Integrative functional and multi-omics profiling reveals divergent natural killer cell responses and tumor cell plasticity in breast and ovarian cancer

Natural killer (NK) cells are key mediators of innate antitumor immunity and promising candidates for cell-based therapies, yet tumor cells frequently evade NK surveillance, particularly in advanced disease. To dissect these evasion mechanisms and identify points of intervention to enhance anti-tumor immunity, we evaluated the cytotoxic response of human primary NK cells toward primary breast cancer (BrCa) and ovarian cancer (OvCa) cells. NK cytotoxicity was assessed by cell surface expression of the CD107a degranulation marker, tumor cell (TC) survival by DAPI exclusion, and TC susceptibility using a fluorogenic protease assay. Responsive and non-responsive NK cells, as well as susceptible and resistant TCs from serial TC:NK co-cultures, were profiled by mini-bulk transcriptomics, low-input proteomics, and secretomics. NK degranulation and killing were consistently higher toward OvCa cells, whereas repeated BrCa exposure induced progressive loss of NK activity. NK receptor-ligand profiling showed stable receptor expression after OvCa co-culture but extensive remodeling upon BrCa contact. Secretome analysis revealed convergence of OvCa and NK secretory programs into a mixed phenotype, whereas BrCa suppressed NK-derived secretion and dominated the BrCa:NK secretome. These data highlighted a heterogenous NK response to different cancer entities and the control of NK activity by TCs. Multi-omics analysis of NK subsets showed glycolytic and proliferative enrichment in degranulating NK cells, whereas non-degranulating NK cells upregulated exhaustion and immunosuppressive markers absent in mono-cultured NK cells. These signatures were consistent across donors, across exposure to six BrCa/OvCa samples, and detected in NK cells from patients with advanced disease, underscoring clinical relevance. Across all six cancer cultures, NK-resistant TC subsets emerged with enhanced survival and a quiescent, metabolically low phenotype. In an orthotopic BrCa model, in vitro- generated resistant cells showed delayed tumor growth. Upon cell cycle re-entry during extended culture, resistant TCs regained NK susceptibility. Control and resistant TCs isolated from tumors two months post-engraftment were equally sensitive to NK killing, and in vivo NK therapy did not preferentially eliminate resistant TCs, indicating a plastic, reversible resistance state. These findings revealed reciprocal reprogramming between NK cells and TCs. NK cells acquired exhaustion, while TCs adopted temporary quiescence to evade killing. Targeting both NK dysfunction and TC plasticity will be essential for effective NK cell-based therapies for solid tumors.