Abstract To improve pathophysiology research, biomarker discovery and drug development, cell culture models should adequately mimic human (patho)physiology and provide reproducible results. This requires comparison between cultured cells/tissue and actual histopathology in the patient, as well as standardization of culture experiments to ensure experimental reproducibility, preferably in a quantitative manner1. 10-15 signal transduction pathways govern major cellular processes, e.g. cell division, differentiation and migration. The past decade we developed tests to quantitatively measure functional activity of signal transduction pathways in individual cell/tissue samples, based on Bayesian computational model inference of pathway activity from measurements of mRNA levels of target genes of the transcription factor associated with the respective signalling pathway. Tests provide quantitative pathway activity scores and are intended to be used for diagnostics and life sciences research2-4. Method: Tests have been developed for androgen (AR) and estrogen receptor (ER), Hedgehog (HH), Wnt, TGFβ, Notch, NFκB, PI3K, JAK-STAT 1/2 and 3, and MAPK pathways. After calibration and freezing of the models, extensive biological test validation was performed on healthy/diseased cell and tissue types, including multiple cancer, fibroblast and immune cell types. Using Affymetrix expression microarray data (GEO database) >900 cell lines of most cell and cancer types were analyzed, as well as primary cultures of most immune cell types. LnCaP (prostate), MCF7, BT474 (breast), HCC827 (lung) and A2780 (ovarian) cancer cell lines were compared across laboratories. Results: Typical expected single or combined pathway activities were confirmed, e.g. ER activity in breast, AR activity in prostate, and Wnt activity in colon cancer; HH activity in soft tissue tumor, NFκB activity in lymphoma, frequently combined with PI3K and/or MAPK and/or JAK-STAT growth factor pathways. Quantitative pathway activities were reproducible within studies, but highly variable between labs, and dependent on culture conditions. Conclusion: Our pathway tests measure signaling pathway activity in many cell and tissue types, and can be used as quantitative readout for cell/tissue culture. Applications are: standardization of cell/tissue culture to ensure reproducibility; comparison between culture-based disease model and patient histopathology; quantitative assessment of drug efficacy on disease models; assessment of toxicity on healthy cell/tissue models. A number of tests have been adapted to qPCR, enabling use on FFPE tissue and small samples. 1 Ben-David U, et al. Nature, 2018;560(7718):325; 2 Verhaegh W, et al. Cancer Res 2014;74(11):2936-45; 3 Verhaegh W, Stolpe A van de. Oncotarget, 2014:5(14):5196-7; 4 Ooijen H. van, et al. Am J Pathol 2018;188(9):1956-1972 Citation Format: Anja Van De Stolpe, Marcia Alves de Inda, Eveline den Biezen-Timmermans, Laurent Holtzer, Henk van Ooijen, Wim Verhaegh. Quantitative measurement of multiple signal transduction pathway activities in cell and tissue culture, including cancer, fibroblast, and immune cell types [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2645.