Snail-induced homotypic cell-in-cell structures are associated with colorectal cancer cell evasion of natural killer cytotoxicity.

Epithelial-to-mesenchymal transition (EMT) is a cellular process associated with cytoskeleton remodeling, aggressive phenotypes, and worse clinical outcomes. The formation of cell-in-cell (CIC) architectures, characterized by the encapsulation of one cell by another, is also associated with a similar set of cellular properties. Still, the relationship between these two phenomena remains poorly understood. Here, we examined the expression of central EMT regulators and the presence of CIC structure in colorectal cancer (CRC) sections, followed by investigating the impact of the EMT program on CIC architecture formation in CRC cell lines using time-lapse microscopy. We found that Snail immunoreactivity, but not Twist1 or Zeb2, was positively associated with CIC structures in CRC tissues. Notably, CRC patients with tumors positive for both Snail immunoreactivity and CIC structures exhibited worse overall survival, supporting a unique role for Snail in the formation of CIC architecture. In a co-culture assay, we showed that Snail-expressing CRC cells predominantly served as the outer cells in homotypic CIC architectures formed with control CRC cells rather than heterotypic CIC structures with an NK cell line, NK92-MI. The Snail-expressing CRC cells resisted NK92-MI cell-mediated contact killing. The formation of homotypic CIC architecture, independent of the RAC1-MLC2 axis, further enhanced the resistance of outer Snail-expressing CRC cells to NK-cell-mediated killing. Moreover, anti-E-cadherin antibody treatment abolished Snail-induced CIC formation. This observation suggests that Snail-expressing CRC cells may evade NK cell-mediated cytotoxicity, at least in part, through the formation of spatial CIC structures and the associated CIC-mediated cellular reprogramming. The detection and targeting of CIC structures may be critical for improving immunotherapeutic outcomes in Snail-dominant CRC patients.