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. 2015 Aug 27;126(9):1106-17.
doi: 10.1182/blood-2014-12-618025. Epub 2015 Jun 22.

Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts

Jerome Paggetti  1 , Franziska Haderk  2 , Martina Seiffert  2 , Bassam Janji  1 , Ute Distler  3 , Wim Ammerlaan  4 , Yeoun Jin Kim  3 , Julien Adam  5 , Peter Lichter  2 , Eric Solary  6 , Guy Berchem  7 , Etienne Moussay  1
Affiliations

Exosomes released by chronic lymphocytic leukemia cells induce the transition of stromal cells into cancer-associated fibroblasts

Jerome Paggetti et al. Blood. .

Abstract

Exosomes derived from solid tumor cells are involved in immune suppression, angiogenesis, and metastasis, but the role of leukemia-derived exosomes has been less investigated. The pathogenesis of chronic lymphocytic leukemia (CLL) is stringently associated with a tumor-supportive microenvironment and a dysfunctional immune system. Here, we explore the role of CLL-derived exosomes in the cellular and molecular mechanisms by which malignant cells create this favorable surrounding. We show that CLL-derived exosomes are actively incorporated by endothelial and mesenchymal stem cells ex vivo and in vivo and that the transfer of exosomal protein and microRNA induces an inflammatory phenotype in the target cells, which resembles the phenotype of cancer-associated fibroblasts (CAFs). As a result, stromal cells show enhanced proliferation, migration, and secretion of inflammatory cytokines, contributing to a tumor-supportive microenvironment. Exosome uptake by endothelial cells increased angiogenesis ex vivo and in vivo, and coinjection of CLL-derived exosomes and CLL cells promoted tumor growth in immunodeficient mice. Finally, we detected α-smooth actin-positive stromal cells in lymph nodes of CLL patients. These findings demonstrate that CLL-derived exosomes actively promote disease progression by modulating several functions of surrounding stromal cells that acquire features of cancer-associated fibroblasts.

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Figures

Figure 1
Figure 1
CLL cells secrete exosomes that rapidly enter stromal cells in culture. (A) A 24-hour coculture assay of primary CLL cells (1 × 106 in upper compartment) and BM-MSCs (2 × 104) was established in 6-well plates containing 0.4-µm pore inserts. BM-MSCs were cultured in the absence (Ctrl) or presence (+CLL) of primary PKH67-labeled CLL cells (green). Images were captured by fluorescence confocal microscopy. Nuclei were stained with 4,6 diamidino-2-phenylindole (DAPI) (blue). Representative image of n = 6 experiments. Scale bar, 20 µm. (B) Electron microscopy image of purified CLL exosomes. Scale bar, 100 nm. (C) Western blot analysis of the fractions collected after sucrose density gradient of the 110 000g pellets obtained using ultracentrifugation of CLL cell supernatants. Positive control was CLL cell lysate. (D) Size analysis of CLL exosomes using tunable resistive pulse sensing (TRPS)-based analysis (qNano). (E) The BM-derived stromal cell line HS-5 and the endothelial cell line HMEC-1 were incubated with 50 µg/mL PKH67-labeled CLL exosomes (MEC-1) for the indicated times before 4 washes and fixation. (Upper) Exosome uptake was followed by fluorescence confocal microscopy. Scale bar, 20 µm. (Lower) Quantification of exosome uptake by ImageJ software. Data are presented as fold change relative to 0 hours (n = 3). (F) HS-5 and HMEC-1 cells were incubated for 4 hours in the absence (Ctrl) or presence of 20 µg/mL PKH67-labeled CLL exosomes (MEC-1) untreated (Exo) or pretreated for 30 minutes with 10 ng/mL heparin (Exo + H). (Left) Images were captured by fluorescence confocal microscopy. Scale bar, 50 µm. (Right) Quantification of exosome uptake by ImageJ software. Data are presented as fold change relative to Ctrl (n = 3). **P < .01, ***P < .001.
Figure 2
Figure 2
Characterization of CLL exosome RNA and transfer of functional miRNAs to target cells. (A) RNA was extracted from CLL cells and exosomes and analyzed using the Agilent Bioanalyzer RNA (left and middle) and small RNA (right) chips. (B and C) Small RNA next-generation sequencing of RNA purified from CLL cells and exosomes (MEC-1). (B) Percentages of the various small RNA categories identified in CLL cells and exosomes. (C) Percentages of the 10 most abundant miRNAs in CLL-exosomes. (D and E) Primary BM-MSCs from healthy donors (HD) were treated with 50 µg/mL CLL exosomes (MEC-1) for the indicated times, and specific miRNAs were quantified by qRT-PCR. Data are presented as fold change (FC) relative to untreated cells (Ctrl). (D) Kinetic quantification of miR-150 (n = 3). (E) Quantification of miR-146a, miR-150, and miR-155 after 72 hours. (F) Quantification of miR-146a and miR-150 in BM-MSCs (2 × 104) cocultured with primary CLL cells (1 × 106 in upper compartment) in 0.4-µm pore inserts for 24 hours by qRT-PCR. Data are presented as FC relative to BM-MSCs alone (Ctrl). (G) qRT-PCR quantification of miR-150 in BM-MSCs incubated with DMEM (Ctrl), DMEM supplemented with 20% fetal calf serum, HD, or CLL plasma for 3 hours (n = 3). (H) HMEC-1 cells were transfected with luciferase reporter plasmids carrying miR-146a or miR-150 antisense sequences (pmiR146aAS or pmiR150AS) and then cultured in the absence (Ctrl) or presence of MEC-1 exosomes (Exo) for 24 hours. Luciferase activity of reporter plasmid was quantified by measuring the light emission (RLU) of both luciferases in 4 replicates per condition. Data are presented as RLU ratio relative to Ctrl (n = 3). *P < .05, ***P < .001.
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).
Figure 4
Figure 4
CLL exosomes rapidly activate kinases and NF-κB in stromal cells. (A) Phospho-kinase antibody array performed on protein lysates from BM-MSCs and HMEC-1 cells either untreated (Ctrl) or treated for 1 hour with 50 µg/mL CLL exosomes (MEC-1; Exo) (left). Cell lysates were hybridized to membranes containing capture antibodies specific for phosphorylated kinases. Quantification of CLL exosome-induced phosphorylated proteins highlighted by red boxes in left panel (right). Data are reported as fold change (FC) relative to Ctrl. (B) Kinetic of CLL exosome-induced AKT phosphorylation in HS-5 and HMEC-1 cells by immunoblot analysis. ACTB was used as loading control (representative of n = 3). (C) HS-5 and HMEC-1 were preincubated for 30 minutes in the absence (−) or presence (+) of PI3K inhibitor wortmannin (Wort, 100 nM) or MEK inhibitor U0126 (10 µM) before culturing for 5 minutes in the absence (−) or presence (+) of 50 µg/mL CLL exosomes (MEC-1). Expression of AKT and ERK1/2 and their phosphorylated forms (p-AKT and p-ERK1/2) was analyzed by immunoblot. ACTB was used as loading control (representative of n = 3). (D) Immunoblot analysis of IKK-α/β and inhibitory NF-κBα phosphorylation in cell lysates of BM-MSCs and HMEC-1 untreated (Ctrl) or incubated with 50 µg/mL CLL exosomes (MEC-1) for the indicated periods of time. ACTB was used as loading control (representative of n = 3). (E) Representative images of nuclear translocation of p65 in exosome-treated cells.HS-5 and HMEC-1 cells were untreated (Ctrl) or treated with CLL exosomes (Exo; 50 µg/mL) for 1 or 2 hours. After fixation and permeabilization, cells were labeled with anti-p65 antibody (green) and DAPI (blue) and analyzed by confocal microscopy (n = 3). Scale bar, 10 µm.
Figure 5
Figure 5
CLL exosomes alter the transcriptome of stromal cells and induce the release of cytokines and proangiogenic factors. (A) Primary BM-MSCs from 2 healthy donors were untreated (Ctrl) or treated with 50 µg/mL CLL exosomes (MEC-1; Exo) for 6 hours, and gene expression was analyzed by microarrays. Functions (IPA) associated with modulated genes are indicated with their respective P values and numbers of associated molecules. Portion radii were calculated according to z score, reflecting activation of the function. (B) CAF signature of BM-MSCs incubated with CLL exosomes. Normalized gene expression values (in log2) were used for unsupervised hierarchical clustering using the TM4MeV software. (C) Unsupervised hierarchical clustering based on gene expression of selected candidates from Figure 5B and supplemental Figure 4A. BM-MSCs were incubated for 72 hours with exosomes produced by healthy donor B cells (B, n = 3), primary CLL cells (CLL, n = 3), or MEC-1 cells (M). qPCR data are reported as fold change (log2 FC) relative to untreated cells. (D) Unsupervised hierarchical clustering based on gene expression of selected candidates from Figure 5B and supplemental Figure 4A. BM-MSCs were cultured for 30 days and stimulated weekly with 50 µg/mL CLL exosomes (MEC-1; Exo) or cocultured with 1 × 106 primary CLL cells (CLL#7 and CLL#8) or Burkitt’s lymphoma Namalwa cell line (Nam) in culture inserts (0.4-µm pores). Medium and cells were changed twice weekly in the upper compartment. qPCR data are reported as fold change (log2 FC) relative to untreated cells. (E) Angiogenesis and cytokine antibody arrays used for the detection of soluble factors in the supernatants of untreated (Ctrl) or CLL exosome-treated (MEC-1; Exo; 50 µg/mL) BM-MSCs after 30 hours culture (left). Quantification of CLL exosome-modulated factors highlighted by red boxes in left panel (right). Data are reported as FC relative to Ctrl. (F) Immunoblot analysis of additional cytokines of interest not present in the arrays. Culture supernatants of BM-MSCs and HMEC-1 untreated (Ctrl) or treated with 50 µg/mL CLL exosomes (Exo) for 24 hours were concentrated and analyzed by immunoblot. (G) HMEC-1 and HS-5 cells incubated for 24 hours in absence (gray shade) or presence (black line) of 50 µg/mL CLL exosomes (MEC-1). Cells were then analyzed by flow cytometry with specific antibodies or isotype controls (dotted line) (representative of n = 3).
Figure 6
Figure 6
CLL exosomes promote cell proliferation, remodeling of the actin cytoskeleton, cell migration, and angiogenesis in vitro and in vivo. (A) Proliferation index of stromal cells after 96 hours of incubation with increasing concentrations of CLL exosomes (MEC-1) assessed using CCK8 assay. Data are reported as fold change (FC) of Ctrl (n = 4). *P < .05, **P < .01, ***P < .001. (B) Microscopy images of wound healing assay showing closure of the scratch when HS-5 or HMEC-1 cells were cultured in the absence (Ctrl) or presence (Exo) of 50 or 100 µg/mL CLL exosomes (MEC-1) in serum-free medium for 18 hours (left). Scale bar, 100 µm. Wound closure (×104 µm2) was quantified from images using WimScratch (Wimasis; n = 4) (right). **P < .01. (C) Representative images of immunofluorescence staining of α-SMA (green) in primary BM-MSCs untreated (Ctrl) or treated (Exo) for 15 days with exosomes produced by healthy donor B cells (B), lymphoblastoid cell line (ST-EAH), or primary CLL cells, captured by confocal microscopy (nucleus stained with DAPI, blue). Scale bar, 50 µm. (D) Representative images of immunohistochemistry staining of α-SMA in paraffin-embedded sections of human lymph nodes from 2 healthy individuals or 2 CLL patients (representative of n = 5). Scale bar, 50 µm. (E) (Upper) C57BL/6 mouse aorta pieces were incubated in vitro in the absence (Ctrl) or presence (Exo) of 100 µg/mL CLL exosomes (MEC-1) for 7 days. Representative microscopy images are shown. Scale bar, 100 µm. (Lower) Quantification of sprouts area and length using WimSprout (Wimasis; n = 4 replicates). **P < .01. (F) (Left) HMEC-1 cells untreated (Ctrl) or treated for 30 minutes with 50 or 100 µg/mL CLL exosomes (MEC-1; Exo) and then seeded on Matrigel for 3 hours. Scale bar, 100 µm. (Right) Quantification of several parameters of the tube formation assay using WimTube (Wimasis; n = 4). *P < .05, **P < .01, ***P < .001. (G) Matrigel plug assay performed by subcutaneous injection of Matrigel mixed with PBS (Ctrl) or 100 µg of CLL exosomes (MEC-1; Exo) in 5 NSG mice. rhIL-8 was used as positive control. (Left) Images depict surgically removed Matrigel plugs after 14 days. Scale bar, 5 mm. (Right) Quantification of hemoglobin content in Matrigel plugs using Drabkin reagent. Data are reported as FC of Ctrl (n = 5). ***P < .001.
Figure 7
Figure 7
CLL exosomes increase CLL cell adhesion and survival in vitro and promote tumor growth in vivo. (A) Primary CLL cells labeled with PKH67 dye were added for 3 hours to cultures of HS-5 or HMEC-1 cells untreated (Ctrl) or pretreated 24 hours with 50 µg/mL of CLL exosomes (MEC-1; Exo). After 5 washes, images were taken via fluorescence microscopy. (Left) Representative images are shown. Scale bar, 100 µm. (Right) Quantification of CLL cell adhesion (n = 3). *P < .05, **P < .01. (B) CLL cells incubated with supernatants of BM-MSCs untreated (SN Ctrl) or treated for 24 hours with 50 µg/mL CLL exosomes (SN Exo). Cell viability was assessed after 6 days using CCK8 assay. Data are reported as the percentage relative to SN Ctrl (n = 3). **P < .01. (C) Primary CLL cells were treated with indicated cytokines (10 ng/mL) and viability was assessed after 6 days using CCK8 assay. Data are reported as the percentage relative to Ctrl (n = 9). *P < .05, **P < .01, ***P < .001. (D and E) PBS (Ctrl) or 100 µg CLL exosomes (Exo) were mixed with 5 × 106 MEC-1-eGFP cells and subcutaneously injected into eight NSG mice. (D) (Left) Representative images of subcutaneous tumors removed from NSG mice. Scale bar, 5 mm. (Right) Quantification of tumor volume in mm3. *P < .05. (E) (Left) Representative images of kidneys (NSG mice). Scale bar, 5 mm. (Center) Representative flow cytometry plots showing MEC-1-eGFP cells (black gate) in kidneys. (Right) Quantification of GFP positive cells in kidneys.*P < .05.
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