title: Immune cell-based screening assay for response to anticancer agents: applications in pharmacogenomics.
abstract: BACKGROUND
  Interpatient variability in immune and chemotherapeutic cytotoxic responses is likely due to complex genetic differences and is difficult to ascertain in humans. Through the use of a panel of genetically diverse mouse inbred strains, we developed a drug screening platform aimed at examining interstrain differences in viability on normal, noncancerous immune cells following chemotherapeutic cytotoxic insult. Drug effects were investigated by comparing selective chemotherapeutic agents, such as BEZ-235 and selumetinib, against conventional cytotoxic agents targeting multiple pathways, including doxorubicin and idarubicin.
  
  
  METHODS
  Splenocytes were isolated from 36 isogenic strains of mice using standard procedures. Of note, the splenocytes were not stimulated to avoid attributing responses to pathways involved with cellular stimulation rather than toxicity. Cells were incubated with compounds on a nine-point logarithmic dosing scale ranging from 15 nM to 100 μM (37°C, 5% CO2). At 4 hours posttreatment, cells were labeled with antibodies and physiological indicator dyes and fixed with 4% paraformaldehyde. Cellular phenotypes (eg, viability) were collected and analyzed using flow cytometry. Dose-response curves with response normalized to the zero dose as a function of log concentration were generated using GraphPad Prism 6.
  
  
  RESULTS
  Phenotypes were quantified using flow cytometry, yielding interstrain variation for measured endpoints in different immune cells. The flow cytometry assays produced over 16,000 data points that were used to generate dose-response curves. The more targeted agents, BEZ-235 and selumetinib, were less toxic to immune cells than the anthracycline agents. The calculated heritability for the viability of immune cells was higher with anthracyclines than the novel agents, making them better suited for downstream genetic analysis.
  
  
  CONCLUSIONS
  Using this approach, we identify cell lines of variable sensitivity to chemotherapeutic agents and aim to identify robust, replicable endpoints of cellular response to drugs that provide the starting point for identifying candidate genes and cellular toxicity pathways for future validation in human studies.
authors.affiliation: Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA.
authors.forename: Amber
authors.initials: A
authors.lastname: Frick
authors.affiliation: Department of Pathology and Laboratory Medicine, School of Medicine, University of North Carolina, Chapel Hill, NC, USA.
authors.forename: Yuri
authors.initials: Y
authors.lastname: Fedoriw
authors.affiliation: Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina, Chapel Hill, NC, USA ; Department of Genetics, School of Medicine, University of North Carolina, Chapel Hill, NC, USA.
authors.forename: Kristy
authors.initials: K
authors.lastname: Richards
authors.affiliation: Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina, Chapel Hill, NC, USA ; Department of Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill, NC, USA.
authors.forename: Blossom
authors.initials: B
authors.lastname: Damania
authors.affiliation: The Hamner Institutes for Health Sciences, Research Triangle Park, NC, USA.
authors.forename: Bethany
authors.initials: B
authors.lastname: Parks
authors.affiliation: Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA.
authors.forename: Oscar
authors.initials: O
authors.lastname: Suzuki
authors.affiliation: Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA.
authors.forename: Cristina S
authors.initials: CS
authors.lastname: Benton
authors.affiliation: Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Chan
authors.affiliation: Environmental Protection Agency, Research Triangle Park, NC, USA.
authors.forename: Russell S
authors.initials: RS
authors.lastname: Thomas
authors.affiliation: Division of Pharmacotherapy and Experimental Therapeutics, Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, NC, USA ; Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina, Chapel Hill, NC, USA.
authors.forename: Tim
authors.initials: T
authors.lastname: Wiltshire
keywords: chemotherapy; cytotoxicity; immunomodulation; precision medicine