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bioRxiv · 10.1101/2025.05.04.651923

Single-Cell Analysis of NK Cell Cytotoxicity in Cancer Therapy Using Microfluidic Droplets

Ozcan, R. S.·Vahedi, F.·Namakian, S.·Ashkar, A. A.·Didar, T. F.
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Abstract

Natural Killer (NK) cells are critical components of the immune system, uniquely capable of detecting and eliminating cancer cells without prior sensitization. Here, a droplet-based microfluidic platform is introduced that enables real-time monitoring and single-cell analysis of NK cell-mediated cytotoxicity against K562 cancer cells. Distinct NK cells are evaluated to quantify key metrics, including the percentage of cytotoxic NK cells, serial killing capacity, killing time per target, NK-target contact duration, and migration velocities. The results demonstrated that expanded NK cells (exNK) exhibited longer attachments, superior cytotoxic activity, serial killing, and rapid killing dynamics, whereas peripheral blood NK cells (pbNK), especially when they were exposed to ascites tumor microenvironment (TME) (pbNK-asc), displayed reduced cytotoxic abilities in all parameters. Interestingly, expanded NK cells exposed to ascites TME (exNK-asc) retained partial functionality, indicating that expansion provides resilience against suppressive factors. In addition, cell velocity analysis further revealed that the presence of a cancer cell increases the migration of NK cells. This single-cell analysis provides novel insights into NK-cancer cell interactions, offering a robust framework for enhancing the efficacy of future immunotherapy applications especially for optimizing off-the-shelf NK cell-based immunotherapies. Table of Contents O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/651923v1_ufig1.gif" ALT="Figure 1"> View larger version (65K): org.highwire.dtl.DTLVardef@1f6e0b0org.highwire.dtl.DTLVardef@11971fborg.highwire.dtl.DTLVardef@18a2cdeorg.highwire.dtl.DTLVardef@87d4d9_HPS_FORMAT_FIGEXP M_FIG A droplet-based microfluidic platform examines how Natural Killer (NK) cells target cancer cells at the single-cell level. By comparing peripheral blood and expanded NK cells under normal and tumor-like conditions, distinct differences in attachment, serial killing, killing time, and migration are revealed. These findings provide insights that could enhance tumor targeting, particularly in off-the-shelf NK cell therapies. C_FIG

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Ozcan, R. S., Vahedi, F., Namakian, S., Ashkar, A. A., Didar, T. F.. 2025-05-09. Single-Cell Analysis of NK Cell Cytotoxicity in Cancer Therapy Using Microfluidic Droplets. https://doi.org/10.1101/2025.05.04.651923

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