Abstract 221: Ex vivo cultures of freshly explanted tumors: a potent translational approach for screening novel targeted agents
Authors: Tijeras-Raballand Annemilai , Serova Maria , Neuzillet Cindy , Albuquerque Miguel , Colnot Nathalie , Bourgoin Pierre , Dokmak Safi , Bouattour Mohamed , Belghiti Jacques , Paradis Valérie , Raymond Eric , Faivre Sandrine , de Gramont Armand 
Published: August 2015

Abstract
Background: In vitro and in vivo models are important screening tools for novel anticancer agents but they do not reliably reflect the complexity of human tumors and are, by essence, incapable of evaluating the intricate interplay of cancer cells in their native stroma. We used a novel approach using freshly explanted human tumor slices to test the pharmacodynamic effects of targeted agents or combination therapies.
Materials and Methods: Tumor samples were obtained from fresh surgical specimens cut in 300 μm thick slices using a Leica microtome and maintained in culture for 24-72 hours in a defined environment that allows for diffusion of oxygen and nutrient in specific culture media. Tumor samples were analyzed by immunohistochemistry and immunofluorescence for various pharmacodynamics (PD) biomarkers.
Results: The first step was to establish the suitable conditions for ex vivo culture, by comparing several O2 levels and medium composition. Tissue quality and viability controls were more reliably assessed by HE staining than by MTT and LDH assays. In our study, 60% of the samples had &gt;80% viable cancer lesions. Thirteen ex vivo hepatocellular tumor explants were treated with the TGF-β inhibitor galunisertib or sorafenib as control. Galunisertib specific target engagement was evaluated by p-SMAD2/3 expression. We showed a significant decrease in p-SMAD2/3 expression after galunisertib exposure that was mostly unchanged after sorafenib treatment. Expression of the non specific early PD biomarkers pAKT and pERK were decreased in 60% and 90% of the samples treated with galunisertib and sorafenib respectively. Expression of the late PD biomarkers MIB1/Ki67 was decreased in 54% and 77% of the samples whereas expression of caspase-3 was increased in 54% and 62% of the samples treated with galunisertib and sorafenib, respectively. Combination of galunisertib and sorafenib exacerbated the results observed using p-SMAD2/3 expression and early PD biomarkers but not those observed using late PD biomarkers. In twelve head and neck tumors, target engagement for SMAC mimetic treatment was evaluated by c-IAP1 expression using cisplatin and carboplatin as controls. We showed a significant decrease in c-IAP1 expression in all samples after exposure to SMAC mimetic. For both mono and combination therapies, we evaluated expression of MIB1 and caspase-3 as well as necrosis as late PD biomarkers. We observed a stronger effect of the SMAC mimetic/cisplatin combination in inducing necrosis (92% vs 70%) whereas the SMAC mimetic/carboplatin combination was more efficient in inducing caspase-3 expression (90% vs 83%). Both combinations displayed similar effect on reducing MIB1 expression.
Conclusion: Ex vivo culture of fresh tumor explants is a valuable tool to assess the pharmacodynamic effects of anticancer agents in tumors and may contribute to evaluate drug effects in the perspective of predictive medicine.
Citation Format: Annemilai Tijeras-Raballand, Maria Serova, Cindy Neuzillet, Miguel Albuquerque, Nathalie Colnot, Pierre Bourgoin, Safi Dokmak, Mohamed Bouattour, Jacques Belghiti, Valérie Paradis, Eric Raymond, Sandrine Faivre, Armand de Gramont. Ex vivo cultures of freshly explanted tumors: a potent translational approach for screening novel targeted agents. [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 221. doi:10.1158/1538-7445.AM2015-221