CAIX role in human breast cancer cell migration and invasion

Abstract

Introduction Carbonic anhydrase IX (CA IX) is a highly overexpressed membrane protein in numerous cancers and is wellrecognized hypoxia marker with a promising diagnostic and therapeutic value [1]. CA IX regulates pH in hypoxic tumor cells and contributes to microenviromental acidosis, cell adhesion, migration and invasion [2]. To get better insight of CA IX role in the processes of cancer cells migration and invasion, we performed a genetic silencing of CA9 gene in triple negative breast cancer cells MDA-MB-231. Then the capabilities of cell migration and invasion were determined using 3D spheroid invadopodia formation method. Materials and methods Stable knockdown MDA-MB-231 lines, named CA9-1 (90% knockdown) and CA9-2 (0% knockdown, negative control), were generated using two Sigma lentiviral Ca9 shRNAs [TRCN0000349591 and TRCN0000319003] plasmids. Protein level was determined using Western blotting. The invadopodia formation of modified cancer cells were evaluated in 3D cancer cell spheroids. Cell suspension was made in a matrigel and collagen mix and placed into 8-well chambers. The drops solidified after incubation for 1 hour at 37 °C, and the high-serum medium was added. After 6 days of incubation spheroids with invadopodia were formed, and they were imaged by fluorescent microscopy. In inverse invasion assay, the cell suspension was placed on inverted transwell chamber containing collagen/matrigel/fibronectin inside. After the cells were attached to the filter, chambers were placed right way up in 24-well plates, containing free serum medium. On top of the matrix the medium with chemoattractant was added. After 7 days of incubation, the cells were stained with Calcein and imaged by confocal microscopy every 10 microns from the base membrane. All experiments were performed both in normoxia (21% O2) and hypoxia (1% O2) conditions. Results and discussion CA9 knockdown statistically significantly affected the invadopodia formation both in normoxia and h