Description
Chimeric antigen receptor (CAR) therapies have transformed hematological cancer treatment by enabling immune cells to specifically target tumour antigens. Natural Killer (NK) cells are emerging as a promising CAR platform due to their favourable safety profile and broader accessibility. However, NK cells exhibit pronounced functional heterogeneity, with only subsets capable of efficient cytotoxic and serial killing. How CAR expression influences this heterogeneity at the single-cell level remains poorly understood, as cytotoxicity bulk assays obscure individual cell behaviour.
To address this, we employ a droplet-based microfluidic platform to analyse CAR-NK cell cytotoxicity at single-cell resolution. Individual CAR-NK cells are co-encapsulated with multiple target cells, enabling imaging-based quantification of killing dynamics. Droplets are subsequently sorted based on cytotoxic activity, allowing downstream single-cell analysis of killer and non-killer populations.
Preliminary results show an approximately 10% increase in serial killers and monokillers among CAR-NK cells compared with untransduced controls, indicating that distinct killing phenotypes persist after CAR engineering. Sorted killer CAR-NK cells exhibit a trend toward lower CD16 expression, consistent with its known shedding during serial killing. Ongoing studies examine the impact of CAR density and different CAR constructs.
This platform will be extended to multi-omics profiling, including single-cell RNA sequencing, to identify molecular signatures underlying cytotoxic potency. These insights will inform NK cell engineering strategies and support the optimization of next-generation CAR-NK immunotherapies.