Figure 1.
The strategy for capture of CD34+ exosomes using anti-CD34 Ab-coated microbeads purchased from Miltenyi (A) and a schema for capture of CD34+ exosomes directly from AML patients' plasma (B).
Figure 2.
Studies with the Kasumi-1 cell line and with exosomes derived from Kasumi-1 culture supernatants.
A) Flow cytometry of Kasumi-1 cells shows expression of blast markers, CD34, CD117 and CD33 and of CD81 (tetraspanin). Dotted lines indicate isotype controls. B) Kasumi-1 exosomes were separated on a continuous sucrose density gradient, and the collected gradient fractions were tested in Western blots for CD34 and CD81. CD34+ and CD81+ exosomes were recovered at the density of 1.10–1.14. C) An electron microscope image of isolated and negatively stained Kasumi-1 exosomes. D) Western blots of isolated Kasumi-1 exosomes (Kas) and of exosomes isolated from plasma of a normal donor (NC). Each lane was loaded with 10 µg exosomal protein.
Figure 3.
Calibration experiments with CD34+ Kasumi-1 exosomes isolated by ultracentrifugation.
A) Isolated exosomes were loaded at increasing protein concentrations onto gels and blotted using anti-CD34 Ab. Intensity of each band was measured in pixels. The graph illustrates a linear relationship between exosomal protein levels and pixel intensity in Western blots. B) Isolated exosomes (20 µg protein) were added to increasing volumes of CD34 microbeads (left). Five or 10 µL of microbeads were sufficient to capture 20 µg of exosomes. Next, a 10 µL aliquot of beads was used to capture increasing concentrations of Kasumi-1 exosomes (right). The graph shows that the capacity of CD34 microbeads to capture up to 80 µg of input exosomes increased linearly. However, at 80 µg only about 40% of exosomal proteins were captured, suggesting that additional microbeads are necessary for capture of all Kasumi-1 exosomes.
Figure 4.
Capture of CD34+ exosomes from total exosomal fractions by CD34+ microbeads.
Isolated Kasumi-1 exosomes (Kas) and total exosome fractions isolated by ultracentrifugation from normal donor plasma (NC) or from CD34+ AML patient plasma (AML) were used for capture with CD34 microbeads. After the 1st capture and removal of beads, 2nd capture was performed with a fresh aliquot of CD34 microbeads. The final unbound fractions were ultracentrifuged to collect remaining exosomes. While the 2nd capture was necessary to recover all CD34+ Kasumi-1 exosomes, a single capture was sufficient to recover all CD34+ exosomes from the bulk of exosomes isolated from the AML plasma. There were no CD34+ exosomes captured from the bulk exosomal fraction isolated from a normal donor's plasma. CD81 expression indicates that the unbound fractions still contain CD34neg exosomes.
Figure 5.
Capture of CD34+ blast-derived exosomes directly from AML patients' plasma.
A) Negatively stained electron microscope images of exosomes captured on CD34 microbeads (* shows a vacant microbead). B) Increasing AML plasma volumes were used for capture of CD34+ exosomes by microbeads and recovered exosomes were studied by Western blots. The graph shows a linear relationship between the input plasma volumes and pixel densities of captured and blotted CD34+ exosomes. C) Exosomes were captured from plasma samples obtained from five CD34+ AML patients and were analyzed by Western blots. The percentage of leukemic blasts in the peripheral circulation of each of the patients is shown. CD81 serves as the exosome marker. D) Removal of platelet-derived exosomes from plasma using anti-CD61 Ab-coated microbeads prior to capture of CD34+ exosomes. Exosomes captured with CD61 microbeads (left: CD61+) and CD34+ exosomes captured after removing CD61+ exosomes (right: CD61neg/CD34+) are shown in a representative Western blot of three evaluated.
Figure 6.
Characteristics of captured exosomes.
A) A representative Western blot profile of the captured CD34+ exosomes and of non-captured CD34neg exosomes which were isolated by ultracentrifugation from the same AML plasma sample. B) After co-incubation of NK cells with CD34+ exosomes captured directly from AML plasma or CD34neg exosomes as described in materials and Methods, NKG2D expression (in MFI) was found to be down-regulated only with CD34+ exosomes (red) compared to CD34neg exosomes (green) or control without exosomes (blue). The gray peak denotes isotype control. In contrast, NKp46 expression was down-regulated by co-incubation with CD34neg exosomes.