Fig. 4: In vitro evaluation of the antitumor effects of CCA-M1EVs on multicellular tumor spheroids (MCTSs).

a. Illustration of in vitro three-dimensional tumor model. b. CLSM images of surface plots of DiD-labeled M1EVs penetration in MCTS (top); the corresponding fluorescence signal intensity across the spheroids (bottom). c. The concentrations of IL-1β, IL-6, TNF-α, and IFN-γ in supernatants prepared from the MCTS culture medium after treatments with PBS, M1EVs, CC-M1EVs, A-M1EVs, and CCA-M1EVs, respectively. d. Quantitative analysis of M2/M1 ratio of the MCTS based on flow cytometry analysis (n = 3). e. Representative flow cytometry analysis images and corresponding quantitative analysis of the intracellular levels of ROS in U87MG cells treated with different EVs designs (n = 3). f. Photographs of MCTSs at a certain time (day 0 and day 7) (left). The volume of MCTSs treated with different formulations at day 7 (right) (n = 3). All images have the same scale of 100 μm. For d, e, and f, data are presented as the mean ± S.D. Statistical significance between multiple groups was calculated using one-way ANOVA with a Kruskal-Wallis test (d, e, and f). ns, not significant