Cytokine preactivation and ADCC: a potent strategy for enhancing human NK cell effector functions against 3D tumor models

The adoptive transfer of NK cells has shown clinical promise in hematologic malignancies, but its efficacy in solid tumors remains limited. Three-dimensional (3D) models reproduce tumor architecture and immunosuppressive microenvironments more accurately than conventional two-dimensional (2D) cultures. Here, we employed colorectal cancer (CRC) and lung cancer 3D tumor models to evaluate the anti-tumor activity of cytokine-induced memory-like (CIML) NK cells and to test whether cetuximab augments these responses through antibody-dependent cellular cytotoxicity (ADCC). Human NK cells were preactivated overnight with interleukin (IL)-12/15/18 (pre-CIML), then co-cultured with tumor spheroids and patient-derived organoids in the presence or absence of cetuximab. Pre-CIML NK cells showed significantly enhanced effector functions compared to control NK cells, including increased degranulation, higher IFN-γ and TNF-α production, and superior cytotoxicity against both spheroids and organoids. Additionally, pre-CIML NK cells that infiltrated tumor spheroids displayed a more uniform distribution within the tumor mass than control NK cells, which may contribute to their improved killing capacity. Cetuximab-mediated ADCC further enhanced NK cell activity against spheroid models, while in organoids, the enhancement was tumor- and context-dependent. Overall, these findings demonstrate that pre-CIML NK cells exhibit robust anti-tumor activity in clinically relevant 3D tumor models, and identify ADCC as an additional, but context-restricted, mechanism by which targeted antibodies such as cetuximab can further enhance NK cell functionality. These findings underscore the translational potential of NK cell-based immunotherapies that combine cytokine preactivation and ADCC induction to overcome current challenges in the treatment of solid tumors.