3D culture for HTS: spheroids in practice
With drug development costs so high and success rates relatively low, there has been a concerted effort to develop new in vitro models that accurately reflect how small molecule drug candidates influence cellular pathways in vivo.
In keeping with this topic, our latest e-book explores how the latest developments in phenotypic screening using 3D cell culture models can help researchers to generate preclinical data that is more likely to reflect the efficacy and toxicity in later stages of the drug development process.
To give you a quick taste of the insight you’ll find inside our e-book, “Phenotypic Screening: A New Dimension”, this blog provides a brief overview of some of the research currently being carried out using 3D cell culture models to better understand cancer development and treatment.
Adding a new dimension to cell culture
While traditional 2D monolayer cell cultures have been exceedingly useful research tools, they often fail to accurately mirror disease states seen in vivo, especially cancer. One of the main weaknesses is that 2D culture plates do not recapitulate the biological complexity that underlies many in vivo processes, such as the interplay between different cells types and the extracellular matrix or the chemical and gene expression gradients found in complex tissues. Fortunately, the ongoing development of 3D culture has begun to remedy this.
As a case in point, researchers at the University of Michigan are currently using 3D spheroids to assess drug response profiles. An important feature in the progression of many diseases is collagen contraction, whereby cells remodel the surrounding extracellular matrix in which they are growing by secreting remodeling factors in response to external cues. The research team at Michigan have developed a 3D spheroid-based collagen contraction assay that more closely models the in vivo diffusion of nutrients and other soluble cues. Just as importantly, the method is amenable to HTS, allowing the researchers to scale up their operations and screen compounds at the speed and throughput necessary to enable effective discovery and validation.
Figure 1. A fluorescently labeled tumor spheroid scanned using acumen Cellista
TTP Labtech’s acumen® Cellista is ideally suited for the rapid (5 minutes/plate) high throughput analysis of tumor spheroids and colonies, without the need to acquire a Z-stack of images.
Cancer research is another area benefiting from the powerful combination of 3D cell culture and HTS. Multi-layered 3D tumor spheroids form spontaneously from isolated metastatic tumor cells and represent an excellent system for compound screening. Tumor spheroids enable researchers to better understand how cancer therapeutics are likely to influence the disease, largely due to the cells establishing appropriate cell-cell interactions, as they would do in vivo. Several research groups have already started putting this model to great use, adapting their existing processes to make 3D culture more amenable to high throughput screening (HTS).
At the Center for Clinical and Translational Research (Virginia Commonwealth University), the team are using their 3D tumor spheroid system to identify specific signaling pathways involved in breast cancer metastasis. This 3D system accurately mimics the activation of multiple signaling pathways and enables the enrichment of cancer stem cells and/or tumor-initiating cells expressing specific markers (e.g. CD4+, ALDH-1+ and CD133), making it a powerful assay. These combined benefits represent an excellent tool for improving the predictive capability of screening assays.
Cancer cell growth and survival
Studies at Horizon Discovery Group plc (UK) investigated the effect of a KRAS mutation on the growth rates and drug sensitivity of a colon cancer cell line grown in 2D and 3D. While the mutation had no effect on the growth rate of cells in conventional 2D adherent cultures, cells grown as 3D colonies in soft agar showed a reduced growth phenotype when the KRAS mutation was knocked-out (Figure 2). Furthermore, cells harboring the KRAS mutation showed enhanced drug sensitivity when grown in 3D compared to those in 2D.
Figure 2. The effect of a KRAS mutation on cancer cell growth in 2D and 3D culture.
Other ongoing research is stimulating the integration of 3D cell culture systems with other technologies used in the drug discovery process. For example, growing multiple cell types simultaneously in the same culture model can be used to study the development of feedback loops and other pathway interactions, a feature lacking in traditional 2D monotypic cultures. The use of such systems, known as organotypic 3D co-culture platforms, is being pioneered at Wayne State University.
How can HTS with 3D systems improve your screening programs?
The ongoing development of HTS workflows using 3D culture systems is allowing researchers to better understand how cellular pathways respond to small molecule drug candidates. This data is more representative of how these molecules will likely perform in vivo, improving the effectiveness and accuracy of pre-clinical compound screening.