Fig 2: Working model.Illustration showing a proposed mechanism by which SELP-PSGL-1 axis mediates GAMs phenotype and GB progression. This image was created with BioRender.com.

Fig 3: SELP inhibits the microglia pro-inflammatory phenotype and promotes their immunosuppressive activity.A ELISA assay showing higher secretion of SELP by human microglia treated with PD-GB CM compared to naïve DMEM. Data represent mean ± s.d. Each dot represents a triplicate. The graph shows the average of three independent repeats. Statistical significance was determined using an unpaired, two-sided Student’s t test. B Real-time PCR showing higher expression SELP mRNA by human microglia treated with PD-GB4 CM compared to naïve DMEM. Data represent mean ± s.d. Each dot represents a triplicate. The graph shows the average of three independent repeats. Statistical significance was determined using an unpaired, two-sided Student’s t test. Representative image (C) and quantification (D) of flow cytometry analysis of PSGL-1 expression by human microglia, showing higher expression when treated with PD-GB4 GB CM compared to naïve DMEM. Data represent mean ± s.d. The graph shows the average of five independent experiments. Statistical significance was determined using an unpaired, two-sided Student’s t test. E tSNE plot of single-cell RNA-seq analysis of microglia and macrophages isolated from patients' GB tumors, showing the expression of PSGL-1 by microglia compared to macrophages. Data obtained from Darmanis et al.56. Treatment with rSELP resulted in elevated mRNA expression of ARG1 (F), reduced expression of iNOS (G) and elevated expression of IL-10 (H) and TGF-ß (I) by human microglia. SELPi or anti-PSGL-1 neutralizing antibody rescued rSELP effects while anti-CD44 or CD24 neutralizing antibody did not affect gene expression. Data represent mean ± s.d. Each dot represents a triplicate. The graphs show the average of three independent studies. Statistical significance was determined using one-way ANOVA test with multiple comparisons adjustment. Phagocytic activity (J) and NO release (K) by human microglia were reduced when treated with rSELP, and were restored when inhibiting SELP or PSGL-1 but not when neutralizing CD44 or CD24. Data represent mean ± s.d. of four wells per group. The graphs are representative of three independent repeats. Scale bars represent 200 µm. Statistical significance was determined using one-way ANOVA test with multiple comparisons adjustment. Source data are provided as a Source Data file.

Fig 4: SELP mediates GB cell invasion, migration, and proliferation.A Real-time PCR showing reduced SELP mRNA levels in shSELP PD-GB4 cells, compared to control WT (Control), and negative control shRNA (shNC). Data represent mean ± s.d. Each dot represents a triplicate. The graph showing the average of three independent experiments. Statistical significance was determined using one-way ANOVA test with multiple comparisons adjustment. Representative image (B) and quantification (C) of flow cytometry analysis of SELP expression showing reduced expression in shSELP PD-GB4 compared to control WT and shNC PD-GB4 cells. Data represent mean ± s.d. The graph shows the average of three independent experiments. Statistical significance was determined using one-way ANOVA test with multiple comparisons adjustment. D Proliferation of iRFP-labeled PD-GB4 cells alone or co-cultured with unlabeled human microglia showing lower proliferation rate of shSELP PD-GB4 compared to control WT and shNC PD-GB4 cells. Cell proliferation was followed and analyzed using the IncuCyte imaging system for 72 h. Data represent mean ± s.d. of triplicate wells. The graph a is representative of three independent repeats. Statistical significance was determined using two-ways ANOVA test with multiple comparisons adjustment. E Wound healing assay using iRFP-labeled PD-GB4 cells co-cultured with microglia, showing lower migration rate of shSELP PD-GB4 compared to control WT and shNC PD-GB4 cells. Wound closure was followed and analyzed by the IncuCyte imaging system for 60 h. Data represent mean ± s.d. of 4 wells per group. The graph is a representative of three independent repeats. Statistical significance was determined using two-ways ANOVA test with multiple comparisons adjustment. F 3D spheroid invasion in Matrigel showing enhanced invasion of iRFP-labeled PD-GB4 GB cells when co-culture with unlabeled human microglia, and reduced invasion following SELP knockdown or treatment with 0.5 µM SELPi compared to untreated control WT or shNC PD-GB4 cells. Sprouting was followed for 72 h. Data represent mean ± s.d. of 5 spheroids per group. The graph a is representative of three independent experiments. Scale bars represent 100 µm. Statistical significance was determined using one-way ANOVA test with multiple comparisons adjustment. Source data are provided as a Source Data file.

Fig 5: SELP-knockdown inhibits tumor growth and prolongs survival in human GB mouse models.A SELP knockdown reduced tumor growth rate of PD-GB4 tumors in mice compared to control WT (control) or shNC GB tumors. Tumor volume was calculated using Radiant software. Data represent mean ± s.e.m. N = 8 control, 9 shNC, and 14 shSELP. One-way ANOVA, Dunn's method, p < 0.001. B Kaplan–Meier curve showing prolonged survival of shSELP PD-GB4 tumor-bearing mice compared to control WT and shNC. N = 8 control, 9 shNC, and 11 shSELP. P values were determined using two sided log rank test. C Representative T1 weighted MRI images of PD-GB4 tumors following Gd-DTPA administration, detected at day 22 post tumor inoculation. Representative images and quantification of immunostaining for proliferating cells (Ki-67), activated microglia (Iba1), and blood vessels (CD31) in the tumors showing reduced proliferation and blood vessel density, and activated microglia in shSELP tumors. Data represent mean ± s.d. N = 5 images per mouse. The graphs show data from a representative mouse per group out of two mice per group. Statistical significance was determined using one-way ANOVA test with multiple comparisons adjustment. Scale bars represent 100 µm. D Tumor growth of control WT (control), shNC, and shSELP U251 tumors in SCID mice demonstrating delayed tumor growth in shSELP U251 tumors. Tumors were detected by MRI imaging (MR solutions, T2 weighted). Tumor volume was calculated using Radiant software. Data represent mean ± s.e.m. N = 9 control, 8 shNC, and 9 shSELP. One-way ANOVA, Dunn’s method, p < 0.001. E SELP knockdown prolonged the survival of U251 tumor-bearing mice compared to control WT and shNC. N = 7 control, 6 shNC, and 6 shSELP. P values were determined using two-sided log rank test. F Representative T2 weighted MRI images of U251 tumors detected at day 19 post tumor inoculation. Representative images and quantification of immunostaining for proliferating cells (Ki-67), activated microglia (Iba1), and blood vessels (CD31) in the tumors showing reduced proliferation and blood vessel density, and activated microglia in shSELP tumors. Data represent mean ± s.d. Each dot represents the average of three images per mouse. N = 3 mice per group. Statistical significance was determined using one-way ANOVA test with multiple comparisons adjustment. Scale bars represent 100 µm. Source data are provided as a Source Data file.