Activity of Carbonic Anhydrase Inhibitors in Breast Cancer Models Aušra Želvytė1, Vilma Petrikaitė1,2 1Department of Biothermodynamics and Drug Design , Institute of Biotechnology, Vilnius University, Vilnius 2Department of Drug chemistry, Lithuanian University of Health Sciences, Kaunas E-mail: ausrazelvyte@gmail.com Introduction Activity in tumor spheroids Carbonic anhydrase IX has an important role in cancer progression by changing intra- and extracellular pH in response to hypoxia. CAIX is one of the main reasons why cancer cells adapt to the toxic conditions of the extracellular environment. It is known that inhibition of CAIX can affect tumor growth, cancer cell migration and invasion. The aim of our study was to test anticancer activity of several selective and non-selective CA inhibitors in 2D and 3D breast cancer models. Three-dimensional cultures (tumor spheroids) were formed from human breast cancer cell lines (MCF-7 and BT-474) in 96-well plates, using magnetic 3D bioprinting method [1]. After 48 hours incubation on magnetic drive spheroids have been formed. Then they were incubated with different concentrations (5 µM, 20 µM, 50 µM) of tested compounds. The medium with CA inhibitors or DMSO was replaced every 2 days. Photos of spheroids were made every 2-3 days using Nikon Eclipse Ti inverted microscope at 4× magnification. Images were analysed with ImageJ software. Carbonic anhydrase inhibitors Several hundreds of CA inhibitors have been synthesized at the Institute of Biotechnology. Compound binding to CAs was measured by isothermal titration calorimetry and thermal shift assay [2]. Several compounds bound to CAIX significantly stronger than CAI and CAII. This selectivity toward the cancer specific CAIX is very important for drug candidates as they could have less side effects. A B MCF-7 (A) and BT-474 (B) spheroids after 9 days of incubation with 20 µM of each inhibitor. The growth of both types of spheroids was mostly affected by EA2-3 and VD12-09, and this effect was concentration and time-dependent. A B Table. Binding constants (Kb, M) of the most selective CA inhibitors   CA I CA II CA III CA VB CA VI CA VII CA IX CA XII CA XIII VD12-09 4.0×104 1.0×106 5.0×102 3.0×106 4.0×105 8.0×106 1.5×109 5.0×106 1.0×107 EA2-3 6.0×104 4.0×106 2.5×104 9.0×104 6.0×106 3.0×109 3.0×107 2.0×107 EA4-2o 5.0×107 2.0×104 5.3×107 8.0×104 1.5×107 6.5×108 9.0×105 Growth kinetics of different tumor spheroids in the presence of different CA inhibitors. Compound VD12-09 was the most effective in 3D cancer model. Conclusions Cell growth inhibition Tested compounds did not show or showed very low antiproliferative and antimigratory activity in cancer cell 2D model in both normoxic and hypoxic conditions. Compound effect on different breast cancer cell lines was tested by using cell viability assay (MTT) in both hypoxic and normoxic conditions. Tested compounds inhibited the growth of cells only in concentrations of 50 µM and 100 µM. The activity of CAIX-selective inhibitors in 3D model (tumor spheroids) depends on the dose of compound and increases after longer incubation period. Obtained results show that tested inhibitors may be useful for further development as anticancer agents. References Souza GR, Molina JR, Raphael RM, et al. Three-dimensional tissue culture based on magnetic cell levitation. Nat Nanotechnol 2010, 5:291-296. Dudutiene V, Matuliene J, Smirnov A, et al. Discovery and Characterization of Novel Selective Inhibitors of Carbonic Anhydrase IX. J Med Chem 2014, 57:9435-9446. Activity of CA inhibitors in different breast cancer cell lines in normoxia and hypoxia.