Natural killer cells (NKs) are lymphocytes of the innate immune system that target and kill cancer cells through the secretion of lytic granules containing granzyme B and perforin. This process requires the formation of a specialized cell-cell interaction region called the immune synapse (IS). Previous reports have established that the formation of the IS largely rely on sequential rearrangement of actin filaments within NKs. In contrast to the well-characterized configurations of actin filaments in NKs during the lytic process, there is a gap of knowledge regarding the organization and role of the tumor cell actin cytoskeleton during NK attack. 

In this project, we showed that the actin cytoskeleton of NK-resistant tumor cells accumulates at the IS. Using the high-throughput The ImageStream®X MKII Imaging Flow Cytometer and applying specific masks and features, we found that the majority of cells belonging to the resistant cell lines respond to NK cell attack by local and fast accumulation of actin near the IS. Furthermore, we showed that actin accumulation is associated with autophagosome clustering at the IS which drives the degradation of granzyme B inside the target cell. Moreover, our data suggest that the ability of cancer cells to respond to NK attack by fast actin remodelling is associated with their EMT status. We extended these data by analysing a large panel of breast cancer cells and showing that mesenchymal cells are significantly more resistant to NK-mediated cell death as compared to epithelial cells. 

Together our data demonstrates that resistant breast cancer cell lines respond to NK cell attack by a tumor actin remodelling and accumulation at the region of the IS. Targeting actin signalling pathways and regulators involved in these processes allowed to increase the susceptibility of resistant cells to NK cell-mediated cell death.