Evaluation of the sensitivities of six triple-negative breast cancer (TNBC) cell lines to 23 different PI3K/AKT/mTOR inhibitors or to a MEK inhibitor (trametinib). Dataset 2 of 2: GR metrics. - Dataset (ID:20365)
|HMS Dataset ID:||20365|
|Dataset Title:||Evaluation of the sensitivities of six triple-negative breast cancer (TNBC) cell lines to 23 different PI3K/AKT/mTOR inhibitors or to a MEK inhibitor (trametinib). Dataset 2 of 2: GR metrics.|
|Screening Lab Investigator:||Sameer Chopra|
|Screening Principal Investigator:||Peter K. Sorger|
|Assay Description:||We measured the sensitivities of six TNBC cell lines to 23 clinical and pre-clinical small molecule inhibitors of kinases in the PI3K/AKT/mTOR signaling pathway and to one inhibitor of MEK. Cells were treated with each drug using a 9-point ½ log dilution series. Live cell counts were measured at the start and end of the 72 hour treatment period using fixed cell microscopy. Drug potency and efficacy were calculated using GR metrics.|
1. HCC70, HCC38, HCC1806 and BT-549 cells were cultured in RPMI-1640 media. BT-20 cells and Hs 578T cells were cultured in EMEM and in DMEM, respectively. All cultures were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. BT-549 cells were additionally supplemented with 0.023 IU/mL insulin.|
2. HCC70, HCC38, HCC1806, BT-549, and BT-20 cells were seeded in Cell Carrier 384 well plates (Perkin Elmer 6007558) at a density of 2000 cells per well using the Multidrop Combi (Thermo Scientific). Hs 578T cells were seeded at 1000 cells per well. Plated cells were grown at 37°C in the presence of 5% CO2 for approximately 18-24 hours at 37°C prior to drug treatment.
3. Stock concentrations of drugs dissolved in DMSO were pin transferred to treatment plates. Drugs were administered at 9 different concentrations in technical quadruplicate for each of three independent experimental replicates. DMSO alone was used as a control.
4. Cells were stained and fixed at the time of drug administration (untreated, time = 0 plate) and after 72 hours of treatment with active drug or DMSO using 15 μL of staining solution [1:5000 LIVE/DEAD Far Red Dead Cell Stain (Invitrogen L10120), 1:10000 Hoechst 33342 trihydrochloride trihydrate 10mg/mL solution in water (Invitrogen H3570), 1:10 OptiPrep (Sigma-Aldrich D1556) in PBS] for 30 minutes inside of an incubator at 37°C in the presence of 5% CO2, followed by 20 μL fixing solution [1:20 37% formaldehyde (Sigma-Aldrich F1635) and 1:5 OptiPrep in PBS] for 30 minutes at room temperature. Following fixation, all reagents were aspirated and replaced with 60 μL of PBS using an EL406 Washer Dispenser (BioTek, Winooski, VT).
5. The plates were imaged using the Operetta High-Content Imaging System (Perkin Elmer). Three fields of view from each well were acquired using the 10x high NA objective lens. The excitation and emission filters used for image acquisition were 360-400 nm and 410-480 nm for Hoechst, and 620-640 nm and 650-700 nm for LDR.
6. Live cell counts were obtained using Columbus software (Perkin Elmer) to analyze the images of each well. Nuclear segmentation was performed using the Hoechst signal intensity. For HCC70 cells: Module: 'Find Nuclei'; method: M; diameter 29 μm; splitting coefficient: 0.45; common threshold: 0.05. For HCC38 cells: Module: 'Find Nuclei'; method: M; diameter 38 μm; splitting coefficient: 0.45; common threshold: 0.05. For HCC1806 cells: Module: 'Find Nuclei'; method: M; diameter 28 μm; splitting coefficient: 0.45; common threshold: 0.05. For BT-549 cells: Module: 'Find Nuclei'; method: M; diameter 33 μm; splitting coefficient: 0.50; common threshold: 0.05. For BT-20 cells: Module: 'Find Nuclei'; method: C; common threshold: 0.0; area > 25μm^2. For Hs 578T cells: Module: 'Find Nuclei'; method: M; diameter 35 μm; splitting coefficient: 0.45; common threshold: 0.05.
7. Additional features were then extracted from images to exclude mis-segmented nuclei and non-viable cells from total live cell counts. Mis-segmented nuclei were of atypical size and shape compared to appropriately segmented nuclei. Non-viable cells were characterized by positive LDR staining and/or small pyknotic nuclei with extremely bright Hoechst signal. Collectively, the image analysis features in Columbus that best identified these characteristics included nuclear area and roundness (module: 'Calculate Morphology Properties'); nuclear Hoechst intensity (module: 'Calculate Intensity Properties'); nuclear LDR intensity (module: 'Calculate Intensity Properties'); Hoechst texture features (module: 'Calculate Texture Properties'; SER Features, spot; scale: 4 px; normalization by: unnormalized); and LDR texture features (module: 'Calculate Texture Properties'; SER Features; scale: 6 px; normalization by: kernel). These extracted features were then used to set filters (i.e. gates) in the module 'Select Population.' Filter settings or gates were tuned interactively for each independent experimental replicate by visually comparing images of cells in DMSO only-treated wells against images of cells in active drug-treated wells and by using histograms in Columbus to visualize gates applied to the distribution of measured/calculated values for each feature, in order to exclude extreme outliers. Identical filters were applied to all wells on a plate.
8. Viable cell counts at time = 0 and time = 72 hours for the dose range of 0.001 to 3.2 μM were used to calculate GR values and GR metrics with the online GR calculator (grcalculator.org/grcalculator/), which is based on the methods reported in Hafner et al. (2016) (PMID: 27135972).
|Assay Protocol Reference:||Hafner M, Niepel M, Subramanian K, and Sorger PK. Designing drug response experiments and quantifying their results. Curr Protoc Chem Biol 2017 Jun 19;9(2):96-116. doi: 10.1002/cpch.19 PubMed PMID: 28628201.|
|HMS Dataset Type:||Analysis|
|Date Publicly Available:||2019-11-12|
|Most Recent Update:||2019-11-12|