Functional profiling of an allogeneic NK cell therapy product in NSCLC patient-derived organotypic tumor spheroids

Allogeneic natural killer (NK) cell therapies offer an off-the-shelf strategy for targeting solid tumors, but their functional activity within complex human tumor microenvironments remains poorly defined. We evaluated an allogeneic NK-cell therapy product design (NK cells) and tested its performance using the Xsphera platform, which incorporates patient-derived organotypic tumor spheroids (PDOTS) embedded in extracellular matrix (ECM) and cultured in microfluidic devices that preserve tumor-immune-stromal architecture ex vivo. Assay conditions were first optimized using LS1034 tumor cell spheroids as target cells and cryopreserved NK cells to identify parameters that maximized NK cell activity, including ECM concentration, effector-to-target (E:T) ratio, exposure duration, and media composition. Under optimal conditions, NK cells induced up to 40% tumor spheroid killing, as quantified by live/dead imaging using Hoechst and propidium iodide staining. We next applied these conditions to PDOTS generated from five non-small cell lung cancer (NSCLC) patients. NK cells were introduced at an estimated 2:1 E:T ratio and cultured for 96 hours. Live/dead imaging revealed significant cytotoxic responses in 2 of 5 PDOTS, with live tumor area reductions of up to 50%. The NK cells expressed activation markers (NKG2D, CD69) and secreted elevated levels of interferon-gamma, tumor necrosis factor, and granzyme B. Transcriptomic profiling of the media revealed enrichment of interferon-stimulated genes and cytotoxic effectors. Notably, these molecular features did not correlate with tumor responsiveness. These findings demonstrate the utility of the PDOTS platform for functionally profiling NK cell potency in physiologically relevant tumor models and support its use in the rational development of next-generation NK cell therapy products for solid tumors.