Healthy BMSC exosomes inhibit HR-MM cell-induced angiogenesis in Matrigel plugs. (A) Schematic of the Matrigel plug assay. A mixture of Matrigel and 4 × 106 RPMI8226-HR cells was admixed with exosomes derived from young BMSCs (young BMSC exosomes; 4 × 107 particles per 200 µL Matrigel) or older BMSCs (older BMSC exosomes; 4 × 107 particles per 200 µL Matrigel). (B) After 3 weeks, the Matrigel plugs were harvested and photographed. Paraffin-embedded sections of Matrigel plugs were stained with hematoxylin and eosin and then subjected to immunostaining for CD138 (brown) and CD31 (red). Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bar, 500 µm. (C) Quantification of vessel density in Matrigel plugs by pixel density. Young and older BMSC exosomes inhibited tumor angiogenesis in Matrigel plugs compared with the control (RPMI8226-HR only) (*P < .01; **P < .001 vs control, Student t test, n = 6). Values represent the mean ± SD. (D) Effect of BMSC exosomes on the viability of HR-MM cells. RPMI8226-HR cells were cultured with young or older BMSC exosomes (2.5 × 107 particles/mL). Cell viability was assayed after 48 h using Cell Counting Kit-8. Values represent the mean ± SD. (E) The formation of tube-like structures was observed using the cell-permeable dye Calcein AM (green). Endothelial tube formation of HUVECs treated with young BMSC exosomes, older BMSC exosomes, or the control (without exosomes). Scale bar, 500 µm. (F) Quantification of tube-like structures by pixel density. Young and older BMSC exosomes significantly inhibited HUVEC tube formation in comparison with the control (control vs young BMSC exosomes; P < .001, control vs older BMSC exosomes; P = .038, Student t test). Values represent the mean ± SD.
