April 1, 2013 (Vol. 33, No. 7)
Gabriel J. Benton, Ph.D.
What do the high cost of cancer drug development and inconsistencies in many cancer research studies have in common? The most direct answer is that the in vitro models currently in use do not accurately represent the disease of interest. Specifically, current methodologies lack either a physiological context and/or reproducible format for assessing tumor cells in vitro.
Currently, the most popular in vitro method for compound screening and pathway analysis involves culturing cancer cells on rigid, tissue culture treated plastic surfaces where the cells adhere nonspecifically and proliferate as a monolayer. In this format, these cells lose both morphology and gene expression profiles associated with tumors in vivo. Cells may be suspended in extracellular matrix hydrogels to construct 3-D cultures, which provide ligands and a malleable surface to promote the physiological cell program; however, the resulting structures are dispersed throughout the gel, exhibiting significant variability in morphology and size, limiting the establishment of physiological gradients and adversely affecting the reproducibility of each assay.
To address issues of reproducibility and to build more physiological tumor systems, well-established methods for multicellular spheroid formation were incorporated into 3-D culture models. Researchers have been using spheroid cultures for over 70 years, and this format has been utilized for several cancer models. Furthermore, it has been documented that the addition of extracellular matrix proteins during spheroid formation promotes the assembly of cell-cell bonds, promoting cell aggregation and spheroid formation for many cancer cell models that were previously thought to be incompatible with this format.
Trevigen has optimized this process, providing the necessary reagents to evaluate your cells using this method. Simply harvest cells, resuspend in spheroid formation ECM, and then culture in a 96-well spheroid formation plate. Spheroids generally form in 48 to 72 hours. Cell number and culture time determines spheroid size, and since each well produces one spheroid, researchers have complete control over spheroid dimensions with virtually no well-to-well variability.
For most tumor models, we recommend spheroids between 400–500 µm in diameter. This is sufficient to establish physiological gradients for nutrients, oxygen, pH, and catabolites due to limitations in diffusion through multicellular layers.
Another effect of these gradients is the establishment of heterogeneous cell populations with necrotic cells in the core, quiescent cells in the deeper layers, and proliferating cells on the spheroid surface; all of these factors reminiscent of an avascular tumor. Once formed, these multicellular tumor cell aggregates can be treated with pharmacological compounds to evaluate the effect on tumor spheroid growth; alternatively, specific genes or pathways may be altered to evaluate its effect on expansion of the in vitro tumor. This process can be monitored in real-time and label-free using image analysis software to measure spheroid area and/or quantitated at an end point using cell viability reagents, such as the MTT assay.
Tumor cell spheroids also represent an opportunity to evaluate processes and modulators of cancer cell dissemination from the tumor, modeling early metastatic events. Using the same ECM-modulated process for spheroid formation, an invasion matrix comprised of basement membrane proteins and collagen I is applied to the tumor spheroid, fully embedding it. The invasion matrix forms a hydrogel network on which the cells can invade out of the tumor spheroid and into the surrounding matrix in a time-dependent manner. Again, the impact of pharmacological compounds and genetic/pathway manipulation can be evaluated to determine the functional impact on this process.
3-D Spheroid Proliferation / Viability
The Cultrex 3-D Spheroid Proliferation/Viability Assay was used to evaluate the efficacy of the anticancer compound etoposide for MDA-MB-231 human breast cancer spheroids in Figure 1. Etoposide is a commonly used chemotherapeutic, anticancer agent that targets rapidly proliferating cancer cells by forming a complex with DNA and topoisomerase II, causing breaks in the DNA and promoting cell death. Here, MDA-MB-231 human breast cancer cells were resuspended at 60,000 cells/mL in spheroid formation ECM, and 3,000 cells (50 µL) were added to each well of the spheroid formation plate.
Spheroids were formed over a 72 hour period, and were treated with varying amounts of etoposide over a 96-hour period. The progression of MDA-MB-231 proliferation and spheroid expansion over the 96-hour period is demonstrated in Figure 1A. Samples treated with etoposide were evaluated for spheroid expansion by evaluating spheroid area (red) and cell viability using the MTT Assay (blue) in Figure 1B. Increasing the dosage of etoposide inhibited expansion of the MDA-MB-231 tumor spheroid, yielding a proportional effect on cell number, as demonstrated by the MTT Assay.
3-D Spheroid Invasion
The Cultrex 3-D Spheroid BME Invasion Assay was used to evaluate the efficacy of the anticancer compound sulforaphane for invasion of MDA-MB-231 human breast cancer spheroids. Sulforaphane is a bioactive compound found in cruciferous vegetables such as broccoli and cauliflower that exhibits anticancer properties in experimental models. It is a phytochemical that belongs to the isothiocyanate group of organosulfur compounds that inhibits histone deacetylase activity.
Here, MDA-MB-231 spheroids were formed as indicated above. In addition, an invasion matrix (50 µL) was added to each well and incubated at 37°C for one hour. The invasion matrix fully embeds the tumor spheroid in a hydrogel network on which the cancer cells can invade out of the spheroid and into the surrounding matrix, see Figure 2A for a time lapse progression of 3-D cell invasion. By assessing the increase in invasion area, sulforaphane demonstrates the ability to inhibit invasion of MDA-MB-231 cells out of the tumor spheroid in a dose-dependent manner, as seen in Figure 2B.
Existing methodologies lack either a physiological context and/or reproducible format for assessing tumor cell functions in vitro. The Cultrex 3-D Spheroid Proliferation/Viability Assay and the Cultrex 3-D Spheroid BME Invasion Assay provide easy, quantitative, high-throughput tools for assessing the effect of anticancer agents on either tumor spheroid proliferation or dissemination.
Gabriel J. Benton, Ph.D.