Journal: Annals of oncology : official journal of the European Society for Medical Oncology

Article Title: Trastuzumab induces antibody-dependent cell-mediated cytotoxicity (ADCC) in HER-2-non-amplified breast cancer cell lines.

doi: 10.1093/annonc/mdr484

Figure Lengend Snippet: Figure 3. Laser scanning confocal microscope images of trastuzumab bound to HER-2 on breast cancer cells. Visualisation parameters were the same for HER-2-amplified (A and B) and HER-2-non-amplified (C–H) cell lines. Cells were not permeabilised. Blue—DAPI (nucleus), green—trastuzumab (cell surface). Scale bar represents 10 lm. A colour version of the figure and control images can be found in supplemental data (supplemental Figure S3, available at Annals of Oncology online).

Article Snippet: In addition, in order to assess the differential expression of HER-2 between normal and malignant breast tissue necessary for a selective ADCC response to trastuzumab in HER-2 non-amplified tumour cells, we examined HER-2 protein levels in tumour and normal tissue samples from patients with HER-2 negative breast cancer. materials and methods cell lines, cell culture, and reagents The breast carcinoma cell lines SKBR3 [American Type Culture Collection (ATCC)], HCC1954 (ATCC), T47D [European Collection of Cell Cultures (ECACC)], EFM19 (German Collection of Microorganisms and Cell Cultures, DSMZ), MCF-7 (ATCC), and the leukaemic cell line K562 (ATCC), were maintained in RPMI 1640, 10% fetal calf serum (FCS) at 37 C with 5% CO2.

Techniques: Microscopy, Control

Journal: Breast Cancer Research : BCR

Article Title: Expression of truncated human epidermal growth factor receptor 2 on circulating tumor cells of breast cancer patients

doi: 10.1186/s13058-015-0624-x

Figure Lengend Snippet: Expression of full-length HER2 and its truncated form on control SKBR3 and SCOV3 cells. a Equal protein amounts of SCOV3 and SKBR3 cells were subjected to SDS-PAGE electrophoresis and transferred to nitrocellulose membrane. Total proteins were detected by immunoblotting (IB) with ( I ): anti-HER2 ECD, ( II ) HER2 ICD (CB11 clone) and ( III ) anti-actin. b Representative Ariol system images from control slides with SKBR3 cells. ( a ) SKBR3 cells stained with HER2 (ECD)/Alexa633/Alexa555 antibodies ( b ) SKBR3 cells stained with HER2 (ICD)/Alexa633/Alexa555 antibodies and ( c ) SKBR3 cells stained with HER2(ECD)/HER2 (ICD)/Alexa633/Alexa555 HER2 antibodies. c Representative confocal laser scanning micrographs (×40) of SKBR3 and SCOV3 cells triple-stained with HER2 (ICD), HER2 (ECD) and pancytokeratin (A45-B/B3) antibodies. ECD and ICD were identified in SKBR3 ( A-D ) cells while low ( E-H ) or no ECD ( I-L ) was identified in SCOV3 cells. ECD extracellular domain, HER2 human epidermal growth factor receptor 2, ICD intracellular domain

Article Snippet: SKBR3 breast cancer cells (obtained from ATCC; the American Type Culture Collection, Manassas, VA, USA), which overexpress HER2, were cultured in RPMI supplemented with 10 % fetal bovine serum (FBS).

Techniques: Expressing, Control, SDS Page, Electrophoresis, Membrane, Western Blot, Staining

Journal: International Journal of Molecular Sciences

Article Title: Human CD3+ T-Cells with the Anti-ERBB2 Chimeric Antigen Receptor Exhibit Efficient Targeting and Induce Apoptosis in ERBB2 Overexpressing Breast Cancer Cells

doi: 10.3390/ijms18091797

Figure Lengend Snippet: Flow cytometric detection of activation cell surface markers, CD69 and CD25 after co-culture with SKBR3 cells. The cell population was gated at lymphocytes. Singlet was gated from the lymphocyte population to remove residual cell clumps following disaggregation and eliminate auto-fluorescence. Subsequently, CD3+ cells were gated from the singlet population, and CD69+ and CD25+ cells were gated from the CD3+ population. CAR-T cells, 72 h post co-culture with SKBR3 cells, showed significantly higher levels of activation markers (CD69+: 34.6%, CD25+: 93.7%) than those by non-transduced T-cells (control) 72 h post co-culture with SKBR3 cells (CD69+: 14.5%, CD25+: 33.6%). CD3+ percentages were consistently high at 98.0% and 96.5% for non-transduced and CAR-T cells, respectively, 72 h post co-culture with SKBR3 cells. The isotype control used were activated T-cells that were not co-cultured with SKBR3 cells. CD3 was stained by fluorochrome PE, CD69 by PE, and CD25 by allophycocyanin (APC) fluorochromes. For each antibody, isotype-matched mouse immunoglobulin γ antibody was used as the isotype control.

Article Snippet: SK-BR-3 (SKBR3) breast cancer cell line (ATCC HTB-30, Manassas, VA, USA) were cultured in a 25 cm 2 plastic flask containing advanced RPMI 1640 media, 10% foetal bovine serum, and 1% penicillin/streptomycin at a seeding density of 1 × 10 5 cells/mL at 37 °C in a humidified 5% CO 2 atmosphere.

Techniques: Activation Assay, Co-Culture Assay, Fluorescence, Control, Cell Culture, Staining

Journal: International Journal of Molecular Sciences

Article Title: Human CD3+ T-Cells with the Anti-ERBB2 Chimeric Antigen Receptor Exhibit Efficient Targeting and Induce Apoptosis in ERBB2 Overexpressing Breast Cancer Cells

doi: 10.3390/ijms18091797

Figure Lengend Snippet: Detection of interferon-γ production upon co-culture of CAR-T cells with SKBR3 cells. The figure shows interferon-gamma (IFN-γ) secretion in the supernatant of the T-cells and ERBB2 overexpressing cancer cell line, SKBR3, after 72 h of co-culture. Only 6.42 pg/mL IFN-γ was produced in the SKBR3 single culture supernatants ( n = 6). SKBR3 co-cultured with non-transduced T-cells showed 25.57 pg/mL IFN-γ production ( n = 6). SKBR3 co-cultured with CAR-T showed IFN-γ at concentrations of 353.63 ± 10.64 pg/mL was produced in the supernatant of this experimental group with a significant p value of p < 0.0001 ( n = 6) compared to that of SKBR3 co-cultured with non-transduced T-cells. The X-axis indicates the experimental groups, while the Y-axis indicates the concentration of the IFN-γ (pg/mL) produced with the scale bar up to 400 pg/mL. The resulting data was reported as a bar chart of the experimental mean ± standard error of the mean (S.E.M.) ( n = 6). The p < 0.0001 was determined with respect to SKBR3 co-cultured with CAR-T compared to non-transduced T-cells by ordinary one-way analysis of variance (ANOVA).

Article Snippet: SK-BR-3 (SKBR3) breast cancer cell line (ATCC HTB-30, Manassas, VA, USA) were cultured in a 25 cm 2 plastic flask containing advanced RPMI 1640 media, 10% foetal bovine serum, and 1% penicillin/streptomycin at a seeding density of 1 × 10 5 cells/mL at 37 °C in a humidified 5% CO 2 atmosphere.

Techniques: Co-Culture Assay, Produced, Cell Culture, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Human CD3+ T-Cells with the Anti-ERBB2 Chimeric Antigen Receptor Exhibit Efficient Targeting and Induce Apoptosis in ERBB2 Overexpressing Breast Cancer Cells

doi: 10.3390/ijms18091797

Figure Lengend Snippet: Detection of efficiency of CAR-T cells in SKBR3 cell lysis. The effectiveness of CAR-T cells and non-transduced T-cells in the lysis of SKBR3 cells was observed by phase contrast microscopy and measured by the CellTiter 96 Cell Proliferation Assay (MTS). ( A ) Phase contrast image of SKBR3 co-cultured with CAR-T cells. The image showed a minimal amount of attached SKBR3 cells on the plate, indicating low cell viability; ( B ) phase contrast image of SKBR3 co-cultured with non-transduced T-cells. The image showed high numbers of attached SKBR3 cells on the plate, indicating high cell viability; ( C ) phase contrast image of SKBR3 single culture. The image showed high numbers of attached SKBR3 cells indicating high cell viability. Cells were visualized by inverted light microscopy. Cells were imaged at 100× magnification (the scale bar represents 200 μm); ( D ) the graph indicates that cell viability of SKBR3 cells co-cultured with CAR-T ( n = 7) was extremely low compared to that of SKBR3 cells co-cultured with non-transduced T-cells ( n = 6) or SKBR3 single culture cells ( n = 6). Statistical analysis showed an extremely low p value of <0.001 for co-culture with CAR-T cells compared to non-transduced T-cells or SKBR3 single culture. The percentage of viable SKBR3 cells that remained attached to the plate was only 14.84% in CAR-T co-cultured with SKBR3 cells compared to 93.2% in SKBR3 cells co-cultured with the non-transduced T-cell. The SKBR3 single culture was observed to show 100% cell viability. The X-axis indicates the experimental groups while Y-axis indicates the percentage of SKBR3 cell viability. MTS data was reported as a bar chart of the experimental mean ± standard error of the mean (S.E.M) with p < 0.001 with respect to the experimental SKBR3 cell viability upon co-culture with CAR-T cells compared to non-transduced T-cells by ordinary one-way ANOVA analysis.

Article Snippet: SK-BR-3 (SKBR3) breast cancer cell line (ATCC HTB-30, Manassas, VA, USA) were cultured in a 25 cm 2 plastic flask containing advanced RPMI 1640 media, 10% foetal bovine serum, and 1% penicillin/streptomycin at a seeding density of 1 × 10 5 cells/mL at 37 °C in a humidified 5% CO 2 atmosphere.

Techniques: Lysis, Microscopy, Proliferation Assay, Cell Culture, Light Microscopy, Co-Culture Assay

Journal: International Journal of Molecular Sciences

Article Title: Human CD3+ T-Cells with the Anti-ERBB2 Chimeric Antigen Receptor Exhibit Efficient Targeting and Induce Apoptosis in ERBB2 Overexpressing Breast Cancer Cells

doi: 10.3390/ijms18091797

Figure Lengend Snippet: Detection of efficiency of CAR-T cells in SKBR3 cell lysis by FITC Annexin V Apoptosis Detection Kit. The FITC Annexin V Apoptosis Detection Kit was used to measure the amount of apoptosis seen in SKBR3 cells following co-culture with CAR-T cells (stained test) and non-transduced T-cells (stained control). SKBR3 single culture (stained SKBR3 control) was designated as the background. The cell population was gated at SKBR3 cells. Singlet was gated from the SKBR3 cells population to remove residual cell clumps following disaggregation and eliminate auto-fluorescence. Subsequently, apoptotic cells were gated from the singlet population. Viable cells in the apoptosis assay are stained FITC−/Propidium iodide− (PI), necrotic cells are stained FITC−/PI+, early apoptotic cells are stained FITC+/PI−, while late apoptotic cells are stained FITC+/PI+. Upon co-culture with CAR-T cells, SKBR3 cells showed increased late apoptotic cells and necrotic cells compared to non-transduced T-cells. Meanwhile, the number of viable SKBR3 cells decreased in co-culture with CAR-T compared to non-transduced T-cells. In comparison, stained SKBR3 control showed lower late apoptotic and necrotic cells. The data is a representative of one technical replicate from cells ( n = 6) that were pooled together prior to flow cytometric analysis.

Article Snippet: SK-BR-3 (SKBR3) breast cancer cell line (ATCC HTB-30, Manassas, VA, USA) were cultured in a 25 cm 2 plastic flask containing advanced RPMI 1640 media, 10% foetal bovine serum, and 1% penicillin/streptomycin at a seeding density of 1 × 10 5 cells/mL at 37 °C in a humidified 5% CO 2 atmosphere.

Techniques: Lysis, Co-Culture Assay, Staining, Control, Fluorescence, Apoptosis Assay, Comparison