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3D tissue cultures provide a more physiologically relevant context for the screening of compounds, compared to 2D cell cultures. Cells cultured in 3D hydrogels also show complex phenotypes, increasing the scope for phenotypic profiling. Here we describe a high-content screening platform that uses human micro-tissues cultured in 3D in standard 384 well assay plates to study the activity of biologically active molecules. Image analysis tools were developed to process 3D image data to measure 854 phenotype-derived parameters. Multiparametric analysis was used to evaluate the effect of compounds on tissue morphology. We applied this screening platform to measure the activity and selectivity of inhibitors of the c-Met and EGF receptor (EGFR) tyrosine kinases in 3D cultured prostate carcinoma cells. c-Met and EGFR activity was quantified based on the invasive phenotypic profiles induced by their respective ligands, HGF and EGF, which were added in parallel assays. c-Met and EGFR inhibitors generally showed the expected inhibitory profile equivalent to that obtained in the absence of cognate ligand. However, one phase-III c-Met inhibitor, displayed an unexpected phenotypic profile characterised by potent inhibition of tumour cell invasion and off-target effects consistent with a previously reported polypharmacology of this compound. The screening method was applied to a novel collection of 70 bi-specific compounds designed to target both c-Met and EGFR. Compounds were identified that induced phenotypic profiles indicative of selective inhibition of either c-Met, EGFR or both targets. In conclusion, this poster describes a fully scalable high-content screening platform that uses phenotypic profiling to evaluate receptor tyrosine kinase inhibitor selectivity and activity in a physiologically relevant setting. Citation Format: Leo S. Price, Tijmen Booij, Maarten Klop. A 3D tissue culture-based utra-high content screening platform that uses phenotypic profiling of cancer tissues to identify selective inhibitors of receptor tyrosine kinases. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1703. doi:10.1158/1538-7445.AM2015-1703

Osteosarcoma is the most common primary bone malignancy with relatively high incidence in young people. Here, we found that expression of Aven was inversely correlated with metastasis-free survival in osteosarcoma patients and is increased in metastases compared to primary osteosarcoma biopsies. Also in chondrosarcoma, the second most common bone sarcoma, expression correlated with increased histological grade. Aven is an adaptor protein that has been implicated in anti-apoptotic signaling and serves as an oncoprotein in acute lymphoblastic leukemia. In human osteosarcoma cells, silencing Aven triggered a G2 cell cycle arrest. Chk1 protein levels were attenuated and ATR-Chk1 DNA damage response signaling in response to chemotherapy was abolished in Aven-depleted osteosarcoma cells while ATM, Chk2, and p53 activation remained intact. Osteosarcoma is notoriously difficult to treat with standard chemotherapy, and we examined whether pharmacological inhibition of the Aven-controlled ATR-Chk1 response could sensitize osteosarcoma cells to genotoxic compounds. Indeed, pharmacological inhibitors targeting Chk1/Chk2 or those selective for Chk1 synergized with standard chemotherapy in 2D cultures. Likewise, in 3D extracellular matrix-embedded cultures Chk1 inhibition effectively sensitized human osteosarcoma cells to chemotherapy. Together, these findings implicate Aven in ATR-Chk1 signaling and point towards Chk1 inhibition as a strategy to sensitize osteosarcomas to chemotherapy. Citation Format: Zuzanna Baranski, Tijmen T.H. Booij, Yvonne de Jong, Jolieke van Oosterwijk, Anne-Marie Cleton, Leo Price, Bob van de Water, Judith V.M.G. Bovée, Pancras C.W. Hogendoorn, Erik Danen. Aven-mediated checkpoint kinase control regulates proliferation and resistance to chemotherapy in osteosarcoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3780. doi:10.1158/1538-7445.AM2015-3780