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Originally published March, 1998

IVD Technology News

Sugar-coating yields designer cell surfaces

In the past, researchers whose work required getting a cell to express an artificial surface group could expect to endure weeks or even months of genetic engineering. Painstaking labor was required to isolate DNA, put it into a vector, run gels, transfect, culture, and so on. But for applications that require only transient expression, genetic engineering may soon have competition. Research into new methods may make it possible for scientists to produce customized cell surfaces in as little as 48 hours.

At the Ernest Orlando Lawrence Berkeley National Laboratory (Berkeley, CA), Carolyn Bertozzi and her colleagues have succeeded in altering the surface properties of cells simply by growing them in media containing customized oligosaccharides. Oligosaccharides are complexes of sugars that can be chemically synthesized to contain other functional groups, such as ketones.

UC Berkeley researchers engineered the synthetic molecule called N-Levulinoylmannosamine (ManLev) to include a ketone marker (left). The marker was found to be expressed on the plasma membranes of cells incubated with ManLev for 48 hours (right). Figure courtesy of Carolyn Bertozzi

Bertozzi is a member of the Berkeley lab's biomolecular materials program and an assistant professor of chemistry at the University of California at Berkeley. Her research uses the cell's own biosynthetic machinery to incorporate unnatural functional groups onto the cell surface.

The importance of this research is readily apparent: it is the surface of a cell that directs most of its interaction with the outside world. Membrane receptors, for example, are involved when a cell recognizes a virus or responds to a drug. Other surface structures can cause one cell to adhere to another, or to migrate out of a capillary. From a diagnostic standpoint, surface markers often delineate the cell's type, function, or disease state.

The ability to manipulate the surface properties of cells may open the door for cell biologists and organic chemists to develop a wide variety of new applications, including potential diagnostics. But first, says Bertozzi, her research team needed to accomplish its primary goal—"to take control of the cell surface."

To do that, Bertozzi and her colleagues needed a chemical group that was normally expressed on the cell surface and that would be functionally resistant to slight alterations. Cells will incorporate many different types of oligosaccharides on their surfaces. Because of the varied nature of oligosaccharides, small chemical changes can go unnoticed by the cell. Bertozzi chose to work with N-acetyl mannosamine (ManNAc), a natural precursor of sialic acid, which is a common component of cell-surface oligosaccharides.

To distinguish their artificial oligosaccharides from naturally occurring ones, Bertozzi's team also needed a marker. Ketones were selected to fill this role because they possess a number of desirable properties. They are not usually found in human cell-surface oligosaccharides, they are harmless, and they react chemically with a functional group called hydrazides. The molecule that the team synthesized, called ManLev, was identical to ManNAc, except that it contained a ketone group.

Bertozzi incubated a number of common cell lines with ManLev for 48 hours. After incubation, she found that the cells were expressing the ketone-containing sialic acid oligosaccharides. Quantification revealed that more than a million copies were being expressed on most cell surfaces. By altering the ratio of ManLev to ManNAc, Bertozzi's team also showed that the degree of expression was dose-dependent.

Since ketones react with hydrazides, any number of labeling techniques and detection methods can be employed. In her initial study, Bertozzi used a biotinylated hydrazide followed by fluorescently conjugated avidin. The tagged cells were then analyzed by flow cytometry. Many different avidin conjugates are commercially available for use in ELISA, confocal microscopy, and other assays.

Although the therapeutic and diagnostic potential of the technology has yet to be exploited, Bertozzi says that her team is exploring several applications.

So far, only homogeneous populations of cells have been modified in this way. Methods for selecting cells from a heterogeneous pool are still under investigation. It has been reported that some cancer cells are more highly sialyated than normal cells (Sell S, Hum Pathol, 21(10):1003—1019, 1990). Bertozzi postulates that "one might use this tool as a prognostic indicator after a tumor has been removed." Bertozzi's team is also experimenting with a method to make ketone-labeled cancer cells stand out under magnetic resonance imaging.

Using Bertozzi's method, it may be possible to make cells susceptible to toxins or immune recognition. Bertozzi has already demonstrated in vitro that ketone-labeled cancer cells become uniquely vulnerable to a toxin-hydrazide compound. With different functional groups, cells could also be engineered to adhere to polymers, metals, and ceramics. Bertozzi speculates that such applications might enable patients to better tolerate biomedical implants such as pacemakers and artificial organs.—Gary Woo