Fig 1: eGFP-Cavin1 was transferred via EVs to recipient LN229 cells and increased LN229 proliferation in orthotopic xenograft glioma mice. (A) Schematic illustration of experimental grouping and process of the mixed glioma xenograft model. U87-eGFP, U87-C, and U87-vC were respectively mixed with an equal number of LN229-RFP-luc and implanted intracranially in nude mice. IVIS detection was performed at day 7, 14, 21, and 28 post-implantation (n = 8), and brains were dissociated at day 21 for histological analysis and confocal imaging. (B) In vivo bioluminescence imaging showing a higher signal intensity in mice implanted with LN229-RFP-luc+U87-C. (C) Analysis of the bioluminescence intensity suggesting a rapidly increasing growth of LN229-RFP-luc which were mixed with U87-C from day 7. (D) Weight analysis indicating a faster weight loss in mice implanted with LN229-RFP-luc+U87-C from day 12 (n = 8). (E) Kaplan-Meier survival curves showing the percent survival of mice implanted with LN229-RFP-luc+U87-eGFP, LN229-RFP-luc+U87-C, and LN229-RFP-luc+U87-vC, respectively (n = 8, p < 0.001; log-rank test). (F) H&E and Ki67 staining of mouse cerebrum with tumor which was harvested at day 21 post implantation (n = 5). H&E staining showing a more heterogeneous composition in the LN229-RFP-luc+U87-C tumor. Scale bar, 100 µm. (G) IHC for Ki67 showing an increased number of Ki67-positive cells in the LN229-RFP-luc+U87-C tumor (mean±SEM, p < 0.0001; p < 0.0001). (H) 3D images generated from the Z-stacks with a slice-distance of 1.6 µm, showing that the number of LN229-RFP-luc cells co-implanted with U87-C were increased and eGFP-Cavin1 was transferred to LN229-RFP-luc (n = 4). (I) Confocal images clearly showing the transfer of eGFP-Cavin1 to LN229-RFP-luc cells (white arrowheads) whereas no transfer of eGFP-vCavin1 was detected in LN229-RFP-luc cells (n = 4).