Breast Cancer Profiling Project, Drug Sensitivity phase II: Fixed-cell GR measures of 33 breast cell lines and PDX lines to 16 small molecule perturbagens from library plate II. Dataset 2 of 2: Calculated dose response metrics - Dataset (ID:20355)
|HMS Dataset ID:||20355|
|Dataset Title:||Breast Cancer Profiling Project, Drug Sensitivity phase II: Fixed-cell GR measures of 33 breast cell lines and PDX lines to 16 small molecule perturbagens from library plate II. Dataset 2 of 2: Calculated dose response metrics|
|Screening Lab Investigator:||Caitlin E. Mills|
|Screening Principal Investigator:||Peter K. Sorger|
|Assay Description:||We measured the sensitivities of two non-malignant breast cell lines and 31 breast cancer cell lines of which twenty were triple negative, six were hormone receptor positive, five were Her2 amplified, to 16 clinically-relevant small molecule perturbagens. 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 from a maximum dose not exceeding 10 µM and then measured cell number and viability after three days of drug exposure.|
1. Cells in mid-log phase of the growth cycle from 33 breast cancer cell lines were plated at appropriate densities to achieve ~40% confluence at the time of treatment; 500-2000 cells/well in 60 µL of their recommended growth medium in five 384-well plates. The growth conditions used are detailed in this file download: Growth Conditions.|
2. The plated cells were grown for 24 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 or using a D300 Digital Dispenser (Hewlett-Packard, Palo Alto, CA).
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), 10 µM EdU (Lumiprobe, catalogue # 10540), 10% OptiPrep (Sigma-Aldrich, catalogue #D1556-250ML) in PBS) for one hour 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. Cells were then permeabilized with 0.5% Triton X-100 for 20 min at room temperature, the EdU was labeled by Click reaction (2 mM CuSO4, 4 µM sulfo-cy3-azide (Lumiprobe, catalogue #B1330), 20 mg/ml ascorbic acid, 20% OptiPrep in PBS) for 30 min at room temperature. Cells were then blocked for one hour in Odyssey blocking buffer (LI-COR Biosciences, catalogue #927-40150), and stained with 1:2000 anti-phospho histone H3 antibody conjugated to Alexa 488 (Cell Signaling Technology, catalogue #3465S), and 2 µg/ml Hoechst 33342 (Thermo Fisher Scientific, catalog #62249) overnight at 4°C. 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, 460-490 nm and 500-550 nm for p-HH3, 520-550 nm and 560-630 nm for EdU, 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: B or C depending on the cell line; default parameters except ‘Individual Threshold’ which was set to 0.25; Channel: Hoechst). For each channel, the intensity was measured in the nuclear mask and in a surrounding ring for local background. The local background intensity was subtracted from the nuclear intensity for each channel; outputs: pH3INT, EdUINT, LDRINT. For each cell, the average Hoechst intensity was multiplied by the nuclear area; output: DNAcontent. Dead cell texture was calculated as LDR texture: module: ‘Calculate Texture Properties’; method: SER Features; scale: 8px; normalized by: Region Intensity SER Spot. 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’.
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 #20354 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 #20354 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, GEC50 is the concentration at half maximal effect, and GR r2 is a measure of the goodness of fit. pval_GR indicates if fit of sigmoidal curve is significantly better than that of a flat line (based on an F-test with cutoff of p = 0.05). 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:||2019-06-27|
|Most Recent Update:||2019-06-27|