Figure 4.
Features and functions of EVs derived from TP53-deficient and control lymphoma B cells. (A-G) EVs were isolated from TP53-deficient (shTP53-EVs) and control (shCTRL-EVs) hMB lymphoma B-cells. (A) Representative TEM images. Thirty thousand and 100 000 magnifications are shown. (B) Nanoparticle tracking analysis (NTA) of the EVs distribution of the particles according to their size and concentration (upper panel). Box plots showing size particle and particle concentration of the isolated EVs (lower panel; n = 6). (C) Immunoblot analysis of the isolated EVs and their corresponding cell lysates using the indicated antibodies. (D) Confocal image showing DiD-labeled EV (red) uptake by GFP+J774A.1 macrophages (green) after 16 hours in culture. Orthogonal view of the same image confirms the intracellular presence of the EVs (right panel). (E) Alemtuzumab-mediated ADCP of control hMB cells cocultured with J774A.1 macrophages and shCTRL-EVs, shTP53-EVs, or vehicle (PBS) (n = 4). (F-I) RAB27A expression was depleted by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) approached in shTP53 hMB cells. (F) Immunoblotting detection of Rab27a protein in shTP53/RAB27A-WT and -KO hMB cells. The corresponding total protein staining of the samples as a protein loading control is shown. (G) Box plots of NTA determinations (left and middle panel) and protein concentration (right panel) of EVs obtained from shTP53/RAB27A-WT and -KO EVs (n = 3). (H) Alemtuzumab-mediated ADCP of different clones of both, shTP53/RAB27A-WT (n = 4) and –KO (n = 3) hMB cells cocultured with J774A.1 macrophages. All the graphics showed the mean plus or minus SD of at least 3 independent experiments. (I) Kaplan-Meier analysis comparing the survival of shTP53/RAB27A-WT and -KO hMB transplanted NSG mice receiving cyclophosphamide and alemtuzumab (CA) as CIT combination. PBS was used as control. The treatment was given IP 10 days after IV hMB cell injection (n = 7-10). (J) Protein determination of EVs derived from CD19+cells isolated from the spleen of sick Eµ-TCL1 WT and Eµ-TCL1/Tp53fl/fl mice (n = 5-8). (K) Concentration of EVs isolated from primary CLL patient cells (n = 2-3) (*P < .05, **P ≤ .01). IP, intraperitoneal; SD, standard deviation.

Features and functions of EVs derived from TP53-deficient and control lymphoma B cells. (A-G) EVs were isolated from TP53-deficient (shTP53-EVs) and control (shCTRL-EVs) hMB lymphoma B-cells. (A) Representative TEM images. Thirty thousand and 100 000 magnifications are shown. (B) Nanoparticle tracking analysis (NTA) of the EVs distribution of the particles according to their size and concentration (upper panel). Box plots showing size particle and particle concentration of the isolated EVs (lower panel; n = 6). (C) Immunoblot analysis of the isolated EVs and their corresponding cell lysates using the indicated antibodies. (D) Confocal image showing DiD-labeled EV (red) uptake by GFP+J774A.1 macrophages (green) after 16 hours in culture. Orthogonal view of the same image confirms the intracellular presence of the EVs (right panel). (E) Alemtuzumab-mediated ADCP of control hMB cells cocultured with J774A.1 macrophages and shCTRL-EVs, shTP53-EVs, or vehicle (PBS) (n = 4). (F-I) RAB27A expression was depleted by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) approached in shTP53 hMB cells. (F) Immunoblotting detection of Rab27a protein in shTP53/RAB27A-WT and -KO hMB cells. The corresponding total protein staining of the samples as a protein loading control is shown. (G) Box plots of NTA determinations (left and middle panel) and protein concentration (right panel) of EVs obtained from shTP53/RAB27A-WT and -KO EVs (n = 3). (H) Alemtuzumab-mediated ADCP of different clones of both, shTP53/RAB27A-WT (n = 4) and –KO (n = 3) hMB cells cocultured with J774A.1 macrophages. All the graphics showed the mean plus or minus SD of at least 3 independent experiments. (I) Kaplan-Meier analysis comparing the survival of shTP53/RAB27A-WT and -KO hMB transplanted NSG mice receiving cyclophosphamide and alemtuzumab (CA) as CIT combination. PBS was used as control. The treatment was given IP 10 days after IV hMB cell injection (n = 7-10). (J) Protein determination of EVs derived from CD19+cells isolated from the spleen of sick Eµ-TCL1 WT and Eµ-TCL1/Tp53fl/fl mice (n = 5-8). (K) Concentration of EVs isolated from primary CLL patient cells (n = 2-3) (*P < .05, **P ≤ .01). IP, intraperitoneal; SD, standard deviation.

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