Abstract B004: NK cells demonstrate robust cytotoxicity against ovarian high-grade serous carcinoma in 2D and 3D co-culture models
Authors: Yang Deyi , Ennis Darren P , Mahalingam Preethika , Burr Joanna , Brady Hugh J M , McNeish Iain A 
Published: February 2025

Abstract
Background: High-grade serous carcinoma (HGSC) is the most aggressive form of ovarian cancer. Relapse is frequent following current standard treatment combining surgery and platinum-based chemotherapy, leading chemoresistance that is ultimately fatal. Novel treatment options, including immunotherapy, are required for HGSC patients. Natural Killer (NK) cells have shown exceptional potential as an “off-the-shelf" adoptive cell therapy against various malignancies. However, there have been few studies specifically examining the interactions of NK cells from diverse sources against HGSC cells. In this study, we optimized a NK/HGSC co-culture system in both 2D monolayer and 3D spheroid models, tested the cytotoxicity of NK92 cell line on both established and primary HGSC cell lines, and subsequently explored the cytotoxicity of umbilical cord blood (UCB)-derived NK cells, harvested from five donors, against primary HGSC cultures in vitro. Method: NK92 cells, at effect-to-target (E:T) ratios of 1:1, 3:1 and 10:1, were added to established (PEO1, PEO4, OVCAR4) and primary (ASC-24-003, Br2-043, Br2-205) HGSC cells in 2D and 3D. Cytotoxicity was measured using both IncuCyte live-cell imaging and cell viability assays. To generate primary NK cells, UCB from 5 healthy donors was processed by Ficoll-Paque layering to isolate mononuclear cells (MNCs). Cord MNCs were depleted of CD3+ cells using magnetic cell separation. To expand UCB-NK, CD3- Cord MNCs were cultured in RPMI 1640 media, 10% FBS and 100IU IL-2 with sub-lethally irradiated feeder cell line K562 engineered to express membrane bound IL-21 for a minimum of 14 days. The expanded UCB-derived NK cells were added to HGSC cell lines (PE20-001, ASC22-030, Br2-043) at E:T ratios of 1:1, 3:1 and 10:1. The cytotoxic effects were measured by cell death assay. Result: NK92 cells at higher E:T ratios (3:1,10:1) significantly reduced viability of established cell lines growing in 2D. The viability of platinum-resistant PEO4 also had significantly decreased at E:T ratio of 1:1. The viability of all primary cells decreased significantly at all E:T ratios in 2D. Live cell imaging assays yielded comparable results using cell confluency. In the UCB-derived NK cell experiments, cells from every donor induced cell death in all HGSC cell lines at all E:T ratios in 2D. In 3D, every donor again induced cell death at all E:T ratios with the exception of PE20-001 cell line treated with donor 1 NK cells at 1:1 ratio. Cytotoxic effects of UCB-derived NK cells were largely induced within 24 hours. Donor 4 and 5 NK cells showed superior cytotoxicity to NK92 cells when added in the same E:T ratio at the same experiment setting. Conclusion: These results demonstrate the potential of adoptive NK cell therapy, especially with donor-derived NK cells, in treating HGSC cell models. Further in-vitro studies exploring inter-donor variability and spheroid penetration, in-vivo studies assessing the efficacy of donor NK in mouse models and genetic arming of stimulatory receptors are under way.
Citation Format: Deyi Yang, Darren P Ennis, Preethika Mahalingam, Joanna Burr, Hugh J M Brady, Iain A McNeish. NK cells demonstrate robust cytotoxicity against ovarian high-grade serous carcinoma in 2D and 3D co-culture models [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B004.