Abstract 3935: A perfused 3D co-culture model of vemurafenib-resistant melanoma
Authors: Cheluvaraju Chaitra , Shuford Stephen , Mattingly Christina , DesRochers Teresa , Gevaert Matthew , Orr David E. , Crosswell Hal E. 
Published: May 2015

Abstract
Background: Vemurafenib(VF) is highly active against melanomas with a BRAFV600E mutation. Although remarkable clinical responses occur, progression ensues, in part due to innate resistance driven by the tumor micro-environment (TME). 2D co-culture screens have demonstrated that the cMet-HGF axis is an important mediator of TME-associated resistance. However, many aspects of the TME including cell-extracellular matrix (ECM), ligand-receptor interactions and signaling are different in 3D versus 2D cultures, thus potentially limiting the clinical utility of a 2D co-culture screen. We report our initial results of a 3D perfused co-culture model of VF-resistant melanoma as a clinically relevant screen for identifying agents and combinations to revert VF resistance. Methods: Media conditions, 3D hydrogel scaffolds and cell ratios were optimized prior to establishing 3D co-cultures. Stromal components and tumor cells were assessed for adaptation to 3D perfused conditions using the 3DKUBE™ segregated co-culture plasticware. Multi-syringe pumps achieved flow rates of 10ul/min. Analysis consisted of cell viability, dsDNA quantification, confocal imaging and immunoassays (secreted HGF and c-Met). Results: Fibroblast HGF production correlated with increasing cell numbers in 3D perfusion, but not in 2D and there was significantly more HGF produced by fibroblasts in 3D perfusion (2504 v 1639 pg/ml, p&lt;.05). Melanoma spheres maintained cell viability better in Matrigel ™ versus Matrigel:Collagen mix and retained 3D structure in perfused co-culture for 7 days. VF showed increased activity in melanoma spheroids in 3D ECM-free static cultures versus 2D monolayer (IC50 0.016uM v 15.79µM). There was a concentration-dependent response but reduced activity of VF against perfused 3D melanoma spheroids embedded in ECM, whereas there was no effect of VF against perfused melanoma spheroids in co-culture. Conclusion: V600E melanoma cells are extremely sensitive to BRAF targeting in 3D ECM free spheroids as compared with 2D monolayers. Clinical resistance is mimicked by a more complex 3D TME comprising ECM, perfusion and segregated co-culture of HGF-secreting fibroblasts in 3D. Mechanistic studies are ongoing to identify specific factors critical for VF resistance, including HGF/cMet axis interrogation. Future Direction: The ultimate goal is to establish clinically relevant 3D perfused co-culture models to identify novel agents or combinations which will overcome TME-mediated resistance to VF and other targeted agents. Future efforts are focused on incorporation of well characterized primary malignant epithelial cells with defined stromal components in the 3D perfused co-culture model in order to better identify combinations of multitargeted agents in both the preclinical(lead compound selection) and the co-clinical process (patient selection).
Citation Format: Chaitra Cheluvaraju, Stephen Shuford, Christina Mattingly, Teresa DesRochers, Matthew Gevaert, David E. Orr, Hal E. Crosswell. A perfused 3D co-culture model of vemurafenib-resistant melanoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3935. doi:10.1158/1538-7445.AM2014-3935