Fig. 3: Endocan induces stable phenotypic alterations of GBM cells regulating their spatial identity.

a Proliferation assay of control and rEndocan treated (10 ng/ml) WTD and KOD cells. **P = 0.0023 for WTD cells and P = 0.2104 for KOD cells, n = 6 biological replicates, unpaired two-tailed Student’s t test. b Schematic figure delineating the steps for the experiment. Briefly, mouse 7080 spheres were implanted into brains of WT and Esm1 KO host, once tumors were formed, they were isolated into single cells, and were followed by labeling Esm1 KO derived spheres with mCherry and WT derived spheres with GFP. Cells were then co-injected into WT host (n = 5 mice per group). The figure was created in BioRender. Kornblum, H. (2024) BioRender.com/n19r944. c Representative IHC staining with anti-GFP (left) and anti-mCherry (right) antibodies of mice brains co-injected with WTD (GFP⁺) and KOD (mCherry⁺) cells (n = 5 mice per group). The boundaries of the staining areas are shown by white lines, injection site indicated by red asterisk. Scale bar, 2 mm. d Representative IHF images of mice brain sections stained with anti-GFP (green), anti-mCherry (red) antibodies and DAPI (blue). n = 3 biological replicates, Scale bar, 50 μm. e Ratio of GFP+ and mCherry+ cells in IHF images as in “D” at different distances from the site of injection. Quantification was performed using ImageJ software where mCherry+ cells and GFP+ cells were divided by DAPI staining. The experiment was performed using n = 3 mice per group. ****P < 0.0001 for 400 µm vs. 0 µm, ***P = 0.0006 for 800 µm vs. 0 µm, and ****P < 0.0001 for 1200 µm vs. 0 µm, unpaired two-tailed Student’s t test. f Representative IHC images of core (upper panel) and edge regions (middle panel) of the tumor obtained as in “D” stained for CD31, HIF1α, ALDH1A3, and YKL-40. % of field positive areas for indicated proteins on lower panel (n = 3 mice). **P < 0.0001 for CD31, **P = 0.0013 for HIF1α, ***P = 0.0083 for ALDH1A3 and ****P = 0.0001 for YKL-40, unpaired two-tailed Student’s t test. Scale bar, 200 µm. g Kaplan–Meier survival analysis of Esm1 WT mice intracranially injected with WTD and KOD cells or the mixture of both at 1:1 ratio. (n = 5 mice per group), *P = 0.047; ns. - nonsignificant (P = 0.31); **P = 0.00642. P values were determined by log-rank test. Source data are available in the Source Data file.