6th Edition of Neurology World Conference 2026

Abstract

Abstract Glioblastoma (GBM) tissue is mechanically heterogeneous, with Young's modulus values varying widely even within a single tumor mass—ranging from less than 1 kPa in necrotic or ischemic-hypoxic regions to over 13 kPa in densely fibrotic zones—while the peritumoral gliosis zone typically exhibits compliance below 1 kPa and normal brain parenchyma measures approximately 2–3 kPa. Yet the functional consequences of these stiffness gradients for invasion and therapeutic resistance remain poorly defined. Here we combined three-dimensional collagen hydrogel engineering, deeplearning-assisted live-cell tracking, bulk and pharmacological transcriptomic analyses, virtual gene-network knockout, and orthotopic xenograft pharmacology to dissect GBM mechanobiology from the molecular to the tissue level. Contrary to the prevailing two-dimensional paradigm, U-251 and U-87 glioma cells migrated significantly faster in soft (0.3 kPa) than in stiff (13 kPa) three-dimensional matrices (P < 0.0001 for maximum speed), adopting spherical, highly motile amoeboid phenotypes with abundant filopodia in compliant environments while assuming elongated mesenchymal morphologies in rigid ones. Stiffness-gradient invasion assays demonstrated a 3.6- to 3.9-fold preferential invasion from rigid tumor-like regions into soft peritumoral zones (P < 0.0001). RNA-sequencing across the complete stiffness spectrum (0.3, 3, and 13 kPa) revealed biphasic mechanotransduction cascades with 358 differentially expressed genes (FDR < 0.05), and Gene Ontology analysis identified cellular protrusion formation as a significantly enriched program in soft ECM. Bulk transcriptomic analysis of 180 glioma and control brain samples (GSE4290) confirmed broad activation of six ECM-hardening programs in GBM, and cell-source deconvolution within 77 GBM samples revealed a functional division of labor: fibroblast-like stromal cells served as the dominant source of type I collagen (standardized β = 0.77), whereas mesenchymal-like malignant cells independently drove collagen crosslinking and integrin–YAP signaling. The filopodia enrichment finding prompted a targeted drug screen; after excluding the Fascin-1 nanobody Nb 3E11 and docetaxel, metformin was selected for its documented anti-filopodia activity, cerebrospinal-fluid exposure (~10– 30% of plasma), and decades of clinical safety. Concentration-gradient RNAsequencing of metformin-treated glioma cells cultured in 0.3 kPa soft ECM (0–15 mM) confirmed dose-dependent FSCN1 suppression, identified Apelin signaling pathway as a top enriched KEGG term among downregulated genes, and unexpectedly revealed apelin (APLN) as the single most dose-dependently downregulated gene (log₂FC = −2.52 to −3.40, P < 10⁻³⁷). Virtual gene-network knockout confirmed APLN as a central hub linking proliferation, VEGFA–CHI3L1 signaling, and cytoskeletal programs. Metformin dose-dependently inactivated YAP1 via Ser127 phosphorylation (P = 0.0007) and suppressed filopodia in a stiffness-modulated manner. In orthotopic xenograft models, temozolomide (TMZ) monotherapy paradoxically reinforced collagen– fibronectin barriers, upregulated α-SMA and lysyl oxidase (LOX), suppressed MMP9, activated nuclear YAP, and elevated APLN protein—collectively building a "matrix fortress." Addition of metformin dismantled this barrier, progressively suppressed APLN protein (AOD P < 0.0001), and triggered a surge of macrophage/microglia infiltration (IBA-1, CD86, CD206 all P < 0.0001). Further addition of the ROCK inhibitor fasudil abolished mechanotransduction signaling (ROCK1, P-MLC2, total YAP all significantly reduced), disrupted macrophage cytoskeletal integrity, reduced immune cell infiltration, and dramatically enhanced Cleaved-caspase3-mediated apoptosis (P < 0.0001 versus control; P = 0.0002 versus TMZ). These findings establish GBM ECM stiffening as a cooperative tumor–stromal process, reframe stiff matrix as a mechanically protective niche rather than a migration highway, and demonstrate that standard chemotherapy inadvertently builds physical barriers blocking both drug diffusion and immune access—barriers that can be dismantled by matrix-softening combination therapy that simultaneously functions as a chemosensitizer. Keywords: Glioblastoma; Extracellular matrix stiffness; Three-dimensional mechanotransduction; Filopodia; Apelin; Matrix-softening therapy; Tumor immune microenvironment; YAP/TAZ signaling