(K-433) Using a 3D microphysiological system to describe characteristics of tissue-resident NK cells and conventional NK cells in the tumor microenvironment

Introduction:: Natural Killer (NK) cells are cytotoxic lymphocytes in the innate immune system that can recognize and eliminate target cells without prior sensitization. Their clinical advantages and broad specificity for tumor cells make them an attractive approach for cancer immunotherapy compared to T-cell based therapies. Tissue-resident NK cells (trNK), which resemble intraepithelial group 1 innate lymphoid cells (ieILC1s), are found in many normal and malignant tissues and have been shown to have enhanced cytotoxicity compared to conventional NK cells (cNKs) in the tumor microenvironment [1]. Given the presence of trNK cells in tumors, we investigated functional differences between cNKs and trNKs in a three-dimensional environment. A microphysiological system was utilized to recapitulate the tumor cell-vascular interface in the tumor microenvironment and analyze the ability of both cell types to infiltrate and eliminate tumor cells. This approach provide insights into the interaction of different NK subsets with the TME and has significant potential to be used in clinical applications for the assessment of NK cell-based cancer immunotherapy.


Materials and Methods:: The injection-molded 3D microphysiological system comprises three microchannels. The central channel contains head and neck cancer cells (FaDu or PCI-13 cells) and normal human fibroblasts embedded within fibrin, and an endothelial cell monolayer. NK cells were isolated from healthy donor blood using RosetteSep NK cell Enrichment Mixture and differentiated in vitro by co-culturing with tumor cells and stimulating with human recombinant IL-15 based on previously established protocols. After staining for surface markers (CD103, CD49a, and CD56), NK cells and ieILC1-like trNK cells were analyzed by flow cytometry and then introduced into the microphysiological system to evaluate their cytotoxicity and infiltration capability in a 3D environment. Florescence images were captured using confocal microscopy.


Results, Conclusions, and Discussions:: In vitro differentiated ieILC1-like trNK cells and activated cNK cells from the same donor were added to the microphysiological system to assess migration capacity and cytotoxicity against tumor cells. Quantitative analysis was performed to determine the number of cells migrating towards tumor cells and their distance of travel. It was observed that a higher percentage of ieILC1-like trNK cells (approximately 45%) migrated into the extracellular matrix and traveled a longer displacement compared to cNK cells. To make the microphysiological tumor environment more representative of a solid tumor microenvironment, immunosuppressive squamous cell carcinoma cells were used under low oxygen conditions. The resulting immunosuppressive TME demonstrated distinct immune responses between ieILC1-like trNK cells and cNK cells. The results of this study demonstrated that this system provides a promising approach to advance our understanding of NK cell biology and the complex interactions within the tumor-immune microenvironment, potentially leading to the development of more effective NK cell-based cancer immunotherapies.


Acknowledgements (Optional): : This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number : HI19C1352).

References (Optional): : [1] Moreno-Nieves UY, Tay JK, Saumyaa S, Horowitz NB, Shin JH, Mohammad IA, Luca B, Mundy DC, Gulati GS, Bedi N, Chang S, Chen C, Kaplan MJ, Rosenthal EL, Holsinger FC, Divi V, Baik FM, Sirjani DB, Gentles AJ, Newman AM, Freud AG, Sunwoo JB. Landscape of innate lymphoid cells in human head and neck cancer reveals divergent NK cell states in the tumor microenvironment. Proc Natl Acad Sci U S A. 2021 Jul 13;118(28):e2101169118