Screening Methodology (NCI-60 HTS384)

NCI-60 One-Concentration Screen

General Description:

As of early 2007, all compounds submitted to the NCI-60 Human Cancer Cell Line Screen are tested initially at a single high concentration (10^-5 M) in the full NCI-60 cell line panel. Only compounds that satisfy pre-determined threshold inhibition criteria in a minimum number of cell lines will progress to the full Five-Concentration Screen. The threshold inhibition criteria for progression to the Five-Concentration Screen was selected to efficiently capture compounds with anti-proliferative activity based on a careful analysis of historical DTP screening data. The threshold criteria may be updated as additional data becomes available.

Interpretation of One-Dose Data:

The One-Concentration Screen data will be reported as a mean graph of the percent growth of treated cells and will be similar in appearance to mean graphs from the Five-Concentration Screen. The number reported for the One-Concentration Screen is growth relative to the vehicle control, and relative to the number of cells at time zero. This allows detection of both growth inhibition (values between 0 and 100) and lethality (values less than 0). This is the same as for the Five-Concentration Screen, described below. For example, a value of 100 means no growth inhibition. A value of 40 would mean 60% growth inhibition. A value of 0 means no net growth over the course of the experiment. A value of -40 would mean 40% lethality. A value of -100 means all the cells are dead. Information from the One-Concentration Screen mean graph is available for COMPARE analysis.

NCI 60 Cell Five-Dose Screen

Compounds that exhibit significant growth inhibition in the One-Concentration Screen are evaluated against the 60-cell line panel at five concentrations.

The sixty human tumor cell lines of the cancer screening panel are grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. To initiate a screen, 40 μl of cells are inoculated into the wells of white 384-well microtiter plates at plating densities ranging from 250 to 2,500 cells/well, depending on the doubling time of individual cell lines. After inoculation, the microtiter plates are incubated at 37°C with 5% CO² and 95% relative humidity for 24 h before the addition of controls and test agents. Prior to a screen, the test agents and controls are solubilized in dimethyl sulfoxide (DMSO) at 400-fold the final test concentration and stored frozen in polypropylene 384-well microtiter plates.

Twenty-four hours after cell line inoculation, acoustic dispensing is used to transfer 100 nl of DMSO (0.25% (v/v), final) into the wells of microtiter plates, each containing a single cell line. Subsequently, 40 μl of CellTiter-Glo are dispensed into the wells, according to the manufacturer's protocol. Luminescence is measured to assess cell viability at the time of drug addition (time zero, Tz). Acoustic dispensing is also used to transfer 100 nl of controls and test agents (400-fold dilution, final) into duplicate microtiter plates for each cell line to achieve technical replicates. Controls in each microtiter plate include vehicle, 100% cytotoxicity (1 μM Staurosporin NSC755774 and 3 μM Gemcitabine NSC613327 [n = 8]), and five concentrations of doxorubicin (NSC123127, 25 μM [n = 2], 2.5 μM [n = 1], 250 nM [n = 2], 25 nM [n = 1], 2.5 nM [n = 2]. Following the delivery of controls and test agents, the microtiter plates are incubated for 72 h at 37°C with 5 % CO² and 95% relative humidity. After 72 h of exposure, 40 μl of CellTiter-Glo are dispensed into the wells of the microtiter plates and luminescence is measured, according to the manufacturer's protocol, to assess cell viability. Using the various measurements (time zero [Tz], vehicle control growth [C], and growth in the presence of test agent at the five concentrations [Ti]), the percentage growth (%G) is calculated at each of the test agent concentrations. Percentage growth is calculated as shown below.

If Ti >/= Tz, the following equation is used:
%G = [(Ti - Tz)/(C - Tz)] x 100
If Ti < Tz, the following equation is used:
[(Ti - Tz) / Tz] x 100

Three response parameters are calculated for each test agent. The GI50 (50% growth inhibition) is calculated from [(Ti - Tz) / (C - Tz)] x 100 = 50, which is the concentration resulting in a 50% reduction in growth compared to the vehicle control-treated cells during the 72-hour exposure period. The concentration resulting in total growth inhibition (TGI) is calculated from Ti = Tz. The LC50 (50% lethal concentration) is calculated from [(Ti - Tz) / (C - Tz)] x 100 = -50. Values are calculated for each of these three parameters if the level of activity is reached; however, if the activity is not reached or is exceeded, the value for that parameter is expressed as greater or less than the maximum or minimum concentration tested.