Developing quantitative imaging of Akt activation in tissue sections

Abstract

The PI3K/mTOR pathway regulates fundamental cellular processes such as proliferation, growth, metabolism and autophagy. PI3K signalling is often constitutively activated in cancer cells and tumours. Chemical inhibitors have been developed that target different components within this pathway. Patients with hyperactivated PI3K signalling would benefit from such targeted treatment. Diagnostic tools will have to be available to select those patients. Here, a novel approach to quantitatively image the activity of Akt is presented. Akt phosphorylation at Serine473 (pAkt) antibody binding was imaged by near infrared immunofluorescence and confocal microscopy. With the help of cells, cell pellets, lysates and tissue sections several control models were established to test the method. The developed control models validated the method for the use on paraffin embedded samples. Staining of breast cancer xenograft models supported the possible application of the method. Treatment of the tumour bearing mice demonstrated a clear reduction of pAkt level upon treatment with PI3K inhibitors. A pilot study of clinical breast cancer samples indicated that the developed method is usable to quantitatively image Akt activity in tissue sections. Together the present results suggest that quantitative imaging of Akt protein and phosphorylation levels could be future supplement in cancer diagnostics.