Journal: bioRxiv

Article Title: The genomic landscape of intrinsic and acquired resistance to cyclin-dependent kinase 4/6 inhibitors in patients with hormone receptor positive metastatic breast cancer

doi: 10.1101/857839

Figure Lengend Snippet: (a) T47D cells were modified via CRISPR-mediated downregulation (RB1) or lentiviral overexpression (AKT1, KRAS G12D, AURKA, CCNE2) to interrogate potential resistance mediators identified in patient biopsy samples. Western blotting with the indicated antibodies is included. (b-f) Modified T47D cells were exposed to escalating doses of CDK4/6i (palbociclib – left, abemaciclib – right) and viability was estimated via cell-titer-glo (CTG) assay. Control (CRISPR non-targeting guide or GFP) cells are plotted along with the resistance driver of interest (RB1 – b, AKT1 – c, KRAS G12D – d, AURKA – e, CCNE2 – f). Parental and variant cell lines are normalized to vehicle control and viability is plotted as a function of increasing CDK4/6i (graphed as triplicate average +/-standard deviation). All variants provoke CDK4/6i resistance (to both palbociclib and abemaciclib) in vitro in T47D cells. Corresponding IC50 values are included in Supplemental Table 7.

Article Snippet: HR+/HER2-human breast cancer cell lines T47D (HT-133) and MCF7 (HTB-22) were obtained from American Type Culture Collection (ATCC).

Techniques: Modification, CRISPR, Over Expression, Western Blot, CTG Assay, Control, Variant Assay, Standard Deviation, In Vitro

Journal: bioRxiv

Article Title: The genomic landscape of intrinsic and acquired resistance to cyclin-dependent kinase 4/6 inhibitors in patients with hormone receptor positive metastatic breast cancer

doi: 10.1101/857839

Figure Lengend Snippet: (a) Breast cancer cell lines (T47D, MCF7, MDA-MB-361) were cultured long-term to resistance in the presence of CDK4/6i (palbociclib, abemaciclib). The resulting cell lines which emerged were subjected to western blotting for putative mediators of drug resistance (RB1, AKT1, KRAS/ERK, AURKA, and CCNE2). (b-c) T47D cells cultured to resistance in the presence of abemaciclib demonstrated low levels of RB1 expression (T47D-AR1) and increased sensitivity to the AURKA inhibitor LY3295668. MDA-MB-361 cells cultured to resistance in the presence of abemaciclib demonstrated high levels of ERK activation (361-AR1) and increased sensitivity to the ERK inhibitor LY3214996. MDA-MB-361 cells cultured to resistance in the presence of palbociclib demonstrated high levels of AURKA (361-PR1) and increased sensitivity to the AURKA inhibitor LY3295668. MCF7 cells cultures to resistance in the presence of palbociclib demonstrated increased levels of CCNE2 (MCF7-PR1) and increased sensitivity to the CHEK1 inhibitor prexasertib.

Article Snippet: HR+/HER2-human breast cancer cell lines T47D (HT-133) and MCF7 (HTB-22) were obtained from American Type Culture Collection (ATCC).

Techniques: Cell Culture, Western Blot, Expressing, Activation Assay

Journal: Frontiers in Oncology

Article Title: BCMA (TNFRSF17) Induces APRIL and BAFF Mediated Breast Cancer Cell Stemness

doi: 10.3389/fonc.2018.00301

Figure Lengend Snippet: Expression levels of BAFF, APRIL, BAFF-R, BCMA, and TACI in breast cancer cell lines and the effect of steroid hormones on APRIL expression levels (A) . The expression of BAFF, APRIL, BAFF-R, BCMA, and TACI investigated by PCR in T47D and MDA-MB-231 cells breast cancer cell lines. Figure presents the results of a single experiment, while table shows the presence or absence of transcripts, based on three different experiments. See Material and Methods for further details and description of the controls. (B) APRIL expression [from data previously reported ( , )] and deposited in GEO under the numbers GSE18146, GSE32666, and GSE32668) after treatment of breast cancer cells for 3 h with steroid hormones, testosterone (Testo) or estradiol (E2), unconjugated or in their membrane impermeable form conjugated to BSA (steroid-BSA). See Material and Methods for details of data extraction and analysis. (C) Effect of testosterone-BSA (T-BSA) on APRIL expression (investigated by PCR) in T47D breast cancer cells in the presence or absence of cyproterone acetate (C) (*denotes p < 0.05).

Article Snippet: The T47D (a luminal A type cell line: ERα+, PR+, and HER2-) and MDA-MB-231 (basal type cell line: triple negative) breast cancer cell lines were purchased from DSMZ (Braunschweig, Germany), were tested as mycoplasma free and were cultured in RPMI supplemented with 10% fetal bovine serum (FBS), at 37°C, 5% CO 2 .

Techniques: Expressing, Membrane, Extraction

Journal: Frontiers in Oncology

Article Title: BCMA (TNFRSF17) Induces APRIL and BAFF Mediated Breast Cancer Cell Stemness

doi: 10.3389/fonc.2018.00301

Figure Lengend Snippet: Effect of APRIL and BAFF on migration and EMT status . (A) Effect of APRIL and BAFF (100 ng/ml) on migration of T47D and MDA cells after 24–48 h of treatment. Figure presents mean ± SE of three independent experiments. (All samples were analyzed with t -test comparing at each time point, treatment vs. control, and *denotes p < 0.05) (B) . Representative photos (Magnification 1000x) of control (untreated) T47D cells and APRIL or BAFF (100 ng/ml) treated cells for 4 days immunostained in suspension for keratins (upper panel, green) and vimentin (lower panel, red). Fluorescence intensity was calculated using Image J. Values represent the ratio of vimentin/keratins expression in these cells. (C) Graphical presentation of EMT status changes obtained by calculating the ratio of vimentin/keratins expression of 50 cells for each treatment condition. All samples were analyzed with t -test (*** denotes p < 0.0001).

Article Snippet: The T47D (a luminal A type cell line: ERα+, PR+, and HER2-) and MDA-MB-231 (basal type cell line: triple negative) breast cancer cell lines were purchased from DSMZ (Braunschweig, Germany), were tested as mycoplasma free and were cultured in RPMI supplemented with 10% fetal bovine serum (FBS), at 37°C, 5% CO 2 .

Techniques: Migration, Control, Suspension, Fluorescence, Expressing

Journal: Frontiers in Oncology

Article Title: BCMA (TNFRSF17) Induces APRIL and BAFF Mediated Breast Cancer Cell Stemness

doi: 10.3389/fonc.2018.00301

Figure Lengend Snippet: Effect of APRIL and BAFF on mammosphere formation and breast cancer stem cell population and of testosterone on the effect of APRIL and BAFF on mammosphere formation (A) . Percentage of primary mammospheres formed after treatment of breast cancer cells (T47D and MDA) with either BAFF or APRIL (100 ng/ml) for 9 days. Results of three independent experiments are expressed as a percentage of control (untreated) cells (mean ± SE, *denotes p < 0.05, at least). (B) Percentage of mammospheres (primary) formed after treatment of breast cancer cells (T47D and MDA) with either BAFF or APRIL (100ng/ml) in the presence or absence of testosterone-BSA, for 9 days. Results of three independent experiments are expressed as a percentage of control (untreated) cells performed (mean ± SE, *denotes p < 0.05). (C) Percentage of mammospheres formed after treatment of T47D cells with either BAFF or APRIL (100 ng/ml) for 9 days (primary mammospheres) compared to secondary mammospheres and a subsequent 7-day period. Results of three independent experiments are expressed as a percentage of control (untreated) cells. (mean ± SE, * p < 0.05) (D) . Percentage of breast cancer stem cells (T47D and MDA) with or without treatment with BAFF or APRIL (100 ng/ml) for 4 days as estimated by assaying them with high green autofluorescence on a population of 20,000 cells, in a BL2-A/BL1-A dot plot. Three independent experiments were performed and the results of a representative one is presented. (E) Comparison of the percentages of breast cancer stem cells as estimated by autofluorescence on a population of 20,000 cells in a BL2-A/BL1-A dot plot and ALDEFLUOR kit with or without treatment with BAFF or APRIL (100 ng/ml) for 4 days. Three independent experiments were performed (mean ± SE, *denotes p < 0.05). (F) Comparison of the percentages of breast cancer stem cells (identified by autofluorescence on a population of 20,000 cells in a BL2-A/BL1-A dot plot) in T47D cells and mammospheres treated or not with BAFF or APRIL (100 ng/ml) for 4 and 12 days respectively. Three independent experiments were performed (mean ± SE, *denotes p < 0.05).

Article Snippet: The T47D (a luminal A type cell line: ERα+, PR+, and HER2-) and MDA-MB-231 (basal type cell line: triple negative) breast cancer cell lines were purchased from DSMZ (Braunschweig, Germany), were tested as mycoplasma free and were cultured in RPMI supplemented with 10% fetal bovine serum (FBS), at 37°C, 5% CO 2 .

Techniques: Control, Comparison

Journal: Frontiers in Oncology

Article Title: BCMA (TNFRSF17) Induces APRIL and BAFF Mediated Breast Cancer Cell Stemness

doi: 10.3389/fonc.2018.00301

Figure Lengend Snippet: Expression levels of pluripotency markers in T47D cells after APRIL or BAFF treatment . (A) Expression levels of ALDH1A1, KLF4, SOX-2, and c-MYC, genes (quantified by Real-Time PCR and expressed as percentage of control) after treatment of T47D cells with APRIL or BAFF (100 ng/ml) for 4 days. Three independent experiments were performed (mean ± SE, *denotes p < 0.05, at least). (B,C) Expression of SOX-2, c-MYC, ALDH1A1, and NANOG proteins (assayed by immunocytochemistry). Representative images of three independent experiments are presented (B) Quantification of the proteins' expression levels was performed in at least 10 cells in three different photos for both control and treated cells using Image J and is expressed as percentage of control (mean ± SE, (C) ).

Article Snippet: The T47D (a luminal A type cell line: ERα+, PR+, and HER2-) and MDA-MB-231 (basal type cell line: triple negative) breast cancer cell lines were purchased from DSMZ (Braunschweig, Germany), were tested as mycoplasma free and were cultured in RPMI supplemented with 10% fetal bovine serum (FBS), at 37°C, 5% CO 2 .

Techniques: Expressing, Real-time Polymerase Chain Reaction, Control, Immunocytochemistry

Journal: Frontiers in Oncology

Article Title: BCMA (TNFRSF17) Induces APRIL and BAFF Mediated Breast Cancer Cell Stemness

doi: 10.3389/fonc.2018.00301

Figure Lengend Snippet: (A) Effect of APRIL and BAFF on breast cancer stem cells in the presence of siBCMA. Percentage increase in the number of breast cancer stem cells transfected or untransfected with siBCMA and treated with BAFF or APRIL (100 ng/ml) for 4 days. (Breast cancer stem cell number was estimated by counting the cells with high green autofluoresence in a BL2-A/BL1-A dot plot). (B) NFk-B activity of untreated (control) and treated with APRIL or BAFF (100 ng/ml, for 24h) T47D cells. Experiments were performed in triplicate. (C,D) Effect of APRIL and BAFF on ALDH1A1 and KLF4 expression in the presence of a JNK inhibitor or sh RNA for JNK1 or JNK2. Expression levels of ALDH1A1 and KLF4 after treatment of T47D cells with APRIL or BAFF (100 ng/ml) for 4 days in the presence of a JNK inhibitor SP600125 (10 μM) (C) or sh RNA for JNK1 or JNK2 (D) . Data are presented as a mean ± SE of three independent experiments (*denotes p < 0.05, at least).

Article Snippet: The T47D (a luminal A type cell line: ERα+, PR+, and HER2-) and MDA-MB-231 (basal type cell line: triple negative) breast cancer cell lines were purchased from DSMZ (Braunschweig, Germany), were tested as mycoplasma free and were cultured in RPMI supplemented with 10% fetal bovine serum (FBS), at 37°C, 5% CO 2 .

Techniques: Transfection, Activity Assay, Control, Expressing