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

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Abstract

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.