalyx to Tune Mechanically Regulated Signaling and Migration in Glioblastoma Multiforme

Introduction: Glioblastoma multiforme (GBM) is one of the most aggressive cancer types that affect the brain and is characterized by a bulky presence of sugar glycocalyx, mechanically tensioning its membrane, and fueling aggression. This glycocalyx is a potent mechanosensor that transmits mechanical and chemical environmental cues, as well as all signals that must navigate this barrier to reach the cell. Any change to the composition of this sugar layer would, in turn, impact the harmonious interaction between the cell and its environment. In more than 95% of cancer cells, specific glycocalyx signatures including increased thickness with 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, as most patients do not survive 8 months post-diagnosis. In a bid to extend the length of survival in patients, modification of the glycocalyx remains a promising therapeutic target worthy of perturbation. GBMs express a complex mixture of glycoproteins, glycolipids, and mucins, and insight into the specific glycan that is homing the critical functional end-group sugar(s) to mediate mechanical forces on its membrane is needed to effectively edit it. To this end, we hypothesize that CRISPR-Cas9-mediated editing of either the chain-initiating enzymes of N-linked glycans (MGAT1), O-linked glycans (COSMC), or glycolipids (UGCG) can edit the glycocalyx in such a way that it quenches the aggressive 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 PX458-GFP and PX459-DsRed vector were generated, and U87-COSMC-KO, U87-MGAT1-KO and U87-UGCG-KO single KO cell lines were created by chemically transfecting the WT U87s with these vectors using lipofectamine 3000TM reagent and FAC-sorting for double positive cells with two different guide CRISPR cassette. To characterize the KOs, the region surrounding the CRISPR target site was amplified from genomic DNA and specific deletions confirmed. Approximately 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 fashioned polyacrylamide gels at a range of stiffnesses (400-60,000 Pa) conjugated with Fibronectin and monitored if the knockouts migrate differently than the controls and if the stiffness effects the migration of the GBMs. Next, we checked to see if the signaling (FAK) is altered in any of the GBM knockouts through use of FAK tyrosine specific activated antibodies (and western blotting) on both the wild type and mutants to check changes in migration profiles and response to mechanical cues.

Results, Conclusions, and Discussions: : Microscopic observation (Figure 1) showed a trend towards a more pro-neural phenotype, as expected, in each of U87-COSMC-KO, and U-87-UGCG-KO and U87-MGAT1-KO. What is more surprising is that U87-COSMC-KO and U87-UGCG-KO showed an increased cellular spread area and pro-neural phenotype. This phenotype is seen more often in healthy human astrocytes, an observation that is expected to correlate with a glycocalyx content and size upon quantification.U87-COSMC-KO and U87-UGCG-KO appear to be the most critical sugars responsible for GBM aggression. However, our ongoing experiments are quantifying the changes in surface topography and glycocalyx architecture as well as signaling and migration associated with the phenotypic changes seen in the U87-COSMC-KO and U-87-UGCG-KO with the hope of identifying mucin or glycolipids as key players in GBM aggression. We plan to inject each of the KOs in mice to study tumor development and survival. Finally, we will check for immune and drug susceptibility for each KOs. This could ultimately lead to new potential therapies that increase immune recognition and in improved patient prognosis in GBM.

Acknowledgements (Optional): This work was funded by NIH-NSFT GM143357, Center for Engineering MechanoBiology, CEMB CMMI: 15-48571, NJIT Faculty Seed Grant and New Jersey Health Foundation (NJHF), Grant number: PC:26-24, to the cellular motion lab.