Profiling Angiogenic Factors in Breast Cancer

To analyze the soluble factors involved in the angiogenesis process, a RayBio^® C-Series Human Angiogenesis Antibody Array C1000 (RayBiotech) was used according to the manufacturer’s instructions. This kit can detect 43 proteins involved in angiogenesis and invasiveness thanks to the antibodies spotted in duplicate onto nitrocellulose membranes. Briefly, at the end of the experiment (96 hours), the conditioned medium of MDA-MB-231 and BT-549 cells untreated or treated with STD and mCHT protocols were collected, centrifugated and stored at -20°C until further use. Membranes were incubated with blocking buffer for 30 minutes at room temperature and then incubated with conditioned medium overnight at 4°C on a rocking platform. After washing the membranes with the appropriate buffers, a biotinylated antibody cocktail was added to the well and incubated for 2 hours at room temperature. Following the washing steps, HRP-Streptavidin solution was added onto the membrane, incubated for 2 hours at room temperature and then detection buffer was added and quickly visualized afterward. The chemiluminescent signal was acquired using G:BOX XT4 Chemiluminescence and Fluorescence Imaging System (Syngene, Cambridge, UK). The signal was then quantified with ImageJ Software (Wayne Rasband National Institutes of Health, USA) and reported as the percentage of untreated control.