Fig. 6: CCA-M1EVs exhibited potent anti-tumor effects against patient-derived xenograft (PDX) model in vivo.

a. Schematic illustration of PDX model, humanized EVs construction, and experimental design for evaluating the efficiency of tumor inhibition upon treatment with PBS, CC-M1EVs, and CCA-M1EVs (human PBMC source) in PDX mice. The mice were given the indicated formulations at day 7, 10, 13, 16 and 19. T₁-weighted MR signals in the brain was determined using magnetic resonance imaging (MRI) at day 7 and 20. 24 h after the final injection, ROS production was detected by DCFH-DA using two-photon fluorescence images and O₂ concentration was measured by PA. Meanwhile, some of the mice in each group were sacrificed, and brains were harvested for TME and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) analyses. The remaining mice were used to monitor survival time. b. In vivo PA images and relative PA signal intensity statistics of GBM PDX mice after i.v. injection of PBS, CC-M1EVs, or CCA-M1EVs, respectively. Here, Ce6 was served as a PA signals for the assessment of the distribution of CC-M1EVs and CCA-M1EVs (n = 5). c. T₁-weighted MRI of GBM PDX tumor-bearing mice at 7 day and 20 day post i.v. injection with various groups, and corresponding quantification of T₁-weighted MRI from the tumor site (n = 5). Images were analyzed with Analyze 11.0. d. The body weight of the mice with different treatments (n = 5). e. Varieties of survival rates of PDX tumor-bearing mice in different groups (n = 5). f. Immunofluorescence imaging of brain histological sections of M2 (CD163, green) and M1 (iNOS, red), and corresponding quantification of M2/M1 ratio. All images have the same scale of 50 μm (n = 3). g. Two-photon fluorescence images of GBM PDX tumor-bearing mice and quantitative analysis of ROS signals in tumor tissues after i.v. injection different treatments. All images have the same scale of 100 μm (n = 3). h. PA images of GBM PDX tumor-bearing mice and quantitative analysis of the oxyhemoglobin saturation levels in tumors with treatment of different extracellular vesicle designs (n = 3). i. TUNEL staining of tumor sections for each group, with corresponding quantification (n = 3). All images have the same scale of 50 μm. For b, c, d, f, g, h, and i, data are presented as the mean ± S.D. Statistical significance between multiple groups was calculated using one-way ANOVA with a Kruskal-Wallis test (b, c, d, f, g, h, and i). Survival analysis was calculated using two-sided Log-rank Mantel-Cox tests (e). ns, not significant