Fig 2: T140-KLA-EVs suppress leukemia development and improves survival of AML xenografted mice. (A) Percentage of circulating leukemia MOLM13-mCherry cells that uptake EVs following a single systemically administered dose of EVs. EV uptake was tracked using CFSE and analyzed using flow cytometry (n = 3 mice). (B) Representative flow cytometry plots illustrating the uptake of CFSE-labelled EVs by circulating cells. mCherry expression is used to distinguish leukemia MOLM13 cells from mouse PBMC. (C) Outline of the animal study for verifying the efficacy of KLA-T140-EVs in leukemia suppression. (D) Representative bioluminescent images of NSG mice implanted with CXCR4-positive luciferase-expressing MOLM13 cancer cells during a course of systemic RBCEV treatments. Colors indicate bioluminescent signals (photon/s) in 2 scales (the images are divided into 2 groups, day 0-6 and day 8-14 from the treatment start date, to avoid oversaturation of signals). (E) Average bioluminescent signals quantified in the mice during the development of leukemia (photons/s), normalized by the signals at the start of the treatments, and presented as mean ± SEM. (n = 7 mice). (F) Representative images of H&E staining of spleen sections from leukemic mice for each treatment condition. Scale bar, 50 µm. Student's one-tailed t-test (A) and Two-way ANOVA test (E): *P < 0.05, ***P < 0.001.

Fig 3: Ligation of RBCEVs with a T140 peptide allows CXCR4-specific cellular targeting. (A) In vitro uptake of control RBCEVs or RBCEVs ligated with CXCR4-binding T140 peptide in three cell lines with varying CXCR4 expression, presented as the percentage of cells that uptake CFSE-labelled RBCEVs, quantified using flow cytometry. (B) Comparison of uptake of T140 or Scr-T140 peptide ligated RBCEVs by MOLM13 cells based on flow cytometry analysis of CFSE. (C) Western blot of hemoglobin A (HBA) to track EV uptake in MOLM13 cells incubated with targeting T140-EVs or non-targeting Scr-T140-EVs. (D) Uptake of T140-ligated RBCEVs by MOLM13 cells pretreated with free T140 peptide. (E) Western blot analysis of HBA indicating RBCEV uptake in MOLM13 cells in the presence or absence of a pretreatment with free T140 peptide. For A, B and D, graphs include data from 3 biological replicates prepared from RBCEVs from independent donors. The graphs present the mean ± SEM. Student's one-tailed t-test: ns - not significant, *** P < 0.001. For C and E, EV uptake was tracked via HBA while GAPDH served as an internal control.