Non-destructive platform for morphologic and cell density characterization of developing and drugged multicellular tumor spheroids

Abstract

In vitro tumor models are important tools for exploring cancer progression and novel therapeutic strategies. These models are often oversimplified and lack many features of the 3D tumor environment, limiting their ability to simulate in vivo tumor behavior. Recapitulating key features of the tumor microenvironment, particularly 3D structure, is critical for the next generation of in vitro cancer diagnostic tools. Emerging 3D tissue-engineered in vitro models, such as multicellular tumor spheroids (MCTSs) and organoids, are a promising solution to this challenge. These cell aggregates have the ability to mimic several key aspects of in vivo tumors, such as 3D structure and pathophysiological gradients, and possess great promise for high-throughput in vitro testing. However, lack of standardization of MCTS fabrication has led to a large spectrum of “spheroid” and ”organoid” models, few of which are able to replicate the necessary sphericity for physiologically-representative behavior. This discrepancy is thought to be a major contributor to the high failure rate in drug discovery, where only a low percentage of drugs investigated in vitro succeed in clinical trials. As an additional challenge, the required size and shape of these in vitro tumor models precludes them from conventional microscopy, thus limiting our ability to use these models to collect data. Advancement of these MCTS models relies heavily on our ability to characterize and assess them. Herein, I propose an image-based analytical tool capable of characterizing mesoscopic (∼ 3mm thick) samples. Based on Optical Coherence Tomography (OCT), a structural imaging modality, this approach will be non-destructive and maintain cell-scale resolution while also boasting a field of view sufficient for full aggregate viewing. Using this tool, we can characterize morphology, cellular density, and cell viability of MCTSs, providing key longitudinal information on tumor model development and response to drug. The non-destructive nature of this technique uniquely positions us for longitudinal investigations within singular aggregates. One such application we sought to explore is the acquired resistance of cancer cells to pharmacologic anti-cancer treatments, a complex issue that can add substantial challenges to treatment and eradication of solid tumors. Recentstudies have shown that following initial treatment, indications of sustained health and increased aggressiveness have been observed in cells, which may serve to promote cell proliferation/migration rather than eliminating the cells as intended. Utilizing OCT/Imaris to assess aggregates made from cells that have been exposed to a drug and later treated again with the same drug, we can non-destructively assess changes in cell density/viability in response to different priming concentrations/drugs. The goal of this study is to establish a platform for studying the effects of drug-exposure on subsequent drug response. Overall, this research developed a non-destructive tool for analysis of tumor spheroid morphology, cell density, and cell viability within dense cellular aggregates. When applied to aggregates that were primed and subsequently treated with the same drug, the tool provided evidence of cell density spikes/dips that mirrored clinical acquired resistance observations. In summary, this tool holds great promise for advancing multicellular tumor spheroid use, especially toward drug screening applications.