Abstract 4303: High-throughput 3D tumoroid models for immunotherapy and drug discovery
Authors: Nguyen Duy , McGhee Alexander , Pedro Diego , Pepe Alfonso , Schaller Matthew , Smolchek Ryan , Famiglietti Jack , Warrington Stephanie , Sawyer W. Gregory 
Published: April 2023

Abstract
The tumor microenvironment (TME) is a complex 3D cellular system comprised of a diverse amalgam of cells. To characterize the TME, we developed a 3D cell culture platform which facilitates imaging-based analysis and cell viability for extended durations. We used this platform to collect in-situ spatiotemporal measurements of cytokine concentrations in the tumor vicinity, track cellular activity, and model local tumor invasion of glioblastoma (GBM) using fast-scanning confocal microscopy. The mechanism by which we achieve long-term 3D culture is perfusion, modeled as flow through a porous medium. Perfusion facilitates regulated transport of nutrients and waste within the soft microgel medium: Liquid-Like Solids (LLS). The interstitial space is tuned to mimic a capillary bed, and surface bioconjugation of the microgels promotes cellular adhesion and migration. To measure cytokine concentrations, GBM tumoroids were grown in LLS and printed into a dispersion of ELISA beads in LLS. The mechanical stability of LLS ensures the tumor and ELISA beads remain stationary without impeding cellular activity. Cytokine on and off-rates were referenced alongside measured bead fluorescence intensities and positions to fit spatiotemporal reaction-diffusion models. Fast-scanning confocal microscopy facilitated in-situ observation of the evolutionary dynamics of tumor progression. Co-culture of patient-derived tumor explants and autologous PBMCs printed into type I collagen-bioconjugated LLS enabled studies of cancer-immune interactions. In-situ cytokine measurements revealed local IL-8 concentrations reached a maximum value of 2 ng ml−1 after 10 hours. A cellular production rate was estimated at 2 molecules cell−1 s−1. Invasive behavior into the proximal space was determined to be super-diffusive; off-lattice agent-based simulations indicated this behavior is a result of the confinement of invasive fronts to the microgel interstitium. The invading glioblastoma cells used anchorage-dependent migration and were guided by geometric cues to traverse the porous bioconjugated LLS network. Cancer-immune interaction studies revealed average CD8+ speeds greater than 2.8 µm min−1 and both chemotaxis and chemokinetic behavior. CD8+ T-cell killing rates were estimated at approximately 3 cancer cells hr−1 initially, monotonically falling over 12 hours to roughly 1 cell hr−1. The development of the in-situ 3D ELISA assay and imaging-based analysis techniques have enabled the tracking of tumor-immune cell interactions, observation of dynamic tumor progression, and local cytokine concentration profiling at appreciable spatiotemporal resolutions. The combination of a physiologically relevant 3D culture platform with the capacity for in-situ qualitative and quantitative observation may lead to new, powerful, preclinical models that allow for interrogation of the TME and decrease the rate of false discovery.
Citation Format: Duy Nguyen, Alexander McGhee, Diego Pedro, Alfonso Pepe, Matthew Schaller, Ryan Smolchek, Jack Famiglietti, Stephanie Warrington, W. Gregory Sawyer. High-throughput 3D tumoroid models for immunotherapy and drug discovery. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4303.