Traction Force Dynamics During Patient-Derived Glioblastoma Neurosphere Invasion in 3D Matrigel

Abstract Purpose Glioblastoma multiforme (GBM) is an aggressive brain tumor with a 5-year survival rate below 7%. Poor outcomes are driven in part by diffuse invasion into surrounding brain tissue, which limits complete surgical resection and promotes recurrence. Improved therapies require a quantitative understanding of the mechanics of GBM invasion. Methods Patient-derived GBM neurospheres were embedded in Matrigel and analyzed using live imaging, three-dimensional traction force microscopy (3D TFM), and targeted cytoskeletal perturbations. Results Invading GBM cells adopted an elongated, protrusion-rich morphology, with F-actin enriched at the cell periphery and microtubules extending along the protrusion shaft, consistent with a mesenchymal-like invasion program. 3D TFM revealed sustained matrix engagement, including progressive bead clustering, increasing cumulative traction forces, and traction hotspots concentrated near protrusion tips. Perturbation studies separated cytoskeletal requirements for invasion and force transmission: actin polymerization was essential for invasion, myosin II activity was required for robust traction generation and efficient invasion, and microtubule polymerization supported directional persistence and maximal traction output. Notably, a low level of invasion persisted under myosin II inhibition despite minimal detectable traction forces, and this residual invasion was not suppressed by pan-MMP inhibition, indicating a traction-poor, MMP-independent invasion component in Matrigel. Conclusions These findings establish a quantitative mechanical framework for GBM neurosphere invasion in 3D Matrigel and define distinct contributions of actomyosin contractility and microtubules to invasive progression.