Cell-specific uptake of mantle cell lymphoma-derived exosomes by malignant and non-malignant B-lymphocytes

Abstract

Mantle cell lymphoma (MCL) is an aggressive and incurable mature B cell neoplasm. The current treatments are based on chemotherapeutics and new class of drugs (e.g. Ibrutinib(®)), which in most cases ends with tumor resistance and relapse. Therefore, further development of novel therapeutic modalities is needed. Exosomes are natural extracellular vesicles, which play an important role in intercellular communication. The specificity of exosome uptake by different target cells remains unknown. In this study, we observed that MCL exosomes are taken up rapidly and preferentially by MCL cells. Only a minor fraction of exosomes was internalized into T-cell leukemia and bone marrow stroma cell lines, when these cells were co-cultured with MCL cells. Moreover, MCL patients' exosomes were taken up by both healthy and patients' B-lymphocytes with no apparent internalization to T lymphocytes and NK cells. Exosome internalization was not inhibited by specific siRNA against caveolin1 and clathrin but was found to be mediated by a cholesterol-dependent pathway. These findings demonstrate natural specificity of exosomes to B-lymphocytes and ultimately might be used for therapeutic intervention in B cells malignancies.

Keywords: B-lymphocytes; Endocytosis; Exosomes; Mantle cell lymphoma.

Fig. 1. Structural and biochemical characterization of Jeko-1 exosomes

(A) Exosomes were isolated from the culture media of Jeko-1 cells and analyzed by transmission electron microscopy (TEM). (B) Immuno-electron microscopy of isolated Jeko-1 exosomes incubated with anti-CD81 followed by IgG 12-nm in diameter gold nanoparticle-conjugated mAb. (C) Measurement of Jeko-1 exosomes mean diameter by nanoparticle tracking analysis system. (D) Western blot analysis of the exosome markers CD81, TSG1 and the intracellular protein Calnexin. (E) Flow cytometry analysis of the exosomal surface proteins CD81, CD63 and the B-lymphocytes marker CD19 on Jeko-1 exosomes. Representative of three independent experiments are shown.

Fig. 2. Characterization of MCL primary exosomes

(A) Exosomes were isolated from the culture media of MCL patient(s) cells and analyzed by transmission electron microscopy (TEM). (B) Measurement of MCL primary exosomes mean diameter by nanoparticle tracking analysis system. (C) Flow cytometry analysis of exosomal surface protein (CD81 or CD63) and the B-lymphocytes marker (CD20) on primary exosomes from serum of patients MCL7, MCL8, and MCL4.

Fig. 3. Rapid uptake of Jeko-1 exosomes by Jeko-1 cells

Kinetics of Jeko-1 exosomes uptake by Jeko-1 cells measured by confocal microscopy and flow cytometry analysis. (A) PKH-26 labeled Jeko-1 exosomes were incubated with Jeko-1 cells, reaction was stopped at different time points (1, 5, 10, 20, 30, 45 and 60 min) and cells were analyzed by confocal microscopy. The cell membrane of Jeko-1 cells was stained with AlexaFluor 488-anti CD44 mAb. Negative control- Jeko-1 cells with no addition of labeled exosomes. Data is representative of three independent experiments. Scale bar equals 25μm. (B) Flow cytometry analysis of Jeko-1 cells, mean fluorescence after incubation with PKH-67 labeled Jeko-1 exosomes for the indicated time points (10, 20, 30, 60, 120 and 180 min). The data are presented as mean ± SD of four independent experiments.

Fig. 4. MCL exosomes specifically internalized into MCL cells

(A) Time course of Jeko-1 derived exosomes internalization to Jeko-1, Jurkat and HS-5 cells. PKH-67 labeled Jeko-1 exosomes were incubated for the indicated time points with co-culture of Jeko-1, Jurkat or HS-5 cells. Internalization was measured by flow cytometry using specific antibodies (PE-anti CD19 for Jeko-1 cells, PerCP-anti CD3 for Jurkat cells and AlexaFluor 647-anti CD105 for HS-5 cells). The data present the percent of cells that uptake labeled exosomes and are expressed as mean ± SD of three independent experiments. (B) Time course of Mino derived exosomes internalization to Mino, Jurkat and HS-5 cells. PKH-67 -labeled Mino exosomes were incubated for the indicated time points with co-culture of Mino, Jurkat or HS-5 cells. Internalization was measured by flow cytometry using specific antibodies (PE-anti CD19 for Mino cells, PerCP-anti CD3 for Jurkat cells and AlexaFluor 647-anti CD105 for HS-5 cells). The data present the percent of cells that uptake labeled exosomes and are expressed as mean ± SD of three independent experiments. (C) PKH-26 labeled Jeko-1 exosomes were incubated for 60 min with co-culture of Jeko-1, Jurkat or HS-5 cells and uptake of exosomes was analyzed by confocal microscopy. After incubation Jurkat and Jeko-1 cells were removed from the HS5 cells and the cell membrane was labeled with AlexaFluor 488-anti CD3 (Jurkat) or AlexaFluor 488-anti CD19 (Jeko-1) HS-5 cells were labeled by AlexaFluor 488-wheat germ agglutinin. Negative control- Jeko-1, Jurkat and HS-5 cells with no addition of labeled exosomes. Data is representative of three independent experiments. Scale bar is 25μm.

Fig. 5. Normal B-cells and MCL cells preferentially uptake MCL exosomes

Exosomes derived from MCL patient’s cells (MCL5 and MCL6) were incubated for the indicated time points (0, 10, 30, 60, 120 and 180 min) with PBMCs of healthy individuals or MCL patients. Internalization of PKH-67 labeled exosomes by healthy (A) PBMCs or (B) MCL patient’s PBMCs was assayed by flow cytometry using specific antibodies to T-lymphocytes (PerCP -anti CD3), B-Lymphocytes (PE- anti CD19), NK cells (APC-anti CD56) and monocytes (APC- anti CD14). The data are expressed as the percentage of cells that taken up labeled exosomes. Results of healthy PBMCs are expressed as mean ± SD of three independent controls. PKH-26 labeled MCL exosomes were incubated for 30 or 60 min with (C) healthy individuals PBMCs or (D) MCL patient’s mononuclear cells. Internalization was assayed by confocal microscopy. The cell membrane was labeled with AlexaFluor 488-anti CD3 (T-lymphocytes), AlexaFluor 488-anti CD19 (B-lymphocytes) or AlexaFluor 488-anti CD14 (monocytes). Negative control- cells without addition of exosomes. Data is representative of two independent experiments. Scale bar is 25μm.

Fig. 6. MCL exosomes internalized by Clathrin and Caveolin1- independent endocytosis pathway

Clathrin heavy chain (CLTC) or Caveoin1 (CAV1) down regulation. qPCR analysis of (A) CLTC and (E) CAV1 mRNA levels 72 h post electroporation of siCLTC or siCAV1 in Jeko-1 cells. Expression was normalized to housekeeping genes eIF3a and eIF3c and depicted as mRNA concentration relative to untreated cells (Mock). Data are presented as mean ± SD of thee independent experiments. (B) Representative Western Blot analysis of Clathrin heavy chain expression 72h post electroporation. α-Tubulin was used as a loading control. (C, F) PKH-67 labeled Jeko-1 exosomes were incubated with untreated (Mock), siLUC electoporated cells, siCLTC or siCAV1 treated cells, 72 h post electroporation for 60 min. Internalization was measured by flow cytometry analysis. The data are expressed as the percent of cells that uptake labeled exosomes as mean ± SD of thee independent experiments. (D) Representative confocal microscopic images of untreated (Mock), siLUC electroporated cells or siCLTC treated cells, 72 h post electroporation, incubated with PKH-26 labeled Jeko-1 exosomes or Transferin CF640 for 60 min or 15 min, respectively. Scale bar is 25μm. (G) Representative confocal microscopic images of untreated (Mock), siLUC or siCAV1 treated cells, 72 h post electroporation, incubated with PKH-26 labeled Jeko-1 exosomes or CholeraToxin-555 for 60 min. The cell membrane was labeled with AlexaFluor 488-anti CD19. Scale bar is 25μm.

Fig. 7. MCL exosomes internalized by cholesterol dependent endocytosis pathway

(A) Jeko-1 cells were pre-treated with 10μM Gefitinib, 80μM Dynasore, or 40μg/ml Nystatin for 3h 30 min and 60 min, respectively. For cholesterol synthesis Jeko-1 cells were cultured in serum free RPMI media supplemented with 0.1% BSA and 20mM Hepes in the presence of 2uM Simvastatin for 16 h. PKH-67 labeled Jeko-1 exosomes were incubated with untreated (control) or treated cells for 60 min and internalization was measured by flow cytometry analysis. The data are expressed as the percent of cells that uptake labeled exosomes as mean ± SD of three independent experiments. (B) PKH-26 labeled Jeko-1 exosomes were incubated with untreated (control) or treated cells (Dynasore or Nystatin) and internalization was measured by confocal microscopy. Jeko-1 cell membrane was labeled with AlexaFluor 488-anti CD44. Scale bar is 25μm.