Building on the pioneering work of Friedrich et al.
] and others, here we demonstrate a novel method for the rapid generation and quantitative analysis of tumor spheroid growth in HT format, with additional applications in further important aspects of the malignant phenotype. The assays can readily be established in any laboratory without specialized equipment and negate the need to purchase preformed spheroids, which may only be available for a limited number of cell lines. Although the quantitation is significantly enhanced by the use of automated cytometry, we have also deliberately exemplified its utility using standard microscopy to broaden its accessibility to the research community.
Recent advances in the development of three-dimensional cultures for cancer drug evaluation have focused mainly on methods that avoid cell surface adhesion and promote cell-cell attachment [12
]. Most are based on cellular aggregation on agarose-coated flat-bottomed plates [26
], poly-Hema-coated round-bottomed or V-bottomed plates [52
] or hanging drops [25
]. Although able to generate reproducibly-sized spheroids, all such methods rely on time-consuming procedures or the need to transfer spheroids from delicate hanging drops to plates for further analysis. More recently, advances in microtechnologies have engineered plates for low binding of cells to the surface [30
In pilot studies we compared different techniques for generating spheroids (bioreactor, agar-coated 96-well plates, poly-Hema-coated plates). We found that the ULA 96-well round-bottomed plate method is the least time consuming and generates the most reproducible spheroids. We have carried out a direct comparison with agar-coated plates and demonstrated that the tumor spheroids grown in ULA plates have equivalent growth rates, comparable gradients of proliferation and increased hypoxia in older spheroids but in general present a more compact and uniform structure. Also ULA plate-generated spheroids have the significant advantage that they are ideally suited to automated image analysis using a Celigo cytometer. Overall, the system (plus the additional simple extensions to key functional assays) represents an important advancement towards the routine use of three-dimensional cultures for preclinical oncology drug development.
HT three-dimensional growth assays must satisfy several key requirements: large scale, speed, simplicity, reproducibility and automation (both for set-up as well as readouts and data analysis). Here, we demonstrate that combining the use of ultra-low attachment round-bottomed plates with a Celigo cytometer, it is possible to utilize three-dimensional tumor spheroid assays in drug discovery projects in HT format. Building on this basic growth kinetic assay, we also successfully optimized de novo three-dimensional tumor spheroid-based functional assays in 96-well plates, for relatively HT formats: in situ three-dimensional tumor spheroid invasion into Matrigel, migration of cells from tumor spheroids on matrix proteins (also enabling detailed phenotypic analysis), and coculture of tumor spheroids with EBs representing tissue invasion with reciprocal angiogenesis.
In contrast to previously reported invasion assays [53
], our method avoids the need to move spheroids and allows true three-dimensional invasion rather than superficial interactions between spheroids placed on top of (or between) layers of matrix proteins. In the complementary assays of cell migration and tissue invasion/angiogenesis, preformed tumor spheroids are easily and quickly transferred onto matrix-coated wells or into coculture with EBs (uniformly generated using the same ULA plates as for spheroids), in a single step using multichannel pipettes. Our methods significantly accelerate and simplify the assays, but at the same time generate highly reproducible results. Furthermore, they combine real-time quantitative kinetic analyses with options for more detailed morphological and molecular investigations: for example, qualitative determinations of modes of cell motility; recovery of spheroids for genetic/phenotypic or pharmacodynamic analyses, target validation using cells in which genes of interest are genetically repressed or mutated.
The assays can easily be modified further according to specific requirements: migration/haptotaxis on different matrix proteins such as collagen, fibronectin, laminin, on endothelial cell monolayers, or (for glioblastomas) on recently described nanofiber scaffolds [15
]: invasion into alternative matrixes (for example, collagen) or, to avoid animal tissue derivates, fully synthetic biopolymers; microenvironment-enriched spheroids incorporating endothelial cells, immune cells and/or stromal cells [10
]. The current spheroid-EB coculture is designed to mimic xenograft tumor transplant systems (human tumors encountering mouse tissue/vasculature), the mainstay of preclinical drug evaluation studies, but human ESCs could be substituted to generate a fully human three-dimensional confrontation culture system.
In addition to optimization and streamlining of spheroid generation and a suite of functional assays, we have successfully automated imaging and quantitation of real-time spheroid growth and also invasion using a Celigo cytometer. We are currently developing further protocols for fully automated imaging and analysis of the migration and the coculture assays. These, together with the tumor spheroid growth kinetic and viability assays will allow a complete analytical package to bridge the gap between simple two-dimensional proliferation assays and in vivo studies. We are currently using these assays in a number of target validation and cancer drug discovery projects where our data suggest that they have the ability to reflect in vivo activity. Once fully validated for a wider range of agents and shown to have predictive value for therapeutic efficacy, we believe such assays will become a mainstay of the preclinical functional tumor assay portfolio and reduce the need for extensive testing in animal tumor models.