Abstract 2936: Induction of hypoxia in 3D human melanoma spheroids leads to c-Met activation and resistance to Vemurafenib.
Authors: Qin Yong , Greene Victoria R. , Chattopadhyay Chandrani , Ekmekcioglu Suhendan , Liu Chengwen , Grimm Elizabeth A. 
Published: August 2014

Abstract
Melanoma is highly heterogeneous in vivo compared to two-dimensional (2D) monolayer adherent cells in culture. Using human A375 and WM793 mouse xenografts, we consistently observed a significant increase of HIF-1α expression in many live tumor cells by Immunohistochemistry. This distinct feature confirms that some melanoma cells exist under hypoxia in vivo. We hypothesize that melanoma cells survive under hypoxic conditions due to unique growth and survival pathways, which are likely to alter their sensitivity to current therapeutics. We employed a three-dimensional (3D) culture system with an inorganic nanoscale scaffolding (SCIVAX NanoCulture Plate, NCP), to mimic part of the in vivo system. Human B-RAF(V600E) mutated melanoma cell lines, 451Lu, A375, and MEL1617, were confirmed to form spheroids with NCP in standard RPMI medium with 5% FBS under normoxic incubator conditions. The majority of melanoma cells in spheroids were identified as hypoxia as indicated by the hypoxic probe LOX-1. The fluorescence of LOX-1 is quenched by oxygen and increased in response to low levels of oxygen, which was consistently negative in parallel monolayer cultures of these same three melanoma cell lines. Furthermore, cells within the melanoma spheroids differentially expressed HIF-1α and VEGF, at both mRNA and protein levels; also both were undetectable in monolayer cultures in all three tested cell lines. Using this system, melanoma spheroids and 2D-monolayer cultures were treated with the B-RAF(V600E) inhibitor, Vemurafenib. Then the cell viability and expression of signaling markers were compared between 2D and 3D cultures. Data from our MTT assays indicated that the 3D melanoma spheroids were more resistant to Vemurafenib than 2D-cultured cells by 20-30% in all three tested cell lines. We then employed a human phospho-kinase array (R&D System) to study multiple kinase pathways related to hypoxia, and found that the phosphorylated c-Met was significantly increased in 3D melanoma spheroids than 2D-cultured cells, and was confirmed by western blot. This data suggested that hypoxia drove c-Met activation in melanoma spheroids. By employing preclinical c-Met inhibitor in 3D culture, we found that it significantly prevent 3D-spheroid formation (40∼60%) and HIF-1α expression. Combined, evidence suggested that hypoxia drove c-Met activation and resistance to Vemurafenib in melanoma cells of 3D spheroids, which may partially reflect the cellular signature of melanoma cells under hypoxia in vivo. Taking advantage of the ability of c-Met inhibitor preventing melanoma spheroid formation, we proposed that combination of c-Met inhibitor and Vemurafenib was a potential therapy strategy to overcome resistant melanoma cells under hypoxia in patients. Further experiments on melanoma spheroids and mouse model to address this concept are in progress.
Citation Format: Yong Qin, Victoria R. Greene, Chandrani Chattopadhyay, Suhendan Ekmekcioglu, Chengwen Liu, Elizabeth A. Grimm. Induction of hypoxia in 3D human melanoma spheroids leads to c-Met activation and resistance to Vemurafenib. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2936. doi:10.1158/1538-7445.AM2013-2936