J7 - Targeting lymphatic endothelial glycolysis

Introduction: : Tumor metastasis is the leading cause of death in patients with solid malignancies. The hypoxic tumor microenvironment encourages the formation of leaky, dysfunctional vasculature which facilitates tumor cell intravasation into the blood and lymphatic system. Glycolysis, as the main source of energy for endothelial cell migration and vessel formation, provides additional targets for vascular stabilization.
As a common metabolic pathway, targets must either be endothelial-specific or disproportionately affect hypoxic, hyper-glycolytic cells. Glutamine is highly consumed by endothelial cells as well as other glycolytic cell types such as immune, stem, and some cancer cells. However, whether or not glutamine directly affects glycolysis is understudied in lymphatic endothelial cells (LECs). An additional target is glucose transporter SLC2A3 (GLUT3), as it is expressed primarily in cells of hematopoietic or neuronal lineages, making it a vascular-specific target.
This study investigates glutamine metabolism and glucose transporter SLC2A3 (GLUT3) as potential candidates to attenuate glycolysis and stabilize lymphatic vasculature in hypoxic conditions which mimic the hypoxic tumor microenvironment. First, we seek to demonstrate the link between our targets and glycolysis in normoxic and hypoxic LECs. In addition to gene and protein expression, we also examine the impact of blocking glutamine uptake/SLC2A3 on LEC tube forming and migratory capabilities in 2D. Once the impact of the chosen targets on LEC glycolysis is confirmed, we will develop a 3D in vitro chip model of tumor lymphatics to observe the impact of reducing glutamine and SLC2A3 on vascular leakage.

Materials and

Methods: : Juvenile Human Dermal Lymphatic Endothelial Cells were acquired from Promocell and expanded in Endothelial Growth Medium MV2 with SupplementMix. To test the impact of glutamine availability on glycolysis, LECs were glutamine starved overnight in glutamine-free DMEM (D5546, Millipore Sigma); the media was then changed to DMEM with glutamine and the cells were incubated for 24 hours in normoxia or hypoxia. Glutamine transport was blocked by incubating the cells in 10μM of V-9302 for 48 hours, while SLC2A3 was knocked down using si-RNA (Dharmacon). Changes in glycolytic activity were measured using the gene expression of glucose transporters SLC2A1 and SLC2A3, as well as the gene and protein expression of Hexokinase II (HK2). Changes in gene expression were quantified using qPCR, while protein was quantified via Western Blot. To further confirm glycolysis, lactate and glutamate levels in media were measured using Promega Lactate and Glutamate-Glo assays.
Changes to cell behavior will be observed using tube formation and migration assays. LECs are stained with CellTracker Red (Thermo Fisher) and seeded on top of Matrigel in Ibidi 15-well μ-slides at a density of 10,000 cells/well. They are cultured for 6 hours in a normoxic or hypoxic incubator then imaged. Vessel analysis will be performed using ImageJ plugin Kinetic Analysis of Vasculogenesis. Migration assay was performed by seeding LECs in an Ibidi 2 well culture insert. After a 4 hour attachment, the insert was removed and cells were imaged every hour using a BioTek Lionheart Automated Microscope; changes in gap size were quantified in ImageJ.

Results, Conclusions, and Discussions:: Gene and protein expression show a consistent trend between glutamine availability and glycolysis-related gene, protein, and metabolite expression. As the concentration of glutamine in the media increases, gene expression of HK2 and SLC2A1 increases in both normoxia and hypoxia, while SLC2A3 also increases after an initial drop (Fig 1a). The inverse is shown when glutamine transport into the cells is blocked using V-9302, with the exception of SLC2A3 (Fig 1 b). This is reflected in HK2 protein expression in hypoxia, which decreases with V-9302 treatment (Fig 1c). Metabolites related to glycolysis also increase with glutamine concentration (Fig 2 a and b). Lastly, preliminary data suggests that SLC2A3 knockdown slows LEC migration. Further exploration into these targets provides a method of normalizing tumor vasculature via endothelial cell metabolism.

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