Breast Cancer Deubiquitinating Enzyme (DUB) Inhibitor Profiling: Fixed-cell GR measures of 21 breast cell lines to 32 small molecule perturbagens. Dataset 2 of 2: Calculated dose response metrics. - Dataset (ID:20351)
|HMS Dataset ID:||20351|
|Dataset Title:||Breast Cancer Deubiquitinating Enzyme (DUB) Inhibitor Profiling: Fixed-cell GR measures of 21 breast cell lines to 32 small molecule perturbagens. Dataset 2 of 2: Calculated dose response metrics.|
|Screening Lab Investigator:||Laura Doherty|
|Screening Principal Investigator:||Peter K. Sorger, Sara Buhrlage|
|Assay Description:||We measured the sensitivities of one non-malignant breast cell line and 20 breast cancer cell lines (six triple negative, six hormone receptor positive, five Her2 amplified, and three established from triple negative patient-derived xenografts) to 32 pre-clinical small molecule deubiquitinating enzyme (DUB) inhibitors. A microscopy-based dose response assay was used to measure drug potency, and to quantify drug efficacy in terms of growth inhibition (GR metrics) and cell death. We treated cells with single drugs over a 9-point ½ log dilution series and then measured cell number and viability after three days of drug exposure.|
1. Cells in mid-log phase of the growth cycle from 21 breast cancer cell lines were plated at appropriate densities to achieve ~40% confluence at the time of treatment: 500-2000 cells/well in 50 μL of their recommended growth medium in four 384-well plates. The growth conditions used are detailed in this file download: Growth Conditions.|
2. The plated cells were grown for 36 hours at 37°C in the presence of 5% CO2 (all MDAMB lines except MDAMB231 were grown in the absence of CO2) and were then treated with the indicated small molecules by pin transfer.
3. Cells were stained and fixed for analysis at the time of drug delivery (one plate) and after 72 hours of incubation (three plates) by adding 15 μL of staining solution (1:1000 LIVE/DEAD Far Red Dead Cell Stain (Thermo Fisher Scientific, catalog #L-34974), 2 μg/ml Hoechst 33342 (Thermo Fisher Scientific, catalog #62249), 10% OptiPrep (Sigma-Aldrich, catalog #D1556-250ML) in PBS) for 30 min at room temperature followed by fixing solution (4% formaldehyde (v/v) (Sigma-Aldrich, catalogue #F1635-500ML), 20% OptiPrep in PBS) for 20 min at room temperature. After fixation, 80 μL of supernatant per well was removed and replaced with 80 μL of PBS with an EL406 Washer Dispenser (BioTek, Winooski, VT).
4. The plates were scanned with a PE Operetta high-throughput plate scanner. Six fields of view covering the full well were acquired with a 10x high NA objective for all wells. 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.
5. Live and dead cell counts were obtained using Columbus software (Nuclear segmentation: module: 'Find Nuclei'; method: C; default parameters except 'Individual Threshold' which was set to 0.25; Channel: Hoechst. Corpse segmentation: module: 'Find Nuclei'; method: B; default parameters except 'Area' which was set to >80 for BT20, >100 for HCC1806, and >50 for all other cell lines; Channel: LDR; filter: Hoechst intensity < 300; output: 'Corpse- Number of objects'. Segmented nuclei were classified as dead based on LDR texture: module: 'Calculate Texture Properties'; method: SER Features; scale: 8px; normalized by: Region Intensity SER Spot; channel: LDR; filter: >0.001; output: 'DeadLDR- Number of objects', or based on size: module: 'Calculate Morphology Properties'; method: Standard Area; population: Nuclei segmented; filter: <60-120 depending on the cell line; output: 'DeadSize- Number of objects'. Live cells were counted as the number of nuclei segmented that did not meet the size or texture criteria for dead cells; output: 'Live-Number of objects'.) The fraction of dead cells was calculated by taking the sum of ('Corpse', 'DeadLDR', 'DeadSize' counts) divided by the sum of ('Corpse', 'DeadLDR', 'DeadSize', 'Live' counts).
6. Consistent with the methods reported in Hafner et al. (2016) (PMID: 27135972), the Mean Normalized Growth Rate Inhibition (GR) Values were calculated according to the following formula: 2^[log2(x(c)/x0)/log2(xctrl/x0)]-1 where x(c) is the mean of the measured 'Live' cell counts after a given treatment, x0 is the mean of the 'Live' cell counts from the day 0 untreated plate grown in parallel until the time of treatment, and xctrl is the mean of the 'Live' cell counts of the DMSO-treated control wells for all technical replicates. See HMS-LINCS dataset #20350 for these results.
7. The Increased Fraction Dead was calculated by subtracting the mean fraction of dead cells in the DMSO-treated control wells from the mean fraction of dead cells in the wells from a given treatment across all technical replicates. See HMS-LINCS dataset #20350 for these results.
8. The mean normalized growth rate inhibition (GR) values for a given cell line / small molecule combination across all tested concentrations were fitted to a sigmoidal curve according to: GRinf+(1-GRinf)/(1+(c/GEC50)hGR) where GRinf is the effect of the drug at infinite concentration, hGR is the GR Hill Coefficient of the fitted curve, and GEC50 is the concentration at half maximal effect. The GR50 value is the concentration at which the GR value is 0.5, GRmax is the maximum effect of the treatment at the highest concentration tested, and GR_AOC is the area over the fitted curve. See Hafner et al. (2016) (PMID: 27135972) for further details.
|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:||2018-08-09|
|Most Recent Update:||2018-08-09|