Methods and data analysis

pharmacomb logo
Combing High Throughput Screening Data

High Throughput Drug Profiling

  1. Cell lines were cultured and passaged approximately every 2-3 days.  Cell media was typically DMEM + 10% FBS + 1% Penicillin Streptomycin.
  2. A Thermo Scientific MultidropTM Combi Reagent Dispenser was utilized to plate 2000 cells on a 384-well microplate with a volume of 25 µL per well.
  3. Plated cells were incubated overnight in a 5% CO2 humidified chamber at 37°C for the cells to attach to the plate.
  4. A V&P Scientific 384 W, 50 nL slotted pin tool and Perkin Elmer JANUS Automated Workstation were used to transfer 50 nL of drugs into each well. Drug concentrations of 4.6 nM, 14 nM, 41 nM, 123 nM, 370 nM, 1.11 µM, 3.33 µM, and 10 µM in DMSO were tested. 100% DMSO was used as the solvent control and 5mM doxorubicin was used as a positive cytotoxicity control.
  5. Drug-dosed cells were incubated for 72 hours in a 5% CO2 humidified chamber at 37°C.
  6. Assay plates were allowed to equilibrate to room temperature before 25 µL of Perkin Elmer ATPlite was added to each well.
  7. Perkin Elmer EnVision Xcite Multilable Plate Reader was used to measure luminescence to determine cell viability.

 

Data analysis

The raw values of the 16 DMSO and 16 doxorubicin treated wells on each assay plate were aggregated and used to calculate z’-factors, as a measure of assay performance and data quality, with a z’-factor > 0.4 representing acceptable data. Raw data values of test wells were normalized to aggregate DMSO and doxorubicin plate control wells and expressed as normalized

percent inhibition (NPI = ((DMSOavg-Test well)/(DMSOavg-Doxorubicinavg) × 100)). 

A nonlinear fit with variable slope (GraphPad Prism 7) of Normalized Percent Inhibition and log10 concentration values was used to define IC50 values for each drug cell line combination.  The data presented in the table is the average IC50 in µM from multiple experiments, while the dose response curves are examples from a single experiment. 

The mean Spearman’s rank correlations for replicates of a cell line was 0.85.

Limitations:

  1. Our assay conditions are typical of other screening centers that carry out HTS cancer screening.  All data are subject to the limitations of the assay system. The system tends to favor drugs that act on rapidly dividing cells. Rapamycin, which is generally regarded as cytostatic, not cytotoxic, does not kill 100% of cells does not yield an accurate IC50.
  2. We grow cells on plastic plates, which ignores the mechanical properties of tissues.
  3. Drugs that act on the NF microenvironment, and not the NF cells such as Imatinib or VEGF inhibitors would not be expected to be active.
  4. Some drugs require metabolic activation in the liver, such as and ifosfamide, which is highly cytotoxic and recommended for treatment of MPNST, requires metabolic activation in the liver.

 

Citing this database:

Guo, J., Grovola, M. R., Xie, H., Coggins, G. E., Duggan, P., Hasan, R., . . . Field, J. (2017). Comprehensive pharmacological profiling of neurofibromatosis cell lines. Am J Cancer Res, 7(4), 923-934. 

https://www.ncbi.nlm.nih.gov/pubmed/28469964