Assess the in vitro potency of candidate compounds via a conventional cell-based toxicity assay (XTT living cell test) in a series of six drug concentrations (ranging from 0.1 nM to 50,000 nM) of a single agent using at least two independent KPC tumor-derived primary cell lines.
In each plate, controls will include:
dimethyl sulfoxide only (vehicle)
200 nM and 800 nM doxorubicin (dose-dependent response control to chemotherapy agent)
0.1% triton (cell-growth negative control)
Conduct drug-treated and control assays in triplicate.
Measure cell viability 72 hours after drug exposure using the XTT assay (Roche) per manufacturer’s instruction.
Express cell viability response to experimental compound as a percentage of dimethyl sulfoxide (or appropriate vehicle) treated control.
The viability of at least one human pancreatic ductal adenocarcinoma cell line (KrasG12D; p53R172H; or p53-/-) will be well tested in response to the compound of interest.
Non-small cell lung cancer derived mouse cells
Plate primary lung adenocarcinoma cells derived from KrasG12D;p53-/- mice at a routine cell density of 5,000 cells per well in 96-well plate.
Allow cells to adhere for 16 hours before replacing the medium and treating the cells with the candidate compound (AE1).
Conduct single drug treatments at increasing concentrations from 0.1 nM to 50,000 nM in 0.5% dimethyl sulfoxide, unless preliminary compound potency data warrants an increase or decrease in concentration range.
Each plate controls will include:
dimethyl sulfoxide only (cell growth in vehicle)
200 nM and 800 nM doxorubicin (known dose-dependent response control)
0.1% triton (cell growth negative control).
Run all treatments in triplicate.
Measure cell viability after 72 hours of drug exposure using an XTT assay (Roche) per manufacturer’s instruction.
Dimethyl sulfoxide-treated wells are considered as 100% viability for each treatment plate.
Express cell viability response to the requestor’s compound as a percentage of the dimethyl sulfoxide-treated control.
Each plate will also include treatment with a standard of care compound (cisplatin or carboplatin) for comparison of response.
The entire assay will be repeated in an additional KrasG12D;p53-/- primary tumor line for comparison.