On The Role Of Experimental Microenvironmental Conditions In Targeted Inhibition Of The Ph-Regulating Carbonic Anhydrase Ix In Colorectal Carcinoma Cells

Tumor hypoxia, causing metabolic changes towards enhanced glycolysis and lactic acid production, is a common microenvironmental feature of solid tumors, and is recognized as a main mechanism both of therapeutic resistance and metastasis. The hypoxia-inducible factor-1α conducts transcription of a number of genes involved in adaption to the hypoxic condition. Among these is the tumor-specific carbonic anhydrase IX (CAIX), a membrane-bound protein that regulates extracellular (pHe) and intracellular (pHi) pH by catalyzing the reversible hydration of carbon dioxide (CO 2 ) to bicarbonate (HCO 3 - ) and protons, enabling tumor cell survival in the acidic microenvironment. To improve therapeutic outcome and reduce metastatic risk, targeted inhibition of CAIX is an attractive strategy. Here we report on effects of a CAIX inhibitor in a panel of colorectal carcinoma (CRC) cell lines, giving specific emphasis to the microenvironmental conditions. Five CRC cell lines were incubated for 24 hours under normoxic (21% O 2 ) or hypoxic (0.2% O 2 ) condition, either in the presence (22 mM HCO 3 - , 5% CO 2 ) or absence (0 mM HCO 3 - , 0% CO 2 ) of HCO 3 - in the culture medium. The cells were treated with 4-(3′-(3”,5”-dimethylphenyl)ureido)phenyl sulfamate (termed S4), considered to be a CAIX-specific inhibitor, and clonogenicity, pHe, and pHi (measured as cells labeled with a pH-sensitive dye) were assessed. In CO 2 /HCO 3 - -buffered medium under normoxia, differential inhibition of clonogenicity following S4 treatment was observed among the five cell lines, correlating with individual CAIX levels: strong suppression in cell lines with high CAIX expression, weaker S4 effect in cell lines with low expression, and no effect in the CAIX-negative cell line. When incubating with this medium under hypoxia, despite significant induction of CAIX, no additional effect of S4 on clonogenicity was observed. Of note, when changing the cell culture medium to CO 2 /HCO 3 - -free condition, S4 strongly inhibited clonogenicity of hypoxic HT29 cells (high CAIX expression) but not of the normoxic counterpart, while HCT116 cells (low CAIX expression) showed no clonogenic response to S4 with either oxygenation status. In all of the five cell lines, hypoxia caused significant acidification (decrease in pHe) of culture media. In parallel with the clonogenic response, S4 treatment significantly counteracted the hypoxia-induced pHe decline in HT29 cultures incubated in the CO 2 /HCO 3 - -free condition. pHi was not altered with any of the tested conditions. This set of data demonstrated that the CO 2 /HCO 3 - buffering system determined CRC cell response to targeted CAIX inhibition. To enable reliable assessment of experimental S4 effects, strict control of the microenvironmental conditions was required. We believe this information may be of note for future evaluation of this specific therapeutic approach. Citation Format: Helga Helseth Hektoen, Kjersti Flatmark, Kirsti Solberg Landsverk, Marie Gron Saelen, Kathrine Roe, Anne Hansen Ree. On the role of experimental microenvironmental conditions in targeted inhibition of the pH-regulating carbonic anhydrase IX in colorectal carcinoma cells. [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 3780. doi:10.1158/1538-7445.AM2014-3780