Fig 2: Soluble ENO1 drives malignant phenotypes in glioblastoma through TLR4-mediated activation of the SPHK1-S1P signaling cascade.A Principal component analysis (PCA) of metabolites and lipids detected by untargeted lipidomics in GBM cells treated with soluble recombinant ENO1 (rhENO1, 1 μg/mL) (n = 6 biological replicates). B KEGG pathway enrichment analysis of differential metabolites with VIP > 1. C Heatmap analysis of differential metabolites with VIP > 1 (n = 6 biological replicates). D Sphingomyelin (SM) was significantly upregulated in rhENO1-treated samples. E Western blot analysis of SPHK1 and ERK phosphorylation levels in serum-starved GBM cells treated with or without rhENO1 (1 μg/mL) for 6 h. F Western blot analysis using anti-phospho-SPHK1, anti-SPHK1, anti-phospho-ERK, and anti-ERK antibodies in serum-starved GBM cells treated with or without rhENO1 (1 μg/mL) and TLR4 inhibitor (TAK242, 10 μM) for 6 h. G Immunofluorescence staining of SPHK1 subcellular localization in GBM cells treated with rhENO1 (1 μg/mL) and TLR4 inhibitor (TAK242, 10 μM). Scale bar = 10 μm. H ELISA quantification of S1P levels in conditioned medium from rhENO1-treated GBM cells. I Western blot analysis of SPHK1 phosphorylation levels in GBM cells treated with increasing concentrations of SPHK1 inhibitor PF-543. J-M Representative images and quantification of J proliferation (scale bar = 100 μm), K colony formation (scale bar = 5 mm), L migration (scale bar = 100 μm), and M invasion (scale bar = 500 μm) in GBM cells treated with DMSO control, PF-543 (10 μM), or S1P (1 μM). Data are expressed as mean ± SEM. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3: ENO1 drives GBM malignant progression through PI3K/AKT signaling activation.A Cluster analysis of differentially expressed genes (DEGs) between rhENO1-treated and control groups (n = 3 biological replicates). B Volcano plot of DEGs displaying: X-axis: log2 fold-change in gene expression; Y-axis: -log10(p-value) for statistical significance; Green dots: non-significant genes; Red/blue dots: significantly up-/down-regulated genes. C KEGG pathway analysis revealing significant association between ENO1 and PI3K- Akt signaling in GBM patient samples. D GSEA enrichment plot demonstrating PI3K- Akt pathway activation correlated with ENO1 expression from the TCGA database. E Western blot analysis of PI3K and Akt phosphorylation in rhENO1-treated GBM cells using anti-phospho-PI3K, anti-PI3K, anti-phospho- Akt, and anti- Akt antibodies. F Western blot analysis of PI3K/Akt phosphorylation in serum-starved GBM cells treated with/without rhENO1 (1 μg/mL) and PI3K inhibitor LY294002. Representative images and quantitative analysis of G colony formation, H proliferation, and migration/invasion I, J in GBM cells treated with or without PI3K inhibition LY294002 and cultured in rhENO1. Scale bars = 5 mm (G), 100 μm (H, J), and 500 μm (I). K Western blot analysis of PI3K/Akt phosphorylation in serum-starved GBM cells treated with/without rhENO1 (1 μg/mL) and TLR4 inhibitor TAK242 (10 μM). Data represent mean ± SEM; ns: not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4: Combinatorial targeting of TLR4/SPHK1 enhances TMZ therapeutic efficacy in GBM.A Experimental workflow of orthotopic glioma mouse model and treatment regimen (n = 6). B Representative in vivo bioluminescence images of Luci-GL261-bearing mice treated with:(i) saline control, (ii) TMZ monotherapy, (iii) TMZ + PF-543 (SPHK1 inhibitor), (iv) TMZ + PF-543 + TAK242 (TLR4 inhibitor) at days 7, 14, and 21 post-implantation. C, D Quantitative analysis of tumor growth using relative flux values (normalized to day 7) across treatment groups. E Kaplan-Meier survival curves showing intracranial tumor progression in the four treatment cohorts; n = 6 mice. F Representative immunohistochemical staining of ENO1, p-SPHK1, and CD163 in tumor sections from each treatment group. Scale bar, 100 μm. G Schematic diagram depicting the mechanistic basis of ENO1 mediating cellular communication between GBM cells and M2-TAMs through an autophagy-dependent secretory pathway and driving TMZ chemoresistance. Data represent mean ± SEM; ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 5: Soluble ENO1 promotes proliferation, migration, and invasion of GBM cells in vitro.A Western blot analysis of ENO1 knockdown efficiency in both intracellular and extracellular fractions of U87MG cells. B Western blot validation of ENO1 overexpression in LN229 cells and conditioned media (CM). Colony formation (C) and EdU proliferation (D) assays of parental LN229 cells treated with CM from ENO1-overexpressing LN229 cells. Scale bars, 100 μm. E, F Metastatic potential assessment using CM from ENO1-overexpressing cells: E Wound healing assay (scale bar = 500 μm). F Transwell migration and invasion assays (scale bar = 100 μm). G Western blot detection of intracellular ENO1 after 24 h treatment with recombinant human ENO1 (rhENO1, 1 μg/mL). H CCK-8 assay evaluating proliferation in rhENO1-treated cells (1 μg/mL). I Transwell migration/invasion of rhENO1-treated GBM cells (scale bars, 100 μm). J Scratch wound healing assay post-rhENO1 treatment (scale bars, 500 μm). Rescue experiments: Proliferation (K, L) and migration/invasion (M, N) capacities of GBM cells treated with shENO1 CM supplemented with rhENO1. Data are expressed as mean ± SEM. ns not significant, *P < 0.05, **P < 0.01, ***P < 0.001.