Advances in establishment and analysis of three-dimensional tumor spheroid-based functional assays for target validation and drug evaluation

Abstract

BackgroundThere is overwhelming evidence that in vitro three-dimensional tumor cell cultures more accurately reflect the complex in vivo microenvironment than simple two-dimensional cell monolayers, not least with respect to gene expression profiles, signaling pathway activity and drug sensitivity. However, most currently available three-dimensional techniques are time consuming and/or lack reproducibility; thus standardized and rapid protocols are urgently needed.ResultsTo address this requirement, we have developed a versatile toolkit of reproducible three-dimensional tumor spheroid models for dynamic, automated, quantitative imaging and analysis that are compatible with routine high-throughput preclinical studies. Not only do these microplate methods measure three-dimensional tumor growth, but they have also been significantly enhanced to facilitate a range of functional assays exemplifying additional key hallmarks of cancer, namely cell motility and matrix invasion. Moreover, mutual tissue invasion and angiogenesis is accommodated by coculturing tumor spheroids with murine embryoid bodies within which angiogenic differentiation occurs. Highly malignant human tumor cells were selected to exemplify therapeutic effects of three specific molecularly-targeted agents: PI-103 (phosphatidylinositol-3-kinase (PI3K)-mammalian target of rapamycin (mTOR) inhibitor), 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) (heat shock protein 90 (HSP90) inhibitor) and CCT130234 (in-house phospholipase C (PLC)γ inhibitor). Fully automated analysis using a Celigo cytometer was validated for tumor spheroid growth and invasion against standard image analysis techniques, with excellent reproducibility and significantly increased throughput. In addition, we discovered key differential sensitivities to targeted agents between two-dimensional and three-dimensional cultures, and also demonstrated enhanced potency of some agents against cell migration/invasion compared with proliferation, suggesting their preferential utility in metastatic disease.ConclusionsWe have established and validated a suite of highly reproducible tumor microplate three-dimensional functional assays to enhance the biological relevance of early preclinical cancer studies. We believe these assays will increase the translational predictive value of in vitro drug evaluation studies and reduce the need for in vivo studies by more effective triaging of compounds.

Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors

“…Scientists have been making an effort to develop standard and rapid protocols for using 3D cultures in drug screening. 71 Others seek simply to develop a reproducible 3D cell culture platform for high throughput screening through the identification and validation of ubiquitous three-dimensionality biomarkers, such as the cytokine family. 72 Table 2 summaries the key advantages and disadvantages of 3D cell culture systems for applications in drug discovery.…”

An Automated Multiplexed Hepatotoxicity and CYP Induction Assay Using HepaRG Cells in 2D and 3D

“…The evolution of microtissue cell culture, where cells aggregate into three-dimensional (3D) biomimetic structures, has the potential to bridge the gap between traditional in vitro and in vivo compound screens. 6 3D spheroids have shown drug responses more similar to those of an in vivo model than the traditional two-dimensional (2D) cell culture, in both cancer therapeutic efficacy [7][8][9] and hepatotoxicity. [10][11][12][13][14] This is particularly important for limited cell models, such as primary human hepatocytes cultured in a 2D monolayer.…”

A three-dimensional microenvironment alters protein expression and chemosensitivity of epithelial ovarian cancer cells in vitro

“…In the past decade, considerable advances have been made to establish cheaper and reliable methodologies for highthroughput phenotypic evaluation of cultured cells in 3D for drug development. 16,17 The continued hesitation to use 3D models for drug development more widely may largely be due to the limited data available detailing the changes in cellular phenotypes following the transition from a 2D to a 3D microenvironment. Many cell lines have not yet been well characterized as 3D models, and so systematic phenotypic evaluation of large numbers of cell lines is required to facilitate the use of those cell lines in 3D drug screening studies.…”