Abstract 3455: Cystatin C decreases proliferation of melanoma cells by affecting mitosis length

Cystatin C is a small secreted protein, produced by most cells in the body and found in significant quantities in all body fluids. It is the major inhibitor of extracellular cysteine protease activity in mammals because of its rapid binding to virtually all known cysteine proteases of families C1 (cysteine cathepsins) and C13 (mammalian legumain). Cystatin C has also been shown to inhibit viral replication within cells and display protective effects against cancer cell invasion. The protein has recently been shown to get internalized into cancer cells, providing an explanation to possible intracellular effects of the inhibitor. The purpose of the present study was to elucidate possible direct effects of cystatin C on cancer cell growth, by use of holometric imaging to analyze proliferation rate as well as to monitor the mitosis phase in real-time. Human A375 melanoma cells were cultured with and without addition of physiological quantities of cystatin C added to the culture medium (1 micromolar final concentration). The cultures were monitored for up to 3 days in a standard incubator using a HoloMonitor M4 instrument. Settings were optimized to allow accurate counting of cells throughout the experiment by analysis of images captured, revealing an inhibitory effect on overall cell proliferation rate by cystatin C addition to the cultures. To analyze the migration and cell division of individual cells, images were captured at short intervals allowing tracking and monitoring of cell morphology with respect to parameters such as thickness/density and area. Parameters could be set to define start of the mitosis phase indicated by a distinct rounding-up of cells and followed until a natural stop at cytokinesis, whereafter the daughter cells could be continuously tracked. These analyses revealed qualitative differences in the normal migration behavior of the A375 cells under study as a result of cystatin C addition. Moreover, the mitosis phase was considerably prolonged in cells grown in cystatin C containing medium, indicating that the proliferation-inhibiting effect of the protease inhibitor may be through its direct action on proteolytic processes in the actual mitosis phase. This finding justifies in-depth studies on additional cancer cell types to reveal how general the anti-proliferative effect of cystatin C is and how it can be explored to possibly control cancer cell growth and spread. The use of real-time holometric imaging of growing cells should be a most useful tool in such studies. Citation Format: Hanna Wallin, Kersti Alm, Magnus Abrahamson. Cystatin C decreases proliferation of melanoma cells by affecting mitosis length [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3455. doi:10.1158/1538-7445.AM2017-3455