LEVs are produced in vivo and internalized by stromal cells. (A) Diagnostic bone marrow plasma is enriched with CD19⁺ LEVs. Imaging flow cytometry identified CD19 PE_Cy7 (LEV) and CD61 FITC (platelets)-positive vesicles in matched diagnostic and remission (day 28 postchemotherapy) ALL patient bone marrow plasma samples (1 × 10⁶ events acquired). The number of CD19⁺ vesicles/mL (LEVs, left) were significantly higher at diagnosis and number of CD61⁺ extracellular vesicles (platelets, right) significantly higher in remission marrow plasma (**P > .05; ***P > .005). (B) SD1 cells produce LEVs within the bone marrow microenvironment that are internalized by mouse BMSCs. LEVs from 1 × 10⁷ SD1 cells or 1 × 10⁶ SD1 cells labeled with the fluorescent membrane label PKH26 were introduced into the left femur of 5 NSG mice. Bone marrow flushes taken 17 days posttransplantation were seeded onto fibronectin-coated glass coverslips and BMSCs were allowed to adhere overnight. Live cell images were taken using bright field and ultraviolet illumination with Red Sedat filter at ×40 magnification. Scale bar is 20 µm. The left image represents an overlay of the images and shows murine BMSCs with internalized PKH26⁺ LEVs as indicated by intracellular red fluorescence (yellow arrow). The figure shows variable, punctate perinuclear red fluorescence within the outer membrane of murine BMSCs and not around the periphery of the recipient cell. The right image shows a cluster of PKH26⁺ SD1 cells (black arrow) and free PKH26⁺ LEVs (yellow arrow) in the extracellular space, produced within the murine bone marrow. Scale bar is 20 µm. (C) Confirmation of LEV internalization by human bone marrow cells in vitro. Isolated SD1 LEVs were labeled using a lipophilic tracer (Dio C₁₈; green). Labeled LEVs were added to cultures of bone marrow cells in glass plates for 24 hours. Cells were fixed using paraformaldehyde and counterstained using Cell Mask (red). Serial images were captured at 0.1-µm intervals in Z using a Spinning disk confocal system based around an Olympus IX71 microscope. Illumination achieved by white light LED and a 300-W Xenon light source for fluorescence and Sedat filters. Composite 3-dimensional image from 81 0.1-µm Z stacks was achieved using IMARIS software (BITPLANE, Oxford Instruments) which revealed the LEVs to be fully internalized by the recipient cells as represented by green fluorescence along the mid-line (indicated by a blue star). Scale bar represents 10 µm. (D) Human CD19⁺ LEVs are detected in peripheral blood plasma in mice engrafted with primary ALL cells. Plasma samples isolated from tail vein bleeds 9 or 20 days after intrafemoral injection of PKH26-labeled human ALL cells were screened for PKH26 and human CD19⁺ vesicles. (Top) Synchronus Image stream acquisition of bright field (left), PKH26 (center), and CD19 PE_Cy7 (right) fluorescence-identified vesicles, which originated from the labeled human ALL cells, 9 days posttransplantation. All transplanted mice (n = 5) showed evidence of PKH26⁺ or human CD19⁺ vesicles in the peripheral blood plasma. Because PKH26⁺ LEVs were generated in vivo by transplanted PKH26⁺ ALL cells, staining is less unform compared with the human CD19⁺ antibody labeling postisolation. The figure shows dual-labeled LEVs from 10 000 acquired events. Scale bar is 7 µm. (Lower): All transplanted mice showed evidence of PKH26⁺ or human CD19⁺ (0.73-5.67% at day 9; 2.23-6.40% at day 20) LEVs in the peripheral blood plasma. (E) Engrafted primary human ALL cells introduced into the mouse bone marrow produce LEVs that are internalized by surrounding mouse BMSCs. Primary ALL cells were labeled with PKH26 (red) and introduced into the femur of 5 NSG mice. Bone marrow flushes 14 days after injection were seeded onto fibronectin and fixed with paraformaldehyde. Cells were counterstained with cell mask green and 4′,6-diamidino-2-phenylindole. The panel shows PKH26⁺ LEVs, visualized as punctate red fluorescent staining within the cell membrane of adherent mouse BMSCs, were internalized by mouse BMSCs in vivo (×160 magnification). Captured using black and white film, the figure shows composite image and individual filters. Scale bar is 10 µm.