Abstract 5057: Activation of stroma-derived matrix metalloproteinases (MMPs) in a model of gastric cancer.

Gastric cancer is the second commonest cause of cancer-related death worldwide. Within these tumors, it is increasingly recognised that signaling between myofibroblasts, an important stromal cell type, and cancer cells results in proliferation, invasion and metastasis. However, little is known of the range of proteins secreted by stromal cells ie their secretome, or the contribution of proteases in shaping the extracellular milieu ie the cancer degradome. Using SILAC labeling and combined fractional diagonal chromatography (COFRADIC) we identified and quantified differential abundance and appearance of neo-N-termini (ie cleavage products) in the secretomes of gastric cancer-associated myofibroblasts (CAMs) compared with myofibroblasts from adjacent tissue (ATMs). There was decreased abundance of many proteins in CAM secretomes, but a small sub-set exhibited increased abundance including matrix metalloproteinases (MMP) -1 and -3. Cancer-restricted proteolytic cleavages included cleavage in the prodomains of MMP-1, -2 and -3 consistent with activation. Western blot, enzyme activity and cancer cell migration assays verified significantly increased abundance of active MMP-1, -2 and -3 in CAM media. To establish the in vivo relevance of these findings we utilized a model in which primary gastric CAMs stimulate growth of MKN45 gastric cancer cells in xenografts in nude mice. MMP activity was imaged by fluorescence molecular tomography (FMT) employing MMPSense750 FAST™, which produces a fluorescent signal following MMP cleavage. CAMs enhanced xenograft growth by 6-fold (mean ± SEM, 29.6±21.1 vs 196.8±32.2 mm3 n=12, p=0.004). Using an experimental design that achieved similar volumes for tumors containing MKN45 cells alone and those containing MKN45+CAM, FMT assessment of tumor-related MMP activity in vivo indicated a two-fold increase in CAM-MKN45 xenografts compared with MKN45 cells alone (0.9±0.4 vs 1.8±0.8 fmol/mm3). Stromal cell secretomes therefore contribute to the remodelling of the cancer cell microenvironment by activation of MMPs with consequences for cell migration and tumor growth. This system has potential for in vivo screening of anti-cancer therapies directed at the inhibition of stroma-stimulated tumor growth. Citation Format: Sandhir Kandola, Christopher Holmberg, Bart Ghesquiere, Francis Impens, Kris Gevaert, J. Dinesh Kumar, Nicole Cash, Peter Hegyi, Nikolina Vlatkovic, Timothy Wang, Graham Dockray, Andrea Varro. Activation of stroma-derived matrix metalloproteinases (MMPs) in a model of gastric cancer. [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 5057. doi:10.1158/1538-7445.AM2013-5057