Journal: Clinical and Translational Medicine

Article Title: Targeting capacity, safety and efficacy of engineered extracellular vesicles delivered by transdermal microneedles to treat plasmacytoma in mice

doi: 10.1002/ctm2.70327

Figure Lengend Snippet: CD38‐EVs targeting tumour cells in vitro. PKH26‐labelled extracellular vesicles (EVs) and CD38‐EVs incubated with RPMI8226 cells (A) and U266 cells (B) for 4, 8 and 12 h, with cell fluorescence measured by flow cytometry (FCM). Kinetics of uptake of EVs and CD38‐EVs within 12 h by RPMI8226 cells (C) and U266 cells (D), measured by relative fluorescence units (RFU). (E) RPMI8226 and U266 cells were incubated with EVs and CD38‐EVs for 8 h and visualised using laser scanning confocal microscope (LSCM) (scale bars: 5 µm). PKH26‐labelled EVs and CD38‐EVs incubated with RPMI8226/HUVECs and RPMI8226/BMSCs (F) and U266/HUVECs and U266/BMSCs (G) co‐culture systems for 12 h, with cell fluorescence measured by FCM. Kinetics of uptake of EVs and CD38‐EVs by myeloma cells, human umbilical vein endothelial cells (HUVECs) and bone marrow stromal/stem cells (BMSCs) in RPMI8226/HUVECs and RPMI8226/BMSCs (H) and U266/HUVECs and U266/BMSCs (I) co‐culture systems, measured by RFU. Data are generated from three independent experiments.

Article Snippet: The HUVECs cell line was grown in ECM medium containing an endothelial cell growth medium bullet kit (Sciencell).

Techniques: In Vitro, Incubation, Fluorescence, Flow Cytometry, Microscopy, Co-Culture Assay, Generated

Journal: The Journal of Biological Chemistry

Article Title: Annexin A2 Is a Molecular Target for TM601, a Peptide with Tumor-targeting and Anti-angiogenic Effects

doi: 10.1074/jbc.M109.066092

Figure Lengend Snippet: Specific binding of TM601 to the cell surface of tumor and vascular endothelial cells. Cell surface binding of TM601 was quantified by measuring the amount of technetium-99m-labeled TM601 bound to the cell surface at 4 °C in the presence of unlabeled TM601 monomer used as competitor. TM601 binds the surface of multiple tumor cells with a high affinity for U87-MG glioma (A) and A549 lung carcinoma (B) and lower affinity for Panc-1 pancreatic carcinoma (C) cell lines. TM601 binds HUVECs (D) in culture with an affinity comparable with Panc-1 cells and shows little surface binding to either normal human astrocytes (E) or normal human dermal fibroblasts (F).

Article Snippet: HUVECs were cultured in vascular endothelial cell medium supplemented with vascular endothelial cell growth supplement, 5% FBS, penicillin, and streptomycin (ScienCell).

Techniques: Binding Assay, Labeling

Journal: The Journal of Biological Chemistry

Article Title: Annexin A2 Is a Molecular Target for TM601, a Peptide with Tumor-targeting and Anti-angiogenic Effects

doi: 10.1074/jbc.M109.066092

Figure Lengend Snippet: TM602 binds annexin A2 expressed on the surface of multiple tumor and vascular endothelial cells. Confluent monolayers of A549, Panc-1, U87-MG, and HUVECs treated with or without trypsin were surface-biotinylated, and biotinylated proteins were isolated using neutrAvidinTM beads. The levels of annexin A2 expressed on the surface of tumor cell lines and HUVECs and the sensitivity to trypsin were determined using an anti-annexin A2 Western blot. A, annexin A2 levels at the cell surface and in total cell lysates for plate-bound (lanes 1, 3, 5, and 7) and trypsinized (lanes 2, 4, 6, and 8) A549 (lanes 1, 2, 5, and 6) and Panc-1 (lanes 3, 4, 7, and 8) cells. B, annexin A2 levels at the cell surface and in total cell lysates for plate-bound (lanes 1, 3, 5, and 7) and trypsinized (lanes 2, 4, 6, and 8) U87-MG (lanes 1, 2, 5, and 6) glioma and HUVECs (lanes 3, 4, 7, and 8). Annexin A2 is expressed at the cell surface in all the aforementioned cell lines. In HUVECs, surface annexin A2 is sensitive to trypsinization (B, lanes 3 and 4). C and D, the association of TM602 with cell surface annexin A2 was validated using an anti-annexin A2 Western blot of neutrAvidinTM pulldowns of lysates from A549, Panc-1, and HUVECs following TM602 surface cross-linking. Annexin A2 specifically associates with TM602 at the cell surface of A549 and Panc-1 tumor cells (C) as well as HUVECs (D).

Article Snippet: HUVECs were cultured in vascular endothelial cell medium supplemented with vascular endothelial cell growth supplement, 5% FBS, penicillin, and streptomycin (ScienCell).

Techniques: Isolation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Annexin A2 Is a Molecular Target for TM601, a Peptide with Tumor-targeting and Anti-angiogenic Effects

doi: 10.1074/jbc.M109.066092

Figure Lengend Snippet: TM601 inhibits VEGF-stimulated tPA activity in human vascular endothelial cell supernatants. The effect of TM601 on VEGF- and bFGF-stimulated activation of secreted tPA in HUVECs was measured. Both VEGF- and bFGF-stimulated tPA activity in serum-starved HUVECs and TM601 treatment significantly decreased the amount of active tPA present in the supernatants of vascular endothelial cells treated with either VEGF or bFGF. *, p < 0.05. Bar, S.D.

Article Snippet: HUVECs were cultured in vascular endothelial cell medium supplemented with vascular endothelial cell growth supplement, 5% FBS, penicillin, and streptomycin (ScienCell).

Techniques: Activity Assay, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Annexin A2 Is a Molecular Target for TM601, a Peptide with Tumor-targeting and Anti-angiogenic Effects

doi: 10.1074/jbc.M109.066092

Figure Lengend Snippet: TM601 inhibits PDGF-CC-induced U373-MG glioma and HUVEC migration in trans-well chamber assays. A, in a trans-well assay using HUVEC or U373-MG cells, PDGF-CC strongly stimulates cell migration, and TM601 significantly inhibits the PDGF-CC-stimulated migration of both vascular endothelial cells and U373-MG glioma cells. *, p < 0.05. Bar, S.D. B, functional importance of annexin A2 in PDGF-CC-stimulated glioma migration was tested using an annexin A2 siRNA knockdown approach. Mock-transfected wild-type U373-MG and annexin A2 knockdown U373-MG glioma cells were used in a trans-well assay testing migration stimulated by PDGF-CC. Wild-type cells demonstrated significant migration stimulated by PDGF-CC that was inhibited by TM601 treatment. Annexin A2 knockdown cells demonstrated very weak migration stimulated by PDGF-CC that was completely abolished by a lower dose of TM601 (25 μm) as compared with wild-type cells. C, Western blot using anti-annexin A2 antibody demonstrates the levels of annexin A2 after annexin A2 siRNA treatment.

Article Snippet: HUVECs were cultured in vascular endothelial cell medium supplemented with vascular endothelial cell growth supplement, 5% FBS, penicillin, and streptomycin (ScienCell).

Techniques: Migration, Functional Assay, Knockdown, Transfection, Western Blot