Application of meso scale technology for the measurement of phosphoproteins in human tumor xenografts

Assay Drug Dev Technol. 2007 Jun;5(3):391-401. doi: 10.1089/adt.2006.044.


In this age of molecularly targeted drug discovery, robust techniques are required to measure pharmacodynamic (PD) responses in tumors so that drug exposures can be associated with their effects on molecular biomarkers and efficacy. Our aim was to develop a rapid screen to monitor PD responses within xenografted human tumors as an important step towards a clinically applicable technology. Currently there are various methods available to measure PD end points, including immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), reverse transcription-polymerase chain reaction, gene expression profiling, and western blotting. These may require relatively large samples of tumor, surrogate tissue, or peripheral blood lymphocytes with subsequent analyses taking several days. The phosphoinositide 3-kinase (PI3-kinase) pathway is frequently deregulated in cancer and is also important in diabetes and autoimmune conditions. In this paper, optimization of the Meso Scale Discovery (MSD) (Gaithersburg, MD) platform to quantify changes in phospho-AKT and phospho-glycogen synthase kinase-3beta in response to a PI3-kinase inhibitor, LY294002, is described, initially in vitro and then within xenografted solid tumors. This method is highly practical with high throughput since large number of samples can be run simultaneously in 96-well format. The assays are robust (coefficient of variation for phospho-AKT 13.4%) and offer significant advantages (in terms of speed and quantitation) over western blots. This optimized procedure can be used for both in vitro and in vivo analysis, unlike an established fixed-cell ELISA with a time-resolved fluorescent end point.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line, Tumor
  • Chromones / therapeutic use*
  • Female
  • Glycogen Synthase Kinase 3 / metabolism*
  • Glycogen Synthase Kinase 3 beta
  • Humans
  • Mice
  • Morpholines / therapeutic use*
  • Neoplasm Transplantation
  • Neoplasms, Experimental / chemistry*
  • Neoplasms, Experimental / drug therapy
  • Phosphoinositide-3 Kinase Inhibitors*
  • Phosphoproteins / analysis*
  • Proto-Oncogene Proteins c-akt / metabolism*
  • Reproducibility of Results
  • Specimen Handling
  • Transplantation, Heterologous


  • Chromones
  • Morpholines
  • Phosphoinositide-3 Kinase Inhibitors
  • Phosphoproteins
  • 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one
  • GSK3B protein, human
  • Glycogen Synthase Kinase 3 beta
  • Gsk3b protein, mouse
  • Proto-Oncogene Proteins c-akt
  • Glycogen Synthase Kinase 3