Abstract 3941: Manipulating the tumor microenvironment for therapeutic benefit: identifying mechanisms of buffer resistance.
Authors: Bailey Kate M. , Wojtkowiak Jonathan W. , Hashim Arig A. Ibrahim , Gillies Robert J. 
Published: August 2014

Abstract
The acidic microenvironment is a common physiological phenomenon of most solid tumors that can be attributed to increased fermentative metabolism combined with poor perfusion. Acidic niches provide a highly selective environment promoting carcinogenesis and malignancy. We previously demonstrated that neutralizing acidosis with buffers reduced spontaneous or experimental metastasis in-vivo. This effect is due to buffering, because by reducing the buffering capacity of lysine five-fold by titrating the solution from pH 10 to pH 8.4 (below the second of three pKa's), we observed a correlating decrease in efficacy in-vivo. In these studies, some tumor types, notably LL/2 and B16-F12 cells are resistant to buffer therapy. To examine the mechanisms that lead to resistance, we first determined their metabolic profiles using a Seahorse XF96® to measure glucose-dependent acidification rates (ECAR) and oxygen consumption rates (OCR). Contrary to our hypothesis, these data showed that the resistant lines, LL/2 and B16-F12 had significantly higher basal OCR and lower glucose-stimulated ECAR, compared to PC3M cells (buffer sensitive). PC3M cells also had low spare glycolytic capacity, suggesting they are at or near maximal glycolysis in their basal state. Glucose consumption assays confirm increased rates of glucose consumption by PC3M cells, confirming a glycolytic phenotype. Hence, resistance to buffer therapy is not simply a consequence of higher metabolic proton production rates. Acidosis has been shown to stimulate lysosomal redistribution and protease secretion in-vitro. Thus, an alternative hypothesis is that protease activation may be a mechanism by which low pH stimulates metastasis in-vivo. To observe protease activity in vivo, MMPsense® 680 and Prosense® 750EX activatable probes, which measure MMP and cysteine cathepsin activity respectively, were injected intravenously into nu/nu mice bearing bilateral LL/2 and PC3M tumors. Mice were provided either tap water or 200 mM lysine, ad lib for the duration of the experiment. Mice were imaged 24 hours following probe's injection using a Perkin-Elmer FMT 2500. LL/2 tumors (buffer resistant) had significantly higher basal cysteine cathepsin activity than PC3M tumors in control mice, and these decreased upon lysine treatment. MMP activity between LL/2 and PC3M tumors was not different in control mice, however, MMP activity increased significantly in LL/2 tumors with buffer therapy. In vitro 3D-DQ-collagen IV degradation assays correlate with protease activity seen in vivo. These data suggest that buffer-sensitive cells rely on acid production from glycolysis for metastatic potential, whereas resistant lines metastasize through a pH-independent proteolytic mechanism.
Citation Format: Kate M. Bailey, Jonathan W. Wojtkowiak, Arig A. Ibrahim Hashim, Robert J. Gillies. Manipulating the tumor microenvironment for therapeutic benefit: identifying mechanisms of buffer resistance. [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 3941. doi:10.1158/1538-7445.AM2013-3941