There's a big difference between a flat layer of cells and a complex, three-dimensional tissue. But until recently, many biologists have glossed over this fact. Alison Abbott discovers what they've been missing.
Let's hear it for the humble petri dish! Many of the seminal findings in cell and molecular biology have come from cultures of cells grown cheaply and conveniently in these familiar, flat receptacles. But the limitations of considering biology in, effectively, just two dimensions are now becoming clear.
Led by cancer researchers, biologists are increasingly turning to three-dimensional cell cultures, where they are discovering patterns of gene expression and other biological activities that more closely mirror what happens in living organisms. “Scientists are starting to realize just how much a cell's context matters,” says Mina Bissell, a pioneer of 3-D cell culture at the Lawrence Berkeley National Laboratory in California.
In mammalian tissues, cells connect not only to each other, but also to a support structure called the extracellular matrix (ECM). This contains proteins, such as collagen, elastin and laminin, that give tissues their mechanical properties and help to organize communication between cells embedded within the matrix. Receptors on the surface of the cells, in particular a family of proteins called the integrins, anchor their bearers to the ECM, and also determine how the cells interpret biochemical cues from their immediate surroundings.
Given this complex mechanical and biochemical interplay, it is perhaps no surprise that researchers will miss biological subtleties if the cells they are studying grow only in flat layers. But providing an appropriate environment in which to culture cells in three dimensions is no easy matter (see 'The matrix, reinvented', below). Some researchers use simple gels consisting of collagen, whereas others make their own gels by extracting ECM material from relevant tissues. Another popular option is the commercially available Matrigel, which consists of structural proteins such as laminin and collagen, plus growth factors and enzymes, all taken from mouse tumours1,2.
Bissell has been experimenting with 3-D culture systems for some three decades. But for years, critics argued that her methods were expensive, cumbersome and unnecessary. Their views changed after the publication of a landmark paper in 1997, in which Bissell's group showed that antibodies against a cell-surface receptor called β1-integrin completely changed the behaviour of cancerous breast cells grown in 3-D culture: they seemed to become non-cancerous, losing their abnormal shapes and patterns of growth3. This result had never been observed in 2-D cultures. Just changing the way a cell interacts with its 3-D environment, Bissell had shown, can radically alter its behaviour.
Since then, Bissell has demonstrated further important differences in the behaviour of cells grown in 2-D and 3-D cultures. For example, in the same breast-cancer system, she has shown that antibodies against β1-integrin also decrease signalling by receptors for epidermal growth factor (EGF); antibodies against EGF receptors similarly depress the activity of β1-integrin4. Again, this reciprocal interaction does not happen in 2-D cultures.
Receptors for growth factors play a key role in the initial development of tumours. But this isn't the only aspect of cancer research to have benefited from the new 3-D perspective. Peter Friedl, a cell biologist at the University of Würzburg in Germany, studies metastasis — the migration of cells away from primary tumours to cause secondary cancers around the body. Over the past couple of years, 3-D studies by Friedl's group and others have revealed unexpected subtleties in the mechanisms that cancer cells use to break out from primary tumours — clues that may help to explain the disappointing clinical performance of a promising class of cancer drugs.
Cancer cells undergoing metastasis normally cut themselves free from a tumour's ECM using protein-digesting enzymes. Yet in clinical trials, drugs that inhibit these enzymes have done little to slow the progress of cancer5. In his 3-D culture system, Friedl blocked the activity of the protein-chopping enzymes in two types of cancer cell, and found that the cells changed into an amoeba-like form, which could squeeze through gaps in the matrix6. “3-D tissue culture is really challenging our assumptions,” says Friedl.
Chris Marshall, a cell biologist at the Institute of Cancer Research in London, has extended this finding, showing that the formation of amoeba-like cells depends on a particular signalling pathway in a range of different tumour cell lines. When this pathway is blocked, drugs that inhibit the protein-digesting enzymes stop the cells from moving through Matrigel7. This result suggests that a combination of drugs might work where inhibitors of the protein-digesting enzymes alone failed.
In another recent paper, 3-D cell culture has improved the prospect of treating cancer with gene therapy. Researchers led by Michael Korn of the University of California, San Francisco, studied the cell-surface receptors to which adenoviruses bind. In 2-D cultures, both normal and malignant breast cells had similar, high levels of the receptors. But in 3-D cultures, only malignant cells carried large numbers of the receptors8. Adenoviruses have been used as 'vectors' to introduce therapeutic genes into target cells, and Korn's findings suggest that they may be particularly suitable for targeting cancerous cells.
Developmental biologists are also getting in on the 3-D act. In 2001, for instance, a team led by Kenneth Yamada of the National Institute of Dental and Craniofacial Research in Bethesda, Maryland, directly compared the growth and development of fibroblasts, collagen-secreting cells that are found in many tissues, in 2-D and 3-D cultures. In three dimensions, the cells moved and divided more quickly, and assumed the characteristic asymmetric shape that fibroblasts have in living tissues9. “At the very least, developmental biologists who have worked with normal tissue culture will have to seriously consider comparing their results to those obtained in 3-D culture,” says Yamada.
Some researchers are now trying to make systematic comparisons of gene activity in 2-D and 3-D cultures. In unpublished work, Linda Griffith, a bioengineer at the Massachusetts Institute of Technology, has used DNA microarrays to look at profiles of gene expression in liver cells. “Our preliminary analysis shows that the expression profile in 3-D is much closer to in vivo expression profiles than the profile we've seen in 2-D,” she says.
If 3-D culture can provide a better model for what happens in the body, it might allow researchers to reduce their use of experimental animals — although experts stress that it is far from a complete alternative. “3-D culture will allow a lot of basic questions to be answered before having to turn to whole-animal research,” says Friedl, whose work has been supported in part by a German research-ministry programme dedicated to reducing animal use. Encouragingly, when Friedl transplanted metastasizing cells into mice and used imaging techniques to track their development, they underwent the same amoeba-like morphological changes seen in 3-D culture6.
In October, 3-D cell culture will receive an important boost when the National Cancer Institute (NCI) in Bethesda, Maryland, launches a new section on the cellular micro-environment, which will rely heavily on 3-D studies. This programme will have an annual budget of some US$40 million, and will include specific funding to spur the development of 3-D culturing techniques.
For Robert Weinberg of the Whitehead Institute for Biomedical Research in Cambridge, Massachusetts, the new NCI programme is a welcome development. In the 1970s and 1980s, he pioneered the study of cancer-causing genes and their associated cell-signalling pathways, mostly using 2-D cultures. “There is a whole dimension of signalling that we purposefully didn't deal with, for simplicity's sake,” says Weinberg. “But now we are ready to move onto the next stage — the more complex level that 3-D culture allows.”
In an article late last year, Weinberg went so far as to describe the study of cancer cells in two dimensions as “quaint, if not archaic”10. And where cancer researchers have led, he predicts, other biologists will follow.
Influential players in industry are already thinking along 3-D lines, says Mihael Polymeropoulos, chief scientific officer of Vanda Pharmaceuticals in Rockville, Maryland, and formerly head of pharmacogenetics at the Swiss-based drugs giant Novartis. “In 10 years, anyone trying to use 2-D analyses to get relevant and novel biological information will find it difficult to get funded,” he predicts.
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Abbott, A. Biology's new dimension. Nature 424, 870–872 (2003). https://doi.org/10.1038/424870a
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