Journal: Molecular Therapy. Nucleic Acids

Article Title: Targeting EGFR/HER2/HER3 with a Three-in-One Aptamer-siRNA Chimera Confers Superior Activity against HER2 + Breast Cancer

doi: 10.1016/j.omtn.2017.12.015

Figure Lengend Snippet: Schematic Illustration of HER2 Aptamer-EGFR siRNA-HER3 Aptamer, H2EH3, and Characterization of H2EH3 (A) Structure of H2EH3. HER2 aptamer was conjugated with HER3 aptamer through 21 bases of EGFR siRNA and 2–4 unpaired base linkers. (B) Western blot detection of the expression levels of HER2, HER3, and EGFR in a panel of breast cancer cell lines. (C) Evaluation of the cytotoxicity of H2EH3 by CCK-8 assay. Breast cancer cell lines, including BT474, SKBR3, MCF7, MDA-MB-231, and Hs587T cells, were treated with varying concentrations of H2EH3 or controls for 72 hr, and cell viability was detected with the CCK-8 agent. Data are the mean ± SD from three independent experiments. (D) Evaluation of EGFR-silencing capability of H2EH3 using western blot. (E) Evaluation of H2EH3-binding capability compared with HER2 aptamer and HER3 aptamer.

Article Snippet: 2′-Fluoro-2′-deoxycytidine-5′-triphosphate, 2′-fluoro-2′-deoxyuridine-5′-triphosphate, and Cy5-labeled 2′-fluoro-labeled HER2 aptamer were purchased from TriLink Biotechnologies (San Diego, CA).

Techniques: Western Blot, Expressing, CCK-8 Assay, Binding Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Targeting EGFR/HER2/HER3 with a Three-in-One Aptamer-siRNA Chimera Confers Superior Activity against HER2 + Breast Cancer

doi: 10.1016/j.omtn.2017.12.015

Figure Lengend Snippet: Analysis of Cell Cycle and Apoptosis (A) Effects of H2EH3 on cell cycle progression of HER2 + HER3 + cells. SKBR3 and BT474 cells were treated with H2EH3 (1 μM, 2 μM) for 24 and 48 hr. The cell cycle was then analyzed by flow cytometry. (B) Quantitative analysis of apoptosis after H2EH3 treatment by flow cytometry. BT474 and SKBR3 cells were treated with the different concentrations of H2EH3 for 72 hr, and cells were stained with Alexa Fluor 488 annexin-V-propidium iodide and analyzed by flow cytometry.

Article Snippet: 2′-Fluoro-2′-deoxycytidine-5′-triphosphate, 2′-fluoro-2′-deoxyuridine-5′-triphosphate, and Cy5-labeled 2′-fluoro-labeled HER2 aptamer were purchased from TriLink Biotechnologies (San Diego, CA).

Techniques: Flow Cytometry, Staining

Journal: Molecular Therapy. Nucleic Acids

Article Title: Targeting EGFR/HER2/HER3 with a Three-in-One Aptamer-siRNA Chimera Confers Superior Activity against HER2 + Breast Cancer

doi: 10.1016/j.omtn.2017.12.015

Figure Lengend Snippet: Evaluation of Binding Specificity and In Vivo Bio-distribution of H2EH3 (A) Western blot analysis of HER2 and/or HER3 gene knockdown. (B) Flow cytometry to quantitatively measure cell binding of H2EH3 upon HER2 and/or HER3 knockdown in BT474 cells. Untreated: solid gray; mock transfected cells: light blue; normal cells stained with H2EH3-Cy5: red line; control siRNA transfected cells stained with H2EH3: green line, HER2-silenced cells stained with H2EH3-Cy5: purple line; HER3-silenced cells stained with H2EH3-Cy5: black line; both HER2- and HER3-silenced cells stained with H2EH3: blue line. (C) Time course whole-body imaging to show the binding profile of H2EH3. Tumor-bearing mice were intravenously injected with Cy5-H2EH3 or non-targeting control aptamer. Cy5 fluorescence of whole body was captured at the time points of 2 and 6 hr with the Xenogen IVIS100 imaging system. (D) Ex vivo organ imaging. Following whole-body imaging, major organs were removed and detected with the Xenogen IVIS100 imaging system. The result is the representative of two independent experiments.

Article Snippet: 2′-Fluoro-2′-deoxycytidine-5′-triphosphate, 2′-fluoro-2′-deoxyuridine-5′-triphosphate, and Cy5-labeled 2′-fluoro-labeled HER2 aptamer were purchased from TriLink Biotechnologies (San Diego, CA).

Techniques: Binding Assay, In Vivo, Western Blot, Flow Cytometry, Transfection, Staining, Imaging, Injection, Fluorescence, Ex Vivo

Journal: Molecular Therapy. Nucleic Acids

Article Title: Targeting EGFR/HER2/HER3 with a Three-in-One Aptamer-siRNA Chimera Confers Superior Activity against HER2 + Breast Cancer

doi: 10.1016/j.omtn.2017.12.015

Figure Lengend Snippet: H2EH3 Significantly Suppresses Tumor Growth in Breast Cancer Xenografts (A) Inhibition of tumor growth by H2EH3 through intratumoral injection. Mice with subcutaneous tumors were intratumorally injected with H2EH3, a mixture of HER2 aptamer, HER3 aptamer, and EGFR siRNA, or PBS every other day for 5 weeks (n = 5 per group). (B) Dissected tumors after treatment though intratumoral injection (n = 5). (C) Quantitation of dissected tumor sizes from (B) (n = 5). (D) Body weight of mice treated with H2EH3 through intratumoral injection (n = 5). (E) Inhibition of tumor growth by H2EH3 through intravenous injection. Mice with orthotopic tumors were intratumorally injected with H2EH3, a mixture of HER2 aptamer, HER3 aptamer, and EGFR siRNA, or PBS every 3 days for 4 weeks (n = 4). (F) Dissected tumors after treatment through intravenous injection (n = 4). (G) Quantitation of dissected tumor sizes from (F) (n = 4). (H) Body weight of mice treated with H2EH3 through intravenous injection (n = 4). *p < 0.05; **p < 0.005. Data represent the mean ± SEM; n = 4–5 for each group.

Article Snippet: 2′-Fluoro-2′-deoxycytidine-5′-triphosphate, 2′-fluoro-2′-deoxyuridine-5′-triphosphate, and Cy5-labeled 2′-fluoro-labeled HER2 aptamer were purchased from TriLink Biotechnologies (San Diego, CA).

Techniques: Inhibition, Injection, Quantitation Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Targeting EGFR/HER2/HER3 with a Three-in-One Aptamer-siRNA Chimera Confers Superior Activity against HER2 + Breast Cancer

doi: 10.1016/j.omtn.2017.12.015

Figure Lengend Snippet: Evaluation of Gene Expression and Apoptosis after H2EH3 Treatment In Vivo Formalin-fixed paraffin-embedded sections of xenograft tumors were stained with antibodies targeting EGFR, HER2, HER3, P21, and cleaved caspase-3. (A) IHC analysis of subcutaneous tumors after intratumoral injection of H2EH3. (B) IHC analysis of orthotopic tumors after intravenous injection of H2EH3. Scale bar, 50 μm.

Article Snippet: 2′-Fluoro-2′-deoxycytidine-5′-triphosphate, 2′-fluoro-2′-deoxyuridine-5′-triphosphate, and Cy5-labeled 2′-fluoro-labeled HER2 aptamer were purchased from TriLink Biotechnologies (San Diego, CA).

Techniques: Expressing, In Vivo, Formalin-fixed Paraffin-Embedded, Staining, Injection