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).

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).

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