Fig. 9

a Immunostaining of clinical histological sections obtained from normal tumor-adjacent tissues and low- (LGG: diffuse astrocytoma, n = 22) and high-grade gliomas (HGG: anaplastic astrocytoma, n = 20; glioblastoma multiforme, n = 22); M1 macrophages (iNOS), M2 macrophages (CD163), and cell proliferation (Ki67) are visualized. Quantitative analysis of the corresponding M2/M1 ratios is shown on the right side. The proliferation marker Ki67 was positively correlated with the M2/M1 ratio. Scale bars: 50 μm. b M2/M1 ratio analysis of 167 HGG and 522 LGG samples acquired from The Cancer Genome Atlas (TCGA) database. Each dot represents a single individual. c Survival curves of glioma patients from TCGA database. The OncoLnc tool was used to explore the correlations of survival with M2/M1 ratios. d In vivo time-lapse two-photon images of the diffusion of M1EVs, M0EVs, EMVs, and PEG NPs across microvascular endothelial cells of the brain at 48 h after i.v. injection (left). Tetramethyl-rhodamine isothiocyanate-Dextran was used to label blood vessels (red). M1EVs, M0EVs, and EMVs were labeled with DiO (green); PEG NPs were labeled with FITC (green); corresponding formulation distributions in tumor tissue are also shown (right). Scale bars: 50 μm. Immunofluorescence images of histological sections showing M2 (CD163, green) and M1 macrophages (iNOS, red) (left), and quantitative analysis of M2/M1 ratios (right) at 48 h after i.v. injection. Scale bars: 50 μm, (n = 3). e CLSM images and surface plots showing DiD-labeled M1EVs penetrating MCTS (top) and the corresponding fluorescence signal intensities across the spheroids (bottom).