Precision medicine’s goal of achieving a better response rate by avoiding ineffective therapies has sparked new approaches, including the testing of patient-derived 3d (PD3Ds) tumor cultures for modeling individual patient response, and the use of various molecular pathology techniques for advanced tumor profiling. Well-established genomics methodologies cannot directly assess cell-signaling activity within the tumor cells defined by the phosphorylation status of cellular signaling pathway networks. Here we present the development of a robust protein profiling strategy utilizing the DigiWest immuno-assay platform, to obtain data on the activation status of key cellular signaling networks implicated in cancer, and on proteins targeted by FDA-approved drugs including a number of targeted cancer therapies for e.g. EGFR, HER2, PI3K, mTOR, ALK and AKT.
We compiled a list of relevant pathway nodes and their phosphorylation sites that yield activity information on RAS/RAF/ERK, PI3K/AKT and mTOR signaling pathways. Based on this, we validated 242 total and phospho-antibodies in a pre-set oncoproteomic DigiWest panel that yields information on the activity of these signaling networks from the receptor level down to transcription factors, apoptosis and proliferation. This oncoproteomic panel can be utilized for elucidating drug response in pre-clinical cell models, in PD3D organoid models or in clinical tumor samples. Exemplary, we show differential effects of PI3K kinase inhibitor copanlisib vs MEK inhibitor trametinib at various levels within their signaling networks. Also, we validated this panel in PD3D organoids that were subjected to screening against common targeted therapies.