DDDR-50. A Chemotherapy-Evoked Microglial Circuit Fuels Invasion in Glioblastoma via S1P–ROCK Signaling

Abstract BACKGROUND & OBJECTIVE Temozolomide (TMZ) remains the clinical workhorse for glioblastoma (GBM), yet edge-initiated invasion and rapid resistance blunt its efficacy. We investigated whether integrated single-cell and spatial transcriptomics could uncover—and therapeutically target—the microenvironmental circuits driving post-therapy invasion. METHODS Orthotopic patient-derived xenografts (PDXs) were profiled using single-cell RNA-seq and spatial transcriptomics. Edge and core regions were balanced by weighted sampling. Differentially expressed edge genes (FDR < 0.05, |log₂FC| > 0.25) underwent GO/MSigDB enrichment and were mapped onto ligand–receptor probabilities to construct a pathway-resolved interactome capturing paracrine crosstalk in the GBM microenvironment. Edge-enriched targets were validated via flow cytometry and immunofluorescence RESULTS During chemotherapy, spatial transcriptomic profiling revealed a distinct injury response program localized specifically to the tumor’s infiltrative edge (P < 0.05), with no comparable activation observed in the tumor core. This was accompanied by a marked increase in microglial infiltration at the leading edge, where only recurrent patient-derived xenograft (PDX) tissues demonstrated a significant elevation in the pro-inflammatory M1 microglia signature (P < 0.0001). Spatial clustering and interactome analyses implicated the sphingosine-1-phosphate (S1P) signaling axis as a central mediator of this treatment-induced invasion, converging on downstream Rac1–ROCK activation. Gene set enrichment further confirmed that invasion-associated programs linked to S1P receptor activation were enriched specifically in edge regions. Functionally, this injury-adaptive S1P–ROCK loop promoted invasive behavior in treated PDXs. Therapeutic inhibition of this pathway with Fasudil, a ROCK inhibitor, effectively suppressed therapy-induced edge invasion in vivo. Fasudil monotherapy extended median survival from 35 to 64 days (P < 0.0001), and its combination with TMZ further improved survival to 71 days (P < 0.0001), underscoring the therapeutic potential of targeting this axis. CONCLUSION Spatially resolved multi-omics identifies a microglia-driven S1P–ROCK axis that fuels chemotherapy-induced invasion. Fasudil—an orally available ROCK inhibitor—interrupts that circuit, significantly prolonging survival in a PDX model and supporting the clinical advancement of ROCK inhibition in GBM.