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Updated: Jun 12, 2026
A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity
Published on: August 9, 2017
Cytotoxicity of activated natural killer cells against pediatric solid tumors
Duck Cho1, David R Shook, Noriko Shimasaki
1Department of Oncology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA.
Purpose:
To develop new therapies for children with solid tumors, we tested the cytotoxicity of natural killer (NK) cells expanded by coculture with K562-mb15-41BBL cells. We sought to identify the most sensitive tumor subtypes, clarify the molecular interactions regulating cytotoxicity, and determine NK antitumor potential in vivo.
Experimental Design:
We tested in vitro cytotoxicity of expanded NK cells against cell lines representative of Ewing sarcoma (EWS; n = 5), rhabdomyosarcoma (n = 4), neuroblastoma (n = 3), and osteosarcoma (n = 3), and correlated the results with expression of inhibitory and activating NK receptor ligands. We also compared expanded and primary NK cells, determined the effects of activating receptor ligation and of chemotherapeutic drugs, and assessed the therapeutic effect of NK cell infusions in xenografts.
Results:
In 45 experiments, EWS and rhabdomyosarcoma cell lines were remarkably sensitive to expanded NK cells, with median cytotoxicities at 1:1 effector/target ratio of 87.2% and 79.1%, respectively. Cytotoxicity was not related to levels of expression of NK receptor ligands, nor was it affected by pretreatment of target cells with daunorubicin or vincristine, but was markedly inhibited by preincubation of NK cells with a combination of antibodies against the NK-activating receptors NKGD2 and DNAM-1. Expanded NK cells were considerably more cytotoxic than unstimulated NK cells, and eradicated EWS cells engrafted in nonobese diabetic/severe combined immunodeficient Il2rgnull mice.
Conclusions:
Among pediatric solid tumors, EWS and rhabdomyosarcoma are exquisitely sensitive to expanded NK cells. The NK expansion method described here has been adapted to large-scale conditions and supports a phase I clinical study including patients with these malignancies.
Insights
Expanded natural killer (NK) cells show potent cytotoxicity against pediatric Ewing sarcoma (EWS) and rhabdomyosarcoma. This NK cell expansion method is scalable and supports a phase I clinical trial for these solid tumors.
Area of Science:
- Immunology
- Pediatric Oncology
- Cancer Therapy
Background:
- Natural killer (NK) cells are crucial for innate immunity and cancer surveillance.
- Developing novel immunotherapies for pediatric solid tumors remains a significant challenge.
- NK cell-based therapies offer a promising avenue for treating difficult-to-treat pediatric cancers.
Purpose of the Study:
- To evaluate the efficacy of expanded NK cells against pediatric solid tumors.
- To identify pediatric tumor subtypes most sensitive to NK cell cytotoxicity.
- To elucidate molecular mechanisms governing NK cell-mediated tumor killing and assess in vivo therapeutic potential.
Main Methods:
- NK cells were expanded using K562-mb15-41BBL coculture.
- In vitro cytotoxicity assays were performed against Ewing sarcoma (EWS), rhabdomyosarcoma, neuroblastoma, and osteosarcoma cell lines.
- NK cell-ligand interactions, effects of chemotherapeutics, and in vivo efficacy in xenograft models were assessed.
Main Results:
- Expanded NK cells demonstrated remarkable in vitro cytotoxicity against EWS (87.2%) and rhabdomyosarcoma (79.1%) cell lines.
- Cytotoxicity was not correlated with NK receptor ligand expression or affected by common chemotherapeutics (daunorubicin, vincristine).
- NK cell-mediated killing was dependent on NKGD2 and DNAM-1 receptor activation and eradicated EWS xenografts in vivo.
Conclusions:
- Ewing sarcoma and rhabdomyosarcoma are highly sensitive to expanded NK cells among pediatric solid tumors.
- The described NK cell expansion method is amenable to large-scale production.
- This approach supports an ongoing phase I clinical study for patients with these malignancies.
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