Journal: International Journal of Molecular Sciences

Article Title: The Impact of Small Extracellular Vesicles on Lymphoblast Trafficking across the Blood-Cerebrospinal Fluid Barrier In Vitro

doi: 10.3390/ijms21155491

Figure Lengend Snippet: Time- and dose-dependent uptake/binding of acute lymphoblastic leukemia (ALL) cell-derived exosomes to blood-cerebrospinal fluid barrier (BCSFB) in vitro model. Fluorescently labeled exosomes were added onto the basolateral side of HiBCPP cells grown in inverted culture as indicated and uptaken/bound exosomes were quantified by fluorescent microscopy. Relative fluorescence intensity (RFI) was measured in 15 fields of view (20× objective). ( A ) Representative observation field of untreated control HiBCPP cells and HiBCPP cells incubated with DiIC18(3) labeled exosomes (red) (16 μg, 48 h). HiBCPP cells were visualized by nuclei staining (DAPI, blue). ( B ) Time-dependent uptake/binding of ALL-derived exosomes (16 μg per filter; SD-1, Nalm-6, and P12, respectively) for indicated time points from basolateral side, and ( C ) dose-dependent uptake/binding of ALL-derived exosomes (48 h; SD-1, Nalm-6, and P12, respectively) for indicated amounts per filter from basolateral side. ( D ) Barrier integrity following exosome incubation was determined by transepithelial electrical resistance (TEER) and showed no significant alterations compared to untreated control. All data shown are box plots with whiskers of at least 3 independent experiments performed in triplicates. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Where indicated, the HiBCPP cells of BCSFB in vitro model were pretreated with chemical inhibitors (Dynasore, 80 μM, Merck Millipore, Darmstadt, Germany; Heparin, 10 μg/mL, Sigma Aldrich, Steinheim, Germany; Cytochalasin D, 5 μg/mL, Sigma Aldrich, Steinheim, Germany; Chlorpromazine, 50 μM, Sigma Aldrich, Steinheim, Germany) for 1 h. After removal of inhibitors, 16 μg of fluorescently labeled exosomes were added onto the basolateral side of HiBCPP cells and incubated for 48 h. Where indicated, the fluorescently labeled exosomes were pretreated with 4 μg/mL anti-integrin-antibody (anti-ITGα5, Immunotools, Friesoythe, Germany; anti-ITGαV Immunotools, Friesoythe, Germany; anti-ITGβ1, Merck Millipore, Darmstadt, Germany; anti-ITGβ3, BD Bioscience, San Jose, CA, USA) for 30 min at 37 °C before incubation for 48 h on HiBCPP cells.

Techniques: Binding Assay, Derivative Assay, In Vitro, Labeling, Microscopy, Fluorescence, Control, Incubation, Staining

Journal: International Journal of Molecular Sciences

Article Title: The Impact of Small Extracellular Vesicles on Lymphoblast Trafficking across the Blood-Cerebrospinal Fluid Barrier In Vitro

doi: 10.3390/ijms21155491

Figure Lengend Snippet: Mechanism of uptake/binding of acute lymphoblastic leukemia (ALL) cell-derived exosomes to blood-cerebrospinal fluid barrier (BCSFB) in vitro model. DiIC18(3) labeled exosomes (16 µg per filter) were added onto the basolateral side of HiBCPP cells grown in inverted culture and incubated for 48 h. The nuclei were stained with DAPI solution. Relative fluorescence intensity (RFI) of exosomes was quantified by fluorescent microscopy (15 fields of view, 20× objective). ( A ) Fluorescently labeled exosomes were pretreated with 4 µg/mL anti-integrin-antibody (anti-ITGα5; anti-ITGαV; anti-ITGβ1; anti-ITGβ3) for 30 min at 37 °C before incubation on HiBCPP cells. ( B ) HiBCPP cells were pretreated with chemical inhibitors (10 µg/mL heparin; 5 μg/mL cytochalasin D; 50 µM chlorpromazine; 80 µM dynasore) for 1 h at 37 °C before incubation. HiBCPP cells were washed with acidic PBS (pH = 2.5) after incubation to strip off adherend exosomes. Data shown are box plots with whiskers of at least 3 independent experiments performed in triplicates. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Where indicated, the HiBCPP cells of BCSFB in vitro model were pretreated with chemical inhibitors (Dynasore, 80 μM, Merck Millipore, Darmstadt, Germany; Heparin, 10 μg/mL, Sigma Aldrich, Steinheim, Germany; Cytochalasin D, 5 μg/mL, Sigma Aldrich, Steinheim, Germany; Chlorpromazine, 50 μM, Sigma Aldrich, Steinheim, Germany) for 1 h. After removal of inhibitors, 16 μg of fluorescently labeled exosomes were added onto the basolateral side of HiBCPP cells and incubated for 48 h. Where indicated, the fluorescently labeled exosomes were pretreated with 4 μg/mL anti-integrin-antibody (anti-ITGα5, Immunotools, Friesoythe, Germany; anti-ITGαV Immunotools, Friesoythe, Germany; anti-ITGβ1, Merck Millipore, Darmstadt, Germany; anti-ITGβ3, BD Bioscience, San Jose, CA, USA) for 30 min at 37 °C before incubation for 48 h on HiBCPP cells.

Techniques: Binding Assay, Derivative Assay, In Vitro, Labeling, Incubation, Staining, Fluorescence, Microscopy, Stripping Membranes

Journal: International Journal of Molecular Sciences

Article Title: The Impact of Small Extracellular Vesicles on Lymphoblast Trafficking across the Blood-Cerebrospinal Fluid Barrier In Vitro

doi: 10.3390/ijms21155491

Figure Lengend Snippet: Exosomal pre-incubation enhances transmigration across blood-cerebrospinal fluid barrier (BCSFB) in vitro model of T-ALL cell line P12 but not of B-cell precursor (BCP)-ALL cell lines in time-dependent manner. CellTracker™ Green labeled ALL cells were added for 6 h onto the basolateral side of untreated HiBCPP cells or HiBCPP cells pre-incubated with 16 μg unlabeled exosomes for indicated time-points. C-X-C motif chemokine ligand 12 (CXCL12) (100 ng/mL) was used as chemoattractant. Migrated ALL cells (SD-1, Nalm-6, and P12, respectively) were quantified in the lower compartment by fluorescent microscopy (10 fields of view, 10× objective). Data shown are box plots with whiskers of at least 3 independent experiments performed in triplicates. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: Where indicated, the HiBCPP cells of BCSFB in vitro model were pretreated with chemical inhibitors (Dynasore, 80 μM, Merck Millipore, Darmstadt, Germany; Heparin, 10 μg/mL, Sigma Aldrich, Steinheim, Germany; Cytochalasin D, 5 μg/mL, Sigma Aldrich, Steinheim, Germany; Chlorpromazine, 50 μM, Sigma Aldrich, Steinheim, Germany) for 1 h. After removal of inhibitors, 16 μg of fluorescently labeled exosomes were added onto the basolateral side of HiBCPP cells and incubated for 48 h. Where indicated, the fluorescently labeled exosomes were pretreated with 4 μg/mL anti-integrin-antibody (anti-ITGα5, Immunotools, Friesoythe, Germany; anti-ITGαV Immunotools, Friesoythe, Germany; anti-ITGβ1, Merck Millipore, Darmstadt, Germany; anti-ITGβ3, BD Bioscience, San Jose, CA, USA) for 30 min at 37 °C before incubation for 48 h on HiBCPP cells.

Techniques: Incubation, Transmigration Assay, In Vitro, Labeling, Microscopy

Journal: BMC Cancer

Article Title: Unveiling the ITGβ1-CLIC1 axis: a new frontier in oral squamous cell carcinoma progression and immune evasion: insights from the tumor-mimetic ECM system

doi: 10.1186/s12885-025-15402-3

Figure Lengend Snippet: Analysis of clinical samples and data from OSCC. A Patients screening process. B Baseline characteristics of patients. C Correlation between ITGβ1 and CLIC1 expression and stage. D Recurrence-free survival analysis

Article Snippet: Specific primary antibodies were used to detect the target proteins: ITGβ1 (1:1000; Wanleibio, Shenyang, China; Rabbit anti-human) and CLIC1 (1:1000; Wanleibio, Shenyang, China; Rabbit anti-human), followed by incubation with secondary antibody (Goat anti-rabbit).

Techniques: Expressing