Abstract 4699: Evaluating extracellular matrix invasion and cytotoxicity of natural killer cells in a novel co-culture assay
Authors: Mony Jyothi Thyagabhavan , Zhang Xiaoyu 
Published: March 2024

Abstract
Background: The complex tumor microenvironment (TME) in solid tumors presents unique challenges for lymphocytes used in cancer immunotherapy. The extracellular matrix (ECM) in TME can limit the recruitment of lymphocytes and restrict access to cancer cells. This study utilized Matrigel to study the kinetics of natural killer cell invasion and cytotoxicity. We hypothesized that the kinetics of tumor cell killing by the NKs would depend on the distance for invasion and rely on matrix-metalloproteinases (MMPs) for remodeling the ECM.
Methods: Increasing volumes (50-100 µL/well) of Matrigel (6 mg/mL) representing increasing distance for invasion was layered over MCF7-red target tumor cells expressing nuclear-localized mKate2 (red fluorescent protein). NK92 (Creative Bioarray) or eGFP-NK92 (BPS Bioscience) were seeded over the solidified Matrigel layer at E:T of 3:1. The broad spectrum MMP inhibitor, Ilomostat (GM6001, 2 µM and 10 µM), was added to the wells to investigate to role of MMPs. Label-free impedance and live cell imaging data was collected in real time on xCELLigence RTCA eSight to evaluate NK cell invasion and cytotoxicity. Percent cytolysis was calculated on the RTCA software from normalized cell impedance readings and imaging data using the formula [(1-(treated/untreated)*100].
Results: Impedance reading increases as the MCF7-red cells settle, attach, and proliferate. The readings plateau as the cells reach confluence. The addition of Matrigel modulates the impedance signature of MCF7 cells. Importantly, Matrigel delays cytolysis by NK92 cells added at 24h. The delay increases with increasing volume of Matrigel representing increasing invasion distance. The time to achieve 60% killing with respect to controls (KT60) was 67, 76 and 89 hours for 50, 75 and 100 µL/well, respectively. Consistent with the role of MMPs in invasion, percent cytolysis calculated independently from normalized impedance readings and live cell imaging data confirmed both delayed and reduced target cell killing by GFP-NK92 cells in the presence of Ilomostat. Interestingly, only a few GFP-NK92 were detected by live cell imaging when there was increased target cell death. These included highly active NK cells that made multiple contacts with different MCF7 red targets in clusters suggesting serial killing activity.
Conclusions: The observations that only a few NK92 cells are detected during the killing of susceptible target cells and that cytolysis is not abrogated by the inhibition of MMPs suggest a potential role for distal effectors like cytokines, independent of invasion. This is a novel 2.5D assay that demonstrates the potential of RTCA eSight platform for studying the invasion and cytotoxicity function of various immune cells through both real-time impedance recording and live-cell time-lapse imaging simultaneously. (For Research Use Only. Not for use in diagnostic procedures. RA45236.3472453704)
Citation Format: Jyothi Thyagabhavan Mony, Xiaoyu Zhang. Evaluating extracellular matrix invasion and cytotoxicity of natural killer cells in a novel co-culture assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4699.