Abstract PO004: Reverse-engineering the tumor microenvironment through microfluidics and bioengineered in vitro models
Authors: Ayuso Jose M. , Farooqui Mehtab , Virumbrales-Munoz Maria , Rehman Shujah , Skala Melissa C. , Beebe David J. 
Published: March 2021

Abstract
Scope: Solid tumors are complex 3D systems where environmental gradients and cellular interactions shape tumor evolution and patient outcome. In this work, we developed bioengineered microfluidic in vitro models to study how environmental factors affect immune exhaustion.
Methods: In this work, we fabricated a microfluidic model that mimicked the tumor architecture to study tumor-immune interactions. Breast cancer cells were cultured as a dense mass and embedded in a 3D collagen hydrogel inside the microfluidic device. The model also included two lateral lumens on the flanks, allowing us to seed endothelial cells and mimicking the cylindrical structure of blood vessels. These biomimetic blood vessels were used to perfuse culture media, antibodies (e.g., anti-PD-1, immunocytokines), or immune cells (e.g., natural killer cells, CD4 T cells). We used a combination of fluorescence and multi-photon microscopy to monitor antibody and immune cell extravasation, migration, and tumor clearance in real-time. We retrieved the cells from the model (i.e., endothelial, tumor, CD4, and natural killer cells) to analyze them by RT-qPCR and functional assays (e.g., proliferation rate, migration speed, natural killer cell killing potential).
Results: The results demonstrated that tumor metabolism rapidly led to nutrient starvation and acidic pH in the inner regions of the model. Consequently, tumor cells showed different metabolism and proliferation rates near the biomimetic vessels compared with the core of the model. We used metabolic inhibitors targeting multiple metabolic pathways (e.g., glycolysis) to selectively destroy tumor cells depending on their location in the model. Real-time microscopy revealed that natural killer cells were able to detect the presence of tumor cells from several hundreds of microns away, exhibiting directional migration towards the tumor cells. Molecular analysis revealed that as natural killer cells reached inner regions, they exhibited progressive signs of immune exhaustion (e.g., upregulation of PD-1, downregulation of granzymes). Interestingly, when retrieved from the model and cultured alone in traditional flasks, natural killer cells remained exhausted for an extended time, highlighting the long-lasting effects of the tumor microenvironment. The use of antibodies (e.g., anti-PD-1), immunocytokines (e.g., IL-2-coupled antibody), or metabolic inhibitors (e.g., IDO-1 inhibitors) partially prevented natural killer cell exhaustion at the core, improving their killing potential.
Conclusions: tumor-immune interactions in the tumor microenvironment are extremely complex. Bioengineered microfluidic models offered a versatile tool to monitor the natural killer cell exhaustion, allowing us to identify multiple molecular factors driving the process. We used this knowledge to test several drugs and antibodies to prevent natural killer cell exhaustion and improve tumor killing.
Citation Format: Jose M. Ayuso, Mehtab Farooqui, Maria Virumbrales-Munoz, Shujah Rehman, Melissa C. Skala, David J. Beebe. Reverse-engineering the tumor microenvironment through microfluidics and bioengineered in vitro models [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO004.