Two morphological changes of cancer cells under natural killer cell killing

Abstract

Abstract Natural killer (NK) cells use granule exocytosis to kill cancer cells, which involves perforin-mediated delivery of granzymes from NK cells to cancer cells. Once inside the cancer cells, granzyme B, a member of serine protease family, activates caspase-3, which activates the serine/threonine kinase Rho-associated coiled coil–containing protein kinase (ROCK) I, an effector of the small GTPase Rho. Activated ROCK I increases the phosphorylation of myosin regulatory light chain and thus stimulates actomyosin contraction, required for apoptotic membrane blebbing. Ultimately, cancer cells under NK cell attack show rounding, shrinkage, and membrane rupture, which are classical morphological changes observed at the onset of apoptosis. Although much has been learned about these morphological changes, a fundamental question of how melanoma cell death is induced by NK cells remains unanswered. Do all melanoma cell lines undergo the classical dying process? If not, is there another dying process? To address this simple issue, we examined the dying of B16 melanoma cell lines under NK cell attack by using time-lapse imaging. Upon contact with NK cells, B16-F10 cells rounded and most of them showed membrane rupture (98 min); however, B16 parent cells showed writhing and delayed membrane rupture (235 min). This morphological difference depended on the expression levels of myosin regulatory light chain 9 (MYL9) but not activating ligands (CD112, CD155, Rae-1, and MULT-1), SPI, FasL, or PD-L1. Taken together, our data show that melanoma cells show two distinct types of morphological changes upon contact with NK cells and suggest that a strategy to decrease MYL9 expression by melanoma cells may improve the efficacy of NK cell–based immunotherapy.