Breast cell line dose response to target inhibition measured by high throughput microscopy - Dataset (ID:20136)

HMS Dataset ID: 20136
Dataset Title: Breast cell line dose response to target inhibition measured by high throughput microscopy
Publication(s) Using Dataset: PMID: 24013279
Project Summary Page(s):
Screening Lab Investigator: Mohammad Fallahi-Sichani
Screening Principal Investigator: Peter Sorger
Assay Description: To investigate how a shallow dose-response curve might arise (as seen in data analyzed in Fallahi-Sichani et al. (2013) Nature Chemical Biology. PMID: 24013279), this assay focuses on drugs inhibiting the PI3K/Akt/mTOR pathway that varied widely in Hill slope (HS) and Emax values independent of proliferation rate. For six compounds with varying HS, target inhibition was measured by immunofluorescence microscopy and cell killing in four breast cell lines (HER2-amplified AU565 and HCC1954 cancer cells, hormone receptor-positive T47D cancer cells, and non-transformed MCF10A cells). The effects of the mTOR inhibitor PP242, PI3K inhibitor GSK1059615, dual specificity mTOR/PI3K inhibitor dactolisib (BEZ235), Akt inhibitors MK2206 and triciribine, and also an EGFR inhibitor gefitinib were probed 6 hr and 24 hr after drug exposure in 9-point dose-response assays using antibodies specific for p-ERK (Thr202/Tyr204), p-Akt (Ser473), p-4EBP1 (Thr37/46), and p-S6 (Ser235/236). A shallow dose-response curve is correlated with high cell-to-cell variability in target (p-4EBP1) inhibition by PP242 and dactolisib as compared to drugs for which HS ~ 1 or HS > 1 (in four of four cell lines tested). This dataset refers to data shown in Figure 5 and Supplementary Figures 5, 7, 8 and 9 of the paper.
Assay Protocol: AU565 and HCC1954 cells were cultured in RPMI 1640 (ATCC) supplemented with 10% FBS (FBS). T47D cells were cultured in RPMI 1640 supplemented with 10% FBS and insulin (0.2 U/ml). MCF10A cells were cultured in DMEM/F12 (Invitrogen) supplemented with 5% horse serum, EGF (20 ng/ml), insulin (10 μg/ml), hydrocortisone (0.5 μg/ml) and cholera toxin (100 ng/ml). Penicillin (50 U/ml) and streptomycin (50 μg/ml) were added to all growth media. All of the compounds were dissolved in DMSO as 10-mM stock solutions. For dose-response experiments, cells were plated in two repli­cates at 7,000 cells per well in 96-well plates (Corning) in full growth medium for 24 h and then treated them with nine doses in serial dilutions (100 pM to 10 uM) of each compound for 6 h and 24 h.

Cells were fixed in 2% paraformaldehyde for 10 min at room temperature and washed with PBS with 0.1% Tween 20 (Sigma-Aldrich) (PBS-T), permeabilized in methanol for 10 min at room temperature, rewashed with PBS-T and blocked in Odyssey Blocking Buffer (LI-CORBiosciences) for 1 hr at room temperature. Cells were incubated overnight at 4°C with rabbit monoclonal antibodies to p-Akt (1:400, Ser473, 4060, Cell Signaling Technology), p-4EBP1(1:400, Thr37/46, 2855, Cell Signaling Technology), p-S6 ribosomal protein (1:400, Ser235/236, 4858, Cell Signaling Technology), mouse monoclonal antibody to p-ERK (1:200, Thr202/Tyr204, 9106, Cell Signaling Technology) and a goat polyclonal antibody to p-Rb (1:400, Ser807/811, sc-16670, Santa Cruz) in Odyssey Blocking Buffer. Cells were washed three times in PBS-T and incubated with mouse, rabbit and goat secondary antibodies labeled with 488, 647 and 568 Alexa Fluors (Invitrogen), respectively, diluted 1:2,000 in Odyssey Blocking Buffer. Cells were washed once in PBS-T, once in PBS and incubated in 250 ng/ml Hoechst 33342 (Invitrogen) and 1:1,000 Whole Cell Stain (blue; Thermo Scientific) solutions. Cells were then washed twice with PBS and imaged with 10× objective on Operetta (Perkin Elmer).

Image segmentation and storage was performed using ImageRail software. Data were analyzed using MatLab software. Intensity of each fluorophore was quantitated for each image.
Assay Protocol Reference: Fallahi-Sichani M, Honarnejad, S Heiser, LM, Gray JW and Sorger PK. Metrics other than potency reveal systematic variation in responses to cancer drugs. Nature Chemical Biology, 2013. doi:10.1038/nchembio.1337. PMID: 24013279. Please refer to Figure 5 and Supplementary Figures 5, 7, 8 and 9.
HMS Dataset Type: Microscopy/Imaging
Date Publicly Available: 2014-01-24
Most Recent Update: 2016-08-24