Journal: Scientific Reports

Article Title: BDNF belongs to the nurse-like cell secretome and supports survival of B chronic lymphocytic leukemia cells

doi: 10.1038/s41598-020-69307-1

Figure Lengend Snippet: NLC secrete BDNF, thereby protecting B-CLL cells from apoptosis. ( a ) Representative western blots showing expression of BDNF in lysates of NLC from two independent patient samples (n = 5). ( b ) Immunofluorescence analysis of BDNF (red) expression in NLC by confocal microscopy. ( c ) Relative expression of BDNF mRNA by normal healthy monocytes (n = 6) and NLC isolated from B-CLL patients (n = 6), as determined by RT-qPCR. ( d ) Representative western blots showing BDNF expression in supernatants of two independent NLC culture (n = 6). ( e ) Representative western blots showing BDNF expression by B-CLL cells cultured alone, with autologous NLC, or with autologous NLC plus an anti-BDNF blocking antibody (anti-hBDNF; 200 ng/mL) for 48 h. The three conditions for each patient assessed on the same western blot, which has been cropped to present only relevant data. The uncropped western blot membranes are shown in Supplementary Fig. ( f ) Quantification of BDNF protein from independent patient samples (n = 5). ( g ) Relative expression of BDNF mRNA by B-CLL cells cultured alone or with autologous NLC for 48 h, as determined by RT-qPCR (n = 7). ( h ) Flow cytometry analysis of cell death, as assessed by annexin V-fluorescein isothiocyanate/propidium iodide dual staining of B-CLL cells cultured for 72 h either alone, with autologous NLC, or with autologous NLC plus an anti-BDNF antibody (200 ng/mL). Cell death was assessed by excluding annexin V/propidium iodide-negative cells. Experiments were performed using n = 9 patient samples. Data are presented as the mean ± SEM from at least three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001). ns not significant. Blots are cropped for clarity; full-length blots are shown in the Supplementary Fig. .

Article Snippet: BDNF was neutralized using an anti-BDNF mouse monoclonal antibody (200 ng/mL; #GF35L, Merck Millipore).

Techniques: Western Blot, Expressing, Immunofluorescence, Confocal Microscopy, Isolation, Quantitative RT-PCR, Cell Culture, Blocking Assay, Flow Cytometry, Staining

Journal: Scientific Reports

Article Title: BDNF belongs to the nurse-like cell secretome and supports survival of B chronic lymphocytic leukemia cells

doi: 10.1038/s41598-020-69307-1

Figure Lengend Snippet: The effects of BDNF on B-CLL pro-survival signaling are similar to those of BAFF, APRIL, and CXCL12 combined. ( a ) Representative western blot showing expression of NTSR2, p-Src, Src, and Bcl-2 by B-CLL cells isolated from patients. Cells were either co-cultured for 48 h with autologous NLC or cultured alone in complete medium supplemented (as indicated) with exogenous human (h) CXCL12 (100 ng/mL), BAFF (2 ng/mL), APRIL (25 ng/mL), or BDNF (100 ng/mL). (b,c,d), full-length blots are shown in the Supplementary Fig. . Quantification of NTSR2 ( b ), p-Src ( c ), and Bcl-2 ( d ) expression in four different patient samples. Data are presented as the mean ± SEM from at least three independent experiments, (* p < 0.05, ** p < 0.01). Blots are cropped for clarity; full blots are shown in the Supplementary Fig. .

Article Snippet: BDNF was neutralized using an anti-BDNF mouse monoclonal antibody (200 ng/mL; #GF35L, Merck Millipore).

Techniques: Western Blot, Expressing, Isolation, Cell Culture

Journal: Scientific Reports

Article Title: BDNF belongs to the nurse-like cell secretome and supports survival of B chronic lymphocytic leukemia cells

doi: 10.1038/s41598-020-69307-1

Figure Lengend Snippet: Inhibiting BDNF in addition to BAFF, APRIL, and CXCR4 reverses NLC-mediated protection of B-CLL cells from apoptosis. ( a ) Representative western blot showing expression of p-Src and Bcl-2 by B-CLL cells isolated from patients. Cells were cultured (for 72 h) alone, with autologous NLC, or with autologous NLC plus single or combined inhibition of BAFF (anti-hBAFF, 100 ng/mL), APRIL (anti-hAPRIL, 500 ng/mL), CXCL12 receptor CXCR4 (AMD3100, 0.5 µg/mL), and BDNF (anti-hBDNF, 200 ng/mL). ( b – d ), full-length blots are shown in the Supplementary Fig. .Quantification of NTSR2 ( b ), p-Src ( c ) and Bcl-2 ( d ) expression in six independent experiments using six different patient samples. ( e ) Flow cytometry analysis of cell death, assessed by annexin V-fluorescein isothiocyanate/propidium iodide dual staining of B-CLL cells cultured (for 72 h) either alone, with autologous NLC, or with autologous NLC and single or combined inhibition of BAFF (anti-hBAFF, 100 ng/mL), APRIL (anti-hAPRIL, 500 ng/mL), CXCL12 receptor CXCR4 inhibition (AMD3100, 0.5 µg/mL), and BDNF (anti-hBDNF, 200 ng/mL). Cell death was assessed by exclusion of annexin V/propidium iodide-negative cells. Experiments were performed using n = 9 different patient samples. Data are presented as the mean ± SEM from at least three independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001). ns not significant. ( f ) Schematic representation of the obtained results. NLC produce and secrete BDNF, which promotes survival of B-CLL cells by activating the Src signaling pathway and upregulating expression of Bcl-2. This newly described member of the NLC secretome appears to exert both complementary (alongside BAFF, APRIL and CXCL12) and independent effects. Here, we propose a model in which BDNF or pro-survival cytokines secreted by NLC within survival centers balance each other out to facilitate survival of B-CLL cells, and argue that simultaneous inhibition of BDNF signaling through the NTSR2-TrkB conditional oncogenic platform, along with inhibition of BAFF, APRIL and CXCR4/CXCL12, could cancel out NLC-mediated protection of B-CLL cells from apoptosis and restore normal cell death.

Article Snippet: BDNF was neutralized using an anti-BDNF mouse monoclonal antibody (200 ng/mL; #GF35L, Merck Millipore).

Techniques: Western Blot, Expressing, Isolation, Cell Culture, Inhibition, Flow Cytometry, Staining