In Vitro Cell Cytotoxicity Evaluation

• Evaluate dose-response to candidate compounds measured at 6 escalating concentrations.
• Available for both:
  • Kras/p53 pancreatic ductal adenocarcinoma derived mouse cells
  • Kras/p53 non-small cell lung cancer derived mouse cells
• Takes about three weeks to complete.
• Includes detailed evaluation (in tabulated and graphical formats) of XTT assay data and sufficient basic consultation with a subject matter expert.

Process

Pancreatic ductal adenocarcinoma derived mouse cells

• 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)
  • gemcitabine
  • 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.