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Biomechanics

Biomechanics - Poster Session E

Poster Q2 - SDF-1α promotes invasive potential via mesenchymal transitions in GBM

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Glioblastoma (GBM) is an aggressive primary brain tumor originating in glial cells, presenting a formidable challenge due to its rapid growth and invasive nature within healthy brain tissue. Current treatment methods are insufficient to cure the disease which underscores the urgent need for deeper insights into its mechanisms. Recent intriguing reports have identified a novel mechanism of infiltration, where GBM cells preferentially invade the sub-ventricular zone (SVZ) in the brain. Upon SVZ contact, the patient's prognosis worsens significantly, leading to patient death. The disruption of this axis may represent a clinically relevant target; however, the mechanisms are poorly understood. Classically, the SVZ is a reservoir for neural stem cells and secretes the chemokine stromal cell-derived factor 1 (SDF-1) to maintain the stem cell niche. Interestingly, SDF-1 has been identified as a potential contributor to malignancy and invasion in various cancers. In this study, we investigate the effect of SDF-1 in facilitating GBM infiltration by examining factors that enable parenchymal migration, including confined migration and mesenchymal transitions.

Materials and

Methods:  U87-MG human GBM cells (U87) were cultured in DMEM with 10% FCS, 1% penicillin/streptomycin, MEM nonessential amino acids, and sodium pyruvate, and experiments were conducted up to passage 5. To investigate the role of SDF-1 on GBM mesenchymal transition, cells were incubated with 100 ng/ml SDF-1 for five days. Western blotting was employed to analyze the expression of ZEB1, a mesenchymal transcription factor, and pFAK, an emergent signaling molecular in mesenchymal transition. Transwell assays and custom-made microchannel platforms were used to measure functional changes that simulate parenchymal migration. Transwell inserts (8-µm pore size) were coated with 100 µg/mL laminin for 3 hours at 37°C. NT cells and cells pretreated with SDF-1 for 5 days were seeded in the top chamber with basal medium, and the bottom chamber contained basal medium with 100 ng/mL SDF-1. After 16 hours, cells were fixed with 4% PFA, washed, stained with propidium iodide, and imaged with a Nikon TE2000E2 microscope. PDMS-based microchannel platforms developed in the lab were functionalized with laminin for 3 hours at 37°C and seeded with cells as previously described. Cell migration through channels was analyzed via time-lapse microscopy and evaluated for changes in cell speed.

Results, Conclusions, and Discussions: The presence of the SDF receptor CXCR4 in GBM cells and the localization of SDF-1 in the sub-ventricular zone SVZ prompted an investigation into whether SDF-1 could induce EMT. Cells were exposed to untreated and SDF-1-treated media over five days, chosen based on the observation that ZEB1 expression peaked after five days. Western blot analysis revealed significant increases in ZEB1 expression in the SDF-treated group compared to the untreated group and an interesting dependency on FAK signaling (Fig 1. A, B). Functional assays were conducted to assess the impact of SDF-1 on GBM cell migration and invasion. In a 2-dimensional (2D) motility experiment, cells pre-treated with SDF-1 exhibited significantly increased motility compared to untreated cells (Fig 1. C). This observation was further supported by a transwell migration assay, where SDF-1 pre-treatment led to more cells traversing through 8 µm pores within 16 hours, indicating enhanced invasive potential (Fig 1. D). We also investigated GBM cell migration under confinement using bioengineered microchannels of varying widths (5 µm and 20 µm) (Fig 1. F). Time-lapse imaging demonstrated that cells treated with SDF-1 migrated faster through both confined conditions than untreated cells. Even under tight confinement (20 µm channels), where cell speed typically decreases, SDF-1-treated cells maintained faster migration rates than untreated cells, suggesting enhanced capability for navigating brain tissue. These findings show that SDF-1 promotes EMT and enhances invasive behaviors crucial for GBM progression. The data underscore the potential of SDF-1 as a therapeutic target in GBM treatment strategies aimed at mitigating tumor aggressiveness and improving patient outcomes. Further research into the molecular mechanisms underlying these effects is crucial and could yield novel therapeutic interventions for this challenging disease.

Acknowledgements (Optional):