Identifying the Mechanism of Glioblastoma Cell Migration in Mouse Brain Slices

Glioblastoma multiforme (GBM) is an aggressive malignant brain tumor with extremely low 5-year survival rates[1]. One characteristic of the disease is the ability of the tumor cells to migrate rapidly and spread throughout healthy brain tissue[2], [3]. In order to develop treatments that effectively target cell migration, it is important to understand the mechanism driving cell migration. One hypothesis for how cell migration occurs is the motor-clutch model, in which intracellular forces are generated by myosin motors pulling on F-actin filaments. Molecular “clutches” bind between the F-actin network and substrate, applying forces to the extracellular matrix and driving the cell forward[4-7]. Although GBM cells have been shown to migrate with a motor-clutch mechanism on in vitro substrates, it is unclear if this is the driving mechanism in brain-like environments. To determine the mechanism behind cell migration in physiologically relevant environment, various cell types, including U251 and patient-derived xenograft lines (PDX) were incubated with 300 µm thick mouse organotypic brain slices. Cells infiltrated into the slice and were then imaged via single photon, dual color, swept-field confocal microscopy. While the motor clutch model would predict that cells pull inward on their substrate, the competing osmotic engine model would predict that cells push outward on the substrate. Imaging interactions between cells and vasculature (Figure 1a) creates an opportunity to discriminate between the two models. Using kymographs of the cell-vasculature interactions (Figure 1b), the maximum speed and direction of pulling can be calculated. Nearly all cells were observed to pull on their environment (Figure 1c), consistent with a motor-clutch mode of migration. Blebbistatin, a drug that reduces non-muscle myosin II motor activity, was incubated with the brain slices with U251 (Figure 1d) and PDX lines (Figure 1e) and found to decrease migration speed at high doses for all cell types. Variable responses were found at low doses, consistent with a motor-clutch mode of migration which predicts a biphasic relationship between migration speed and motor to clutch ratio. Targeting the molecular “clutch” may reduce migration speed and disease progression, but it is not known which molecules constitute the clutch in GBM cells. Talin1, a molecule that connects integrins to the actin cytoskeleton, was knocked out via CRISPR/Cas9 in U251 cells. These cells were found to migrate slower in brain slices, demonstrating that integrins are a component of the molecular clutch. Furthermore, Cyclo-RGD, a drug that targets RGD-binding integrins, was found to decrease migration in some PDX lines, indicating that some lines partially use integrins as a clutch. In order to investigate the role of CD44 in the clutch, CRISPR/Cas9 knockouts of CD44 were developed and led to a partial reduction in migration speed. IM7, an antibody that binds to CD44, was added to brain slices containing U251 and slices containing various PDX lines. While this led a decrease in migration speed in U251, there was no significant effect in the PDX lines. Since these results suggest both integrins and CD44 playing a role in the clutch, studies were done to target both potential clutches at the same time. When cRGD was added to slices containing CD44 knockouts, cRGD further decreased migration speed. Adding cRGD to brain slices containing talin knockouts did not further decrease migration speeds as expected. When both IM7 and cRGD were added to the brain slices, the slowest migration was found with the combination. These findings collectively suggest that both integrins and CD44 act as independent clutches and that targeting clutches would be most effective by simultaneous targeting of integrins and CD44. Assessing the mode of migration in glioblastoma cells. a) An example of a GBM cell (Mayo PDX GBM 44, green) imaged by confocal microscopy migrates along vasculature (red). The cell can be seen pulling the vasculature as it moves forward. b) kymograph of the leading edge of the cell demonstrates that the cell exerts a pulling force on the vasculature. c) Calculated speeds of vasculature movement show that in 5 different PDX lines and U251 vascular is pulled towards the cell at 0-7 nm/sec. d) RMC of U251 cells in mouse brain slices decreases with increasing doses of blebbistatin. e) Response of PDX lines to varying doses of blebbistatin.