Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting and suppression of HER3-positive breast cancer by T lymphocytes expressing a heregulin chimeric antigen receptor

doi: 10.1007/s00262-017-2089-5

Figure Lengend Snippet: Construction and transient expression of HRG1β-CAR in HEK-293T cells. a Lentiviral vector construct of HRG1β-CAR. SP signal peptide, ECD extracellular domain, TM transmembrane portion, ICP intracellular portion. b Agarose gel electrophoresis of PCR and restricted DNA products. Left M, 1 kb DNA marker, right M, DL5000 DNA marker, lane 1, two restricted DNA products: pLenti6.3/V5-DEST plasmid (9387 bp) and HRG1β-CAR gene(1312 bp), lane 3, two restricted DNA products: pENTR3C plasmid (2723 bp) and HRG1β-CAR gene(1312 bp), Lane 2 and 4, PCR product: HRG1β-CAR gene (1312 bp). c HRG1 was detected by flow cytometry in HEK-293T cells transduced with HRG1β-CAR. d CD3ζ was detected by western blotting in HEK-293T cells transduced with HRG1β-CAR. Data are representative images of 3 independent experiments

Article Snippet: Flow cytometry HRG1β-CAR-T cells were detected by staining with rabbit anti-human HRG1 antibody (Abnova, Taibei, China) and PE-conjugated donkey anti-rabbit IgG (BioLegend) as a secondary antibody.

Techniques: Expressing, Plasmid Preparation, Construct, Agarose Gel Electrophoresis, Marker, Flow Cytometry, Transduction, Western Blot

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting and suppression of HER3-positive breast cancer by T lymphocytes expressing a heregulin chimeric antigen receptor

doi: 10.1007/s00262-017-2089-5

Figure Lengend Snippet: Expression of HRG1β-CAR in activated T lymphocytes. a PBMCs were isolated from healthy volunteers’ blood. After isolation, cells were labeled with anti-CD3-PE, anti-CD4-FITC, anti-CD8-FITC, and detected by flow cytometry. b CD3-positive T cells were obtained from PBMC by magnetic bead separation, 1 week after anti-CD3/CD28 activated culture, and were subjected to flow cytometry analysis. c Expression of HRG1β was detected by flow cytometry in activated T-lymphocytes transduced with HRG1β-CAR. Data are representative of PBMCs from five donors

Article Snippet: Flow cytometry HRG1β-CAR-T cells were detected by staining with rabbit anti-human HRG1 antibody (Abnova, Taibei, China) and PE-conjugated donkey anti-rabbit IgG (BioLegend) as a secondary antibody.

Techniques: Expressing, Isolation, Labeling, Flow Cytometry, Transduction

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting and suppression of HER3-positive breast cancer by T lymphocytes expressing a heregulin chimeric antigen receptor

doi: 10.1007/s00262-017-2089-5

Figure Lengend Snippet: Specific lysis by HRG1β-CAR-T cells in vitro. a Breast cancer cell lines and an immortalized mammary epithelial cell line, MCF-10A, were stained, respectively, with monoclonal antibodies specific for the HER2/HER3/HER4 antigen and analyzed by flow cytometry. b ELISA for cytokine production by HRG1β-CAR and control T cells after overnight incubation with the indicated target cells. c Antigen-specific killing of target cells by control T or HRG1β-CAR-T cells was determined using CFSE/PI double staining at the indicated E:T ratio. Data are represented as the mean ± SEM of n = 3 replicates or representative of three independent experiments from three donors. **P < 0.01, ***P < 0.001 (Student’s t test)

Article Snippet: Flow cytometry HRG1β-CAR-T cells were detected by staining with rabbit anti-human HRG1 antibody (Abnova, Taibei, China) and PE-conjugated donkey anti-rabbit IgG (BioLegend) as a secondary antibody.

Techniques: Lysis, In Vitro, Staining, Bioprocessing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Control, Incubation, Double Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting and suppression of HER3-positive breast cancer by T lymphocytes expressing a heregulin chimeric antigen receptor

doi: 10.1007/s00262-017-2089-5

Figure Lengend Snippet: HER3 knockdown confers resistance of breast cancer cells to cytolysis by HRG1β-CAR-T cells. a–c SK-BR-3 cells were transfected with indicated siRNAs, and subject to qRT-PCR (a), western blotting (b) and flow cytometry (c) analysis. d Lysis of target cells by control T or HRG1β-CAR-T cells after 4-h incubation at increasing E:T ratios. Data are represented as the mean ± SEM of n = 3 replicates or representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test)

Article Snippet: Flow cytometry HRG1β-CAR-T cells were detected by staining with rabbit anti-human HRG1 antibody (Abnova, Taibei, China) and PE-conjugated donkey anti-rabbit IgG (BioLegend) as a secondary antibody.

Techniques: Knockdown, Transfection, Quantitative RT-PCR, Western Blot, Flow Cytometry, Lysis, Control, Incubation

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Targeting and suppression of HER3-positive breast cancer by T lymphocytes expressing a heregulin chimeric antigen receptor

doi: 10.1007/s00262-017-2089-5

Figure Lengend Snippet: T-lymphocytes expressing HRG1β-CAR exert antitumor activity in vivo. a–c Nude mice (n = 10) were injected subcutaneously with SK-BR-3 cells in the right back to form xenograft tumors, followed by random grouping and weekly tail vein administration of control or HRG1β-CAR-T cells. Tumor volume was monitored and plotted (a). Mice were killed on day 42, and tumors were excised and weighed (b, c). d Xenograft tumors were established and mice were treated as described in (a–c, n = 6 for each group). The survival of mice was monitored and plotted. e–g Nude mice were inoculated with SK-BR-3 cells expressing luciferase (n = 3 for each group), followed by tail intravenous treatment with PBS, control T and HRG1β-CAR-T cells on day 14. Bioluminescent imaging was performed on indicated days (e), and region-of-interest (ROI) bioluminescence emission measurement was calculated for each group (f). Mice were killed on day 35, and tumors were excised, sectioned and subject to immunohistochemical staining for CD3 (g). The numbers of CD3-positive cells in 9 independent microscope fields were plotted. Magnification, ×400. Scale bar = 100 μm. *P < 0.05, **P < 0.01 (Student’s t test)

Article Snippet: Flow cytometry HRG1β-CAR-T cells were detected by staining with rabbit anti-human HRG1 antibody (Abnova, Taibei, China) and PE-conjugated donkey anti-rabbit IgG (BioLegend) as a secondary antibody.

Techniques: Expressing, Activity Assay, In Vivo, Injection, Control, Luciferase, Imaging, Immunohistochemical staining, Staining, Microscopy