Figure 3
Figure 3. Proteomic characterization of CLL exosomes and transfer of proteins to target cells. (A and B) LC-MS/MS analysis of proteins extracts from CLL exosomes. (A) Subcellular localization of proteins identified in CLL exosomes (UniProt database). (B) Molecular functions associated with proteins identified in CLL exosomes (IPA). Portion radii were calculated according to the number of molecules associated with the functions. Cellular functions are indicated with their respective P values. (C) Phenotyping of individual CLL exosomes (MEC-1) was performed by flow cytometry. Exosomes were labeled with PKH67 and only green fluorescence-positive events were selected for analysis. The size of exosomes was confirmed using 100- and 200-nm beads. Exosomes were stained with indicated monoclonal antibodies (solid line) or with respective isotype controls (dotted line). (D) MEC-1 cells (105 in 100 µL) were treated with 2 µg/mL rituximab alone (Ctrl) or in combination with 50 or 100 µg/mL CLL exosomes (Exo) or with 30% (v/v) CLL plasma for 1 hour at 37°C. The binding of rituximab to CLL cells was followed by flow cytometry using an anti–rituximab-specific antibody. Data are presented as mean fluorescence intensities (MFIs; n = 3). ***P < .001. (E) Immunoblot analysis of proteins from CLL exosomes purified by Optiprep cushion. Lysate from CLL cells served as the control. (F) HMEC-1 and HS-5 cells were untreated (Ctrl, gray shade) or incubated with 50 µg/mL PKH26-labeled CLL exosomes (MEC-1; black line) for indicated periods of time, and the transfer of HLA-DR was followed by flow cytometry using specific antibody or isotype control (dotted line) (left). Results were confirmed using immunoblot (right). (G) C57BL/6 and NSG mouse BM cells were untreated (Ctrl) or incubated with CLL exosomes (MEC-1; 50 µg/mL) ex vivo for 24 hours. The transfer of human HLA-DR protein was followed using flow cytometry (representative of 3 animals). (H) HMEC-1 and HS-5 cells were untreated (gray shade) or incubated with 50 µg/mL CLL exosomes (MEC-1; black line) for 24 hours. Cells were analyzed by flow cytometry using specific antibodies or isotype controls (dotted line) to show the transfer of proteins from CLL exosomes to target cells (representative of n = 3).

Proteomic characterization of CLL exosomes and transfer of proteins to target cells. (A and B) LC-MS/MS analysis of proteins extracts from CLL exosomes. (A) Subcellular localization of proteins identified in CLL exosomes (UniProt database). (B) Molecular functions associated with proteins identified in CLL exosomes (IPA). Portion radii were calculated according to the number of molecules associated with the functions. Cellular functions are indicated with their respective P values. (C) Phenotyping of individual CLL exosomes (MEC-1) was performed by flow cytometry. Exosomes were labeled with PKH67 and only green fluorescence-positive events were selected for analysis. The size of exosomes was confirmed using 100- and 200-nm beads. Exosomes were stained with indicated monoclonal antibodies (solid line) or with respective isotype controls (dotted line). (D) MEC-1 cells (105 in 100 µL) were treated with 2 µg/mL rituximab alone (Ctrl) or in combination with 50 or 100 µg/mL CLL exosomes (Exo) or with 30% (v/v) CLL plasma for 1 hour at 37°C. The binding of rituximab to CLL cells was followed by flow cytometry using an anti–rituximab-specific antibody. Data are presented as mean fluorescence intensities (MFIs; n = 3). ***P < .001. (E) Immunoblot analysis of proteins from CLL exosomes purified by Optiprep cushion. Lysate from CLL cells served as the control. (F) HMEC-1 and HS-5 cells were untreated (Ctrl, gray shade) or incubated with 50 µg/mL PKH26-labeled CLL exosomes (MEC-1; black line) for indicated periods of time, and the transfer of HLA-DR was followed by flow cytometry using specific antibody or isotype control (dotted line) (left). Results were confirmed using immunoblot (right). (G) C57BL/6 and NSG mouse BM cells were untreated (Ctrl) or incubated with CLL exosomes (MEC-1; 50 µg/mL) ex vivo for 24 hours. The transfer of human HLA-DR protein was followed using flow cytometry (representative of 3 animals). (H) HMEC-1 and HS-5 cells were untreated (gray shade) or incubated with 50 µg/mL CLL exosomes (MEC-1; black line) for 24 hours. Cells were analyzed by flow cytometry using specific antibodies or isotype controls (dotted line) to show the transfer of proteins from CLL exosomes to target cells (representative of n = 3).

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