Patient-derived cell models are more and more replacing traditional cell line-centric approaches in cancer research. We have established a platform of patient-derived 3D microtumors (PDMs) that we employ for compound efficacy testing studies, alone or in co-cultures with autologous immune cells from the same patient (either TILs or PBMC-derived). This platform has been established for diverse tumor entities, and for testing small molecules, immunotherapies and combo treatments. Assay options include cytotoxicity/killing assays, tumor infiltration assays, and add-on focused proteomic pathway profiling using DigiWest or RPPA (Reverse Phase Protein Arrays).

In the current study, PDM cultures were derived from fresh patient tumor biopsy materials, grown in multititer plates, and treated with several compounds at different doses and times. Protein lysates were analyzed via RPPA using well-validated antibodies against 109 protein markers (in their total and phospho forms) of key signaling pathways (e.g. MAPK, PI3K/AKT/mTOR).

Pathway activation signatures were generated from RPPA data. Changes upon treatment of the individual pathways were correlated to mechanisms of drug action, with efficacy or resistance, and for treatment response prediction. As such, multiplexed protein and pathway profiling using RPPA can provide meaningful biological marker information and valuable insights into the biology of drug action in truly patient-derived cell models.