Circulating tumor cell clusters exhibit enhanced immune evasion from natural killer cells

Abstract During cancer metastasis, the ability to evade immunosurveillance is key to the survival of circulating tumor cells (CTCs). Recent studies have found that in addition to single CTCs, multicellular clusters of CTCs can be detected in patient blood. Strikingly, CTC clusters show higher metastatic potential than single CTCs, but the molecular mechanism is unclear. We hypothesize that CTC clusters adopt immune evasive strategies, which enhance their metastatic ability. In mouse models of breast cancer, CTC clusters show enhanced metastatic growth over single cells in immunocompetent mice, but this difference is abrogated in immunodeficient mice lacking NK cells or mice treated with NK cell-depleting antibodies. Adoptive transfer of NK cells into NK cell-deficient hosts selectively suppresses metastatic growth by single CTCs, suggesting that single CTCs are more sensitive to NK cells than clusters. Analysis of cell line and patient RNA-seq data reveal that CTC clusters have elevated expression of cell-cell adhesion and epithelial genes as compared to single cells, which is in turn associated with decreased expression of NK cell-activating NKG2D ligands. Furthermore, modulation of tumor cell epithelial status alters NK ligand expression and sensitivity to NK cell killing, but this effect is attenuated by the addition of an NKG2D-blocking antibody. Taken together, we propose a model in which the epithelial characteristics of CTC clusters promotes their resistance to NK cell attack and thus their overall metastatic success. These findings reveal that NK cells play an important role in determining the fate of CTCs of different epithelial/mesenchymal states, and illuminate our understanding of immune evasion mechanisms by CTCs.