National Cancer Institute - Developmental Therapeutics Program- National Cancer Institute (DTP-NCI)

Developmental Therapeutics Program- National Cancer Institute (DTP-NCI)

The evolution of strategies at the National Cancer Institute (NCI) illustrates the changes in screening that have resulted from advances in cancer biology. The Developmental Therapeutics Program (DTP) operates a tiered anti-cancer compound screening program with the goal of identifying novel chemical leads and biological mechanisms. The DTP screen is a three phase screen which includes: an initial screen which first involves a single dose cytotoxicity screen with the 60 cell line assay. Those passing certain thresholds are subjected to a 5 dose screen of the same 60 cell-line panel to determine a more detailed picture of the biological activity. A second phase screen establishes the Maximum Tolerable Dosage and involves in vivo examination of tumor regression using the Hollow fiber assay. The third phase of the study is the human tumor xenograft evaluation. Active compounds are selected for testing based on several criteria: disease type specificity in the in vitro assay, unique structure, potency, and demonstration of a unique pattern of cellular cytotoxicity or cytostasis, indicating a unique mechanism of action or intracellular target. Some of the approved cancer treatment drugs developed with DTP involvement are: Fluorouracil (1962, NSC-19893), Bleomycin (1973, NSC-125066), Doxorubicin (1974, NSC-123127), cis-Platinum (1978, NSC-119875), Carboplatin (1989, NSC-241240), Taxol (1992, NSC-192573) and Erbitux (2004, NSC-632307). Drug testing data are represented as a mean graph that displays growth inhibition in a standard bar graph representation. The mean graph is constructed by projecting bars to the right or left of the mean, depending on whether an individual cell line is more or less sensitive than the average line in the panel. Further the length of each bar is proportional to the relative sensitivity of the cell lines. Thus, each agent can be represented by a characteristic fingerprint of cell-line responsiveness, indicated by the bar graph presentation. These data are presented and analyzed in this section. The broad screen provides a basis for comparison with other compounds, many of which have a known mechanism of action. The NCI COMPARE program is an online database and comparison tool which analyzes both one-dose and five-dose data cytotoxicity data for the 60 cell line panel for similar activity profiles with all the compounds screened previously by the DTP. A compound is entered into the program as a seed, and the computer database elicits a list of those agents that have similar patterns of cellular cytotoxicity. A correlation coefficient is also expressed relating the closeness of the seed to those agents listed by the computer program. Close correlations between agents demonstrates biological and pharmacological importance and implies a common intracellular target despite dissimilarity in structure. A high correlation of cytotoxicity with compounds of known biological mechanism is often predictive of the drugs mechanism of action and thus a tool to aid in the drug development and testing. It also tells if there is any unique response of the drug which is not similar to any of the standard prototype compounds in the NCI database.

Methodology of the in-vitro Cancer Screen The protocol for the NCI-60 screen is as follows. The human tumor cell lines are grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening experiment, cells are inoculated into 96 well microtiter plates in 100 µL at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates are incubated at 37°C, 5% CO2, 95% air and 100% relative humidity for 24 h prior to addition of experimental drugs. After 24 h, two plates of each cell line are fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition (Tz). Experimental drugs are solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate is thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 µg/mL gentamicin. Additional four, 10-fold or ½log serial dilutions are made to provide a total of five drug concentrations plus control. Aliquots of 100 µl of these different drug dilutions are added to the appropriate microtiter wells already containing 100 µl of medium, resulting in the required final drug concentrations. Following drug addition, the plates are incubated for an additional 48 h at 37°C, 5% CO2, 95% air, and 100% relative humidity. For adherent cells, the assay is terminated by the addition of cold TCA. Cells are fixed in situ by the gentle addition of 50 µl of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 minutes at 4°C. The supernatant is discarded, and the plates are washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (100 µl) at 0.4% (w/v) in 1% acetic acid is added to each well, and plates are incubated for 10 minutes at room temperature. After staining, unbound dye is removed by washing five times with 1% acetic acid and the plates are air dried. Bound stain is subsequently solubilized with 10 mM trizma base, and the absorbance is read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology is the same except that the assay is terminated by fixing settled cells at the bottom of the wells by gently adding 50 µl of 80% TCA (final concentration, 16% TCA). Using the seven absorbance measurements, the percentage growth is calculated at each of the drug concentrations levels. Percentage growth inhibition is calculated as: x 100 for concentrations for which Ti>/=Tz x 100 for concentrations for which Ti