Abstract 35: 3D in vitro system for immuno-oncology: Real-time imaging of drug delivery, tumoroids, and immune cell activity

A Liquid Like Solid (LLS) 3D culture system in a convenient microtiter plate format enables long-duration culture of patient derived microtumors (>30 days), in situ confocal imaging, 3D cytotoxic drug studies, inclusion of T cells, and measurements of T cell migration, infiltration, and killing. Introduction: Cancer is a disease in 3-dimensions and there is a desperate need for new tools and infrastructure to study immuno-oncology treatment methods in 3D. Fabrication of microtumors using 3D printing in LLS media comprised of soft granular microgels (3-5 μm crosslinked polyacrylamide (PAA) microgel particles: 7.5% PAA) facilitate precise arrangement of detailed assemblies of extra-cellular matrix components and cells (1), including: epithelial cells (breast ATCC MCF10A), stem cells (bone marrow MSC), cancer cells (breast ATCC MCF7, prostate ATCC-PC3, osteosarcoma ATCC-MG63, melanoma ATCC-A375, primary glioblastoma, and osteosarcoma), fibroblasts (ThermoFisher dermal fibroblasts C0045C), endothelial cells (ThermoFisher HUVECs C0035C), and CD4+/CD8+ T cells (PBMC and Jurkat E6-1). Methods: Long term culture was enabled through the design, development, and validation of a modular perfusion system that uses passive negative pressure within a 96-well microtiter plate format to transport liquid growth medium, drugs (doxorubicin and puromycin), antibodies (aPD1 J43 clone, aCD3, aCD28), growth factors, FBS, and metabolic waste without disturbing the spatial organization and positioning of the experiments (i.e. 3D orientation is preserved and the packed bed of microgel particles remain solid). The perfusion velocities are precisely controlled to between 1-100 nm/s by setting the negative pressure, and complete exchange of liquid media can be tailored from hours to days. Results: Real-time imaging in these 3D assays is performed using in situ confocal microscopy under controlled perfusion. Cell motility, adhesion, and dynamic rearrangement of fibroblasts and endothelial cells within a 3D co-culture of microtumors evolved over the first 72 hours. Immunohistochemistry with Ki-67 and PCNA staining indicated active cell proliferation of the tumoroids after 28 days of continuous culture. Tracking of activated CD8+ T cells revealed super-diffusive motion in the presence of 3D tumors within a range of 250 µm. Activated T cell migration speeds have been measured to be between 1.3 and 2.0 μm/s in the 3D LLS media, and preliminary estimates of T cell migration forces are on the order of 1 nN. Conclusions: This integrated system of 3D bioprinting, perfusion culture plates, and confocal microscopy enables in situ 3D studies of cancer biology, immunotherapy, and drug treatment regimens and provides unique insights and measurements of immune cell invasion dynamics in 3D microtumors. References: 1. Battacharjee et al. Science Advances Sept. 2015 1:8 Citation Format: Eric O. McGhee, Alex J. McGhee, Derek L. Hood, Kylie E. Van Meter, Juan M. Uruena, Duane A. Mitchell, Catherine T. Flores, Steven C. Ghivizzani, C Parker Gibbs, Padriac P. Levings, Colin J. Anderson, W Gregory Sawyer. 3D in vitro system for immuno-oncology: Real-time imaging of drug delivery, tumoroids, and immune cell activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 35.