Abstract B32: Microfluidics to study solid tumor-NK cell interactions: From migration and cytotoxicity to therapeutic antibodies
Authors: Ayuso Jose Maria , Truttschel Regan , Gong Max M. , Humayun Mouhita , Gillette Amani , Patankar Manish , Skala Melissa C. , Beebe David J. 
Published: February 2019

Abstract
Introduction: Solid tumors develop a complex microenvironment; that enables immune surveillance escape. In solid tumors, cancer cells form a dense mass where antibody and immune cell penetration is significantly hindered. Additionally, tumor cell metabolism leads to hypoxia, nutrient starvation and acidic pH; that dampens immune response. Therefore, to translate the success of immunotherapies in hematological cancers to solid tumors; there is need for new in vitro models that recapitulate this complex tumor microenvironment (TME). In this context, microfluidics offers an opportunity to generate sophisticated in vitro systems, combining multiple cell types and capturing in vivo spatial organization. Here, we present a microfluidic model that mimics the TME of solid tumors and apply it to study adoptive immunotherapies and therapeutic antibodies.
Materials and Methods: In our model, a spheroid of MCF7 breast cancer cells was embedded in a 3D collagen I matrix. Multiple luminal structures were generated through the matrix adjacent to the spheroid and lined with endothelial cells (e.g., HUVECs, IPSC-EC) to form blood vessels. Activated immune cells (e.g., NK-92 cells) and/or therapeutic antibodies (e.g., IL-2 conjugated anti-EpCAM) were perfused through these blood vessels. Antibody diffusion, cell migration and cytotoxicity were subsequently measured by fluorescence and confocal microscopy.
Results and Discussion: A small percentage of NK cells extravasated from the blood vessel into the matrix. Once within the extracellular matrix, NK cells exhibited rapid migration; however, we did not observe a directional response towards the tumor spheroid. In fact, the NK cells that reached the spheroid followed a random, tortuous path. Taken together, these initial results point out that immune extravasation and “homing” (i.e., the capacity to track and migrate towards tumor cells) could be engineered to improve treatment efficacy.
After several days in culture, the presence of NK cells induced significant cytotoxicity in the MCF7 spheroid. NK cell-mediated cytotoxicity occurred at the surface of the tumor spheroid; whereas tumor cells in the core remained unaffected. Different therapeutic antibody formulations were perfused through the adjacent lumen to enhance NK-mediated cytotoxicity. Fluorescently-labelled antibodies diffused through the blood vessel, reaching and coating the surface of the spheroid in a few hours. However, penetration of the core was dramatically slower; after 3 days, only the most outer cell layers of the spheroid were stained. The antibody remained attached to the tumor cell membrane for multiple days, showing no signs of internalization. Therefore, this model could be used to study the efficacy of antibody-dependent therapy, in particular, short and repeated cycles of antibody/immune cell injection.
Conclusion: The TME can dramatically limit the immune response against solid tumors. The presented microfluidic model was used to study NK cell extravasation, migration and tumor cytotoxicity in 3D. This microfluidic model provides a TME that more closely mimics in vivo conditions compared with standard assays. Therapeutic antibodies were also evaluated; demonstrating this model can be applied to find the optimal protocol for adoptive immunotherapies combined with therapeutic antibodies.
Citation Format: Jose Maria Ayuso, Regan Truttschel, Max M. Gong, Mouhita Humayun, Amani Gillette, Manish Patankar, Melissa C. Skala, David J. Beebe. Microfluidics to study solid tumor-NK cell interactions: From migration and cytotoxicity to therapeutic antibodies [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B32.