Development of microwell-based technology for anti-tumor efficacy evaluation of cytotoxic lymphocytes in a single cell level

Abstract

Cytotoxicity exerted by cytotoxic lymphocytes against cancer cells is an essential cellular function for successful cancer immunotherapy. Adoptive cell therapy using ex vivo engineered/expanded immune cells demonstrated poor efficacy against solid tumor, despite its great success in treating various hematopoietic malignancies. To improve adoptive cell therapy, cytotoxicity assays assessing detailed information on cytotoxic lymphocyte-cancer cell interactions are needed. Standard cytotoxicity assays commonly performed only provide population level information, whereas live cell imaging-based cytotoxicity assays can assess single cell level heterogeneity, yet suffer from low throughputs. To address this issue, we developed microwell-based cytotoxicity assays that allow single cell-level evaluation of lymphocyte cytotoxicity by live cell imaging with improved throughputs. First, single hematological cancer cell arrays with immobilized hematological cancer cells were fabricated to assess single cell level natural killer (NK) cell cytotoxicity against hematological cancer cells. Depending on microwell surface adhesiveness and inter-microwell distances, distinct modes of NK-hematological cancer cell interactions that result in different NK cell cytotoxicity are observed. For microwell arrays coated with bovine serum albumin, which prevents cell adhesion, NK cells stably contacted cancer cells with rounded morphologies, whereas for microwell arrays coated with fibronectin, which triggers integrin signals, NK cell contacting cancer cells exhibited dynamic behaviors with elongated morphologies and constantly explored extracellular spaces by membrane extension. In addition, fibronectin on extracellular spaces facilitate NK cells detachment from leukemic cells after killing by providing anchorage for force transmission, and promote cytotoxicity and serial killing. Next, we sought to demermine the effects of extracellular adhesion provided by extracellular matrix of tumor microenvironment on immune cell cytotoxicity by devising microwell arrays coated with proteins either preventing or promoting cell adhesion. Solid tumor cells in bovine serum albumin-coated microwells did not attach to the surfaces and exhibited a round morphology, but solid tumor cell in fibronectin-coated microwells adhered firmed to the substrates with a flat shape. Live cell imaging was performed to examine dynamic cell-cell interactions and immune cell cytotoxicity at a single cell level. Both NK cells and T cells showed higher cytotoxicity against round tumor cells in bovine serum albumin-coated microwells compared to flat tumor cell in fibronectin-coated microwells, suggesting that extracellular adhesion-meditated firm adhesion of tumor cells made them more resistant to immune cell-mediated killing. Additionally, NK cell and T cells in fibronectin-coated microwells exhibited divergent dynamic behaviors, indicating that two distinct subsets of cytotoxic lymphocytes respond differentially to extracellular adhesion cues during target cell recognition. Taken together, microwell-based cytotoxicity assays developed in this study would not only be useful to evaluate lymphocyte cytotoxicity in a single cell level but also to study the role of signaling molecules in extracellular spaces on lymphocyte cytotoxicity.