Fig 2: Generation of fused vasculature and brain organoids (fVBOrs) with blood–brain barrier (BBB) structure.(A) Schematic view of the method for generating fVBOrs. EBs, embryonic bodies; NE, neuroepithelium; VP, vascular progenitor; VO, vessel organoid; BOr, brain organoid. (B) fVBOrs at different developmental stages. Scale bar, 500 µm. V, VOr; B, BOr. (C) Immunostaining of CD31 and DCX for labeling vessels and neurons, respectively, in day (D) 40 fVBOrs. Scale bar, 200 µm. (D) Immunostaining of CLDN5 for labeling tight junctions in fVBOrs. Scale bar, 200 µm. D1, enlarged area. (E) Immunostaining for markers of astrocytes (S100/GFAP), neurons (MAP2), endothelial cells (CD31), and vessel structures (GFP) in fVBOrs. Orange arrows indicate astrocytes end feet. Scale bar, 20 µm. (F, G) Confocal fluorescence images showing the transport of rhodamine-labeled angiopep-2 (Angiopep-2–Rhod), rhodamine–scramble peptide (Scramble–Rhod) in fVBOrs. Scale bar, 200 µm. Bottom, z-stack images of rhodamine signals.

Fig 3: Analysis of IHC staining of SFT/HPC. (A) Heatmap of IHC expression of BCL2, CD99, MMP9, CD34, WNT5A, and CLDN5, displayed with grades and locations of SFT/HPCs. p-values are for the correlation between location and IHC expression. (B) Linear correlation analysis of IHC expression of MMP9 and WNT5A. Circles represent groupings according to SFT/HPC location. (C) Correlogram of IHC expression of CLDN5, CD34, WNT5A, and MMP9 according to location and grade of SFT/HPC. Red circles indicate a positive correlation between the paired variables and blue circles a negative correlation. The intracranial location was coded as a positive value, and the extracranial location as a negative value.

Fig 4: SFN-Cys regulated proteomic expression and inhibited cell migration and invasion in U373MG and U87MG cells.a The SFN-Cys-regulated protein expressions in U87MG cells were identified by HPLC–MS/MS. Red, upregulation; black, downregulation. b Gene Expression Profile was performed in predicted SFN-Cys-targeted proteins in either tumor (T) or normal (N) tissue among different cancers by GEPIA. c The Protein–Protein Interaction network in SFN-Cys-targeted proteins was predicted by String server. d The Overall Survival Map of predicted SFN-Cys-targeted proteins in different cancers by GEPIA. e The Correlation Analysis among SFN-Cys-targeted TUBA1C, CLDN5, S100A4, MAP1LC3B, Bnip3, Nix, and LAMP1 in GBM by GEPIA server (p < 0.05). Migration assay without matrigel (f) or invasion assay with matrigel (g) was performed separately to detect the numbers of migratory or invasive cells in U87MG and U373MG cells after cells were treated with 0, 10, and 20 µM SFN-Cys for 24 h. h The area covered by migratory cells was recorded by Leica DMIRB microscope at ×40 magnification at 0 and 24 h. The relative closure (0 h vs. 24 h) was measured by Image J. At least three independent experiments were performed. *p < 0.05. The values are expressed as means ± SD (n = 3).

Fig 5: CLDN5 and CD34 expression in icSFT/HPC and exSFT/HPC. (A–F) Representative exSFT/HPC case. H&E staining showed typical microscopic features of exSFT/HPC (A). IHC staining showed moderate to strong expression of CLDN5, indicating well-preserved tight junctions of the tumor vasculature (B), and diffuse positivity for CD34 in exSFT/HPC (C). Double immunofluorescence showed strong expression of CLDN5 (D), and diffuse expression of CD34 (E). Merged image showing tumor vessels with well-preserved coexpression of CLDN5 and CD34 in exSFT/HPC (F) (D–F, scale bar, 100 µm). (G–L) Representative icSFT/HPC case. H&E staining showed typical microscopic features of icSFT/HPC (G). IHC showed some vessels with significantly decreased expression of CLDN5 (black arrowheads) (H), compared to densely labeled CD34 in tumor vessels of icSFT/HPC (I). Double immunofluorescence revealed some tumor vessels where the expression of CLDN5 was markedly decreased (J) but the expression of CD34 was maintained (gray arrows) (K). Merged image showing good contrast between vessels stained for only CD34 (gray arrows) and other vessels with coexpression of CLDN5 and CD34 (yellow arrowheads) (L) (J–L, scale bar, 100 µm).