Where physics and biology meet

The physical and life science communities tend to know relatively little about the intricacies of each others disciplines. But two Californian research establishments have overcome their cultural differences to collaborate on the development of new biomedical technologies that will have a tremendous impact in clinical, drug development, and research applications.
Joe Naiman

The Scripps Research Institute is a nonprofit organization with a long history of breakthroughs in biomedicine. The Palo Alto Research Center, or PARC, uses its mathematical, physical, and information expertise to bring new technology into commercial development. The two establishments realized that, by combining their expertise in physical and biological disciplines, they could develop biomedical technologies, for example for improved disease detection and drug development. The Scripps-PARC Institute for Advanced Biomedical Sciences was formed in 2002 with the objective of developing new instrumentation and information systems to accelerate discovery in the life sciences. Bringing together engineering and physical sciences with the life sciences provides us with the integrated competencies for the next breakthroughs, explains Peter Kuhn, associate professor for cell biology at Scripps and life sciences director for the Scripps-PARC Institute. The moment we bring [the new biomedical systems] online, we will open new windows of science, which is truly amazing. All the applications that we were dreaming of a little while ago are really within reach.

Partnership of equals
PARC became an independent company from Xerox in January 2002 (Fig. 1), and includes top researchers in information and physical sciences. I wanted to start a formal effort in bioengineering at PARC, recalls Richard Bruce, the director of PARCs efforts in the partnership. We have engineers, but we dont really have the biology side of things. On the other hand, scientists at Scripps investigate biological and chemical aspects of more than 40 diseases, and the San Diego institute (Fig. 2) has earned international recognition for its research in immunology, molecular and cellular biology, chemistry, neuroscience, and vaccine development. This makes the partnership a complementary relationship. PARC engineers gain access to numerous Scripps researchers involved in various aspects of the biological sciences. The two institutions bring top-notch, world-class competencies in biomedical sciences for Scripps and engineering and physical sciences for PARC. Both institutions have a common desire to strive for innovations and breakthroughs, says Kuhn.

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ISSN:1369 7021 Elsevier Ltd 2005

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Fig. 1 Palo Alto Research Center. (Credit: Deanna Horvath. Courtesy of PARC.)

The partnership includes approximately half a dozen research groups consisting of between two and 15 people apiece, although it is hard to pin down exact numbers. For example, approximately 15 people at Scripps are directly involved, with another 35 people indirectly involved.

All the applications that we were just dreaming of a little while ago are really within reach
The question was, could we work together to develop new instruments? says Bruce. The answer, so far, has turned out to be yes. The Scripps-PARC collaboration involves three main projects: a blood test for screening cancers; enthalpy arrays to detect molecular interactions; and a new set of algorithms for the analysis of mass spectrometry data.

Cancer cell screening

Cancer cells have a low concentration in the bloodstream. Roughly one cancerous cell is found per ten million white blood cells. The scanning procedure uses a 10 ml sample of blood from the patient. The cells are placed on a flat substrate and a fluorescently-tagged antibody, which binds to a specific protein, is added to label the target cancer cells. Currently, a digital microscope is used to scan the sample and detect any fluorescence, but the field of view is small and stepping is required to scan the entire sample. The method is very slow, scanning 50 million blood cells in 24 hours. This situation could be about to change. The Scripps-PARC team has developed a fiber array scanning technology (FAST) cytometer that uses lasers, optomechanical engineering, and imaging to scan samples and detect candidate cancerous cells. The FAST cytometer (Figs. 3 and 4) scans at a rate of ~25 million cells per minute approximately 1000 times faster than digital microscopy. However, the cytometer would not eliminate microscopy entirely. A microscopist is still needed to determine if the candidate cells located by FAST are tumor cells. The major innovation in the cytometer is a 50 mm wide and 2 mm deep asymmetrical fiber-optic bundle, which provides a wide field of
Fig. 2 Aerial photograph of The Scripps Research Institute. (Courtesy of TSRI.)

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view for collecting fluorescence from the antibody probes. As the laser moves along the surface of the substrate, it passes over cells with the fluorescent dye, and that fluorescence is captured. The fluorescence is transmitted to a collimating lens system through a circular aperture at the other end of the fiber-optic bundle (Fig. 4). The 50 mm wide field of view of the fiber-optic bundle allows the laser beam to flip back and forth across the sample, traveling at rates of 10 m/s. The position of the laser beam is recorded when fluorescence is detected, giving a map of all the candidate cells identified.

The question was, could we work together to develop new instruments?

While a microscope provides images of cells that are read by a pathologist who uses their experience and expertise, the cytometer does not provide the same resolution. The coarse fluorescence map produced by the FAST cytometer is used to direct an automated microscope to image a much smaller number of objects that include the cancerous cells. Hence, high-resolution microscopic images are collected much more quickly for subsequent identification by a pathologist, who determines if the fluorescence signals correspond to cancerous cells. In this way, the screening of several million cells has been reduced to just a few hundred candidate cells in minutes. The researchers from Scripps and PARC looked at nearly two dozen breast cancer patients who also had secondary cancerous sites in other organs. They found cancerous cells in >90% of the cases. We have shown that we can detect tumor cells in the blood of cancer patients, says Kuhn. The next step is to expand the project to scan for other types of cancer and use larger samples to get better statistical data. known not to react. During the isothermal mixing and merging of the
Fig. 3 Photograph of a sample substrate being inserted into the FAST cytometer for scanning.

Detecting molecular interactions

Conventional assays used in drug discovery require a fluorescent label to be bound to molecules to detect their interactions with a target, which may modify the interaction. Calorimetry, on the other hand, allows scientists to analyze the thermodynamics of molecular interactions without interfering with the reactivity of the molecules. The current dominant method is isothermal titration calorimetry, but large sample sizes and long measurement times are required. Scripps-PARC researchers have fabricated an enthalpy array using microscale technology that interfaces with automated laboratory equipment (Fig. 5). The array consists of 96 calorimetric detectors, and 250 nl samples are deposited onto the array with a fluidic dispenser. Each detector cell in the array has two adjacent and identical detector regions: the first unit contains the molecular solutions being tested for an interaction, and the second unit contains reference solutions that are

two biomolecular solutions of interest, the temperature is measured by a thermistor. The comparative evaporation rates are measured and the differences plotted as a function of time, so that differential temperature measurements are obtained for the sample and the reference specimen. If a molecular interaction occurs, it can be detected through the resulting heat exchange. The temperature differential provides data in considerably less time and at considerably less cost than conventional assay methods. While isothermal titration calorimetry requires an hour to produce a ten-measurement titration, the enthalpy array requires approximately one minute for a measurement and, thus, can provide 10 000 measurements or 1000 titrations per day. The team has measured the enthalpies of reaction for various biological interactions, including protein-ligand binding, enzymatic reactions, and organelle activity.

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Searching mass spectrometry data

Mass spectrometry measures the mass of a proteins fragments, allowing the sequence of amino acids that makes up the protein to be identified. By comparing this sequence to a database, the protein and the gene that encodes it can often be identified. However, a significant proportion of proteins, especially those involved in biochemical signaling pathways or cell-to-cell communication, are modified after being synthesized by the cell. Phosphate groups or chains of carbohydrates called glycans, for example, are added to the amino acid chain to regulate the proteins activity and function. This can make the process of identification using mass spectroscopy much more complex. Intensive data and manual interpretation is acceptable for researchers, but not for laboratories conducting high-throughput proteomics for drug design. The laboratory of Scripps professor John Yates has multiple mass spectrometers and has developed methods to survey proteins present in biological samples and their phosphorylation state. But the computers process spectra that may or may not include proteins. The Scripps-PARC work has developed screening software that can eliminate spectra that are unlikely to represent proteins, allowing the computation to focus on spectra containing proteins. Glycans present another problem, as multiple different carbohydrate structures have the same mass and so cannot be discriminated using

mass spectroscopy. The team has put together an algorithm that combines database inputs with a rules-based approach that incorporates understanding gained from previous research work. This enables the possible glycan structures to be identified.

Different cultures
The collaboration is not without its obstacles. The Internet and phone conversations allow contact between the San Diego and Palo Alto offices when personal interaction is not warranted, but samples and equipment must often travel between the two facilities, which are ~800 km apart. For example, in the case of the cancer detection project, patients are analyzed at Scripps Clinic and the collected samples are sent to PARC for screening. Also, as part of the enthalpy array research, Scripps produces proteins and sends them to PARC for characterization.

A meeting takes a little bit longer, because theres a lot of translating that has to go on
A group meeting for the cancer detection project typically consists of an optical engineer, a systems engineer, a physicist, a cell biologist, an oncologist, a pathologist, and a clinical oncology scientist, as well as

Fig. 4 Schematic of the FAST cytometer optical system. (Reprinted with permission from Curry, D. N., et al., Ann. Int. Conf. IEEE Eng. Med. Biol. Proc. (2004) 26 (II), 1267. 2004 IEEE.)

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research or a clinical application, he says. Bruce thinks that physical scientists enjoy advancing technology. But perhaps there isnt so much distance between the two motivations. Theres a particular level of excitement in being able to create technology that benefits people, feels Bruce. Being able to work on a human level like this, its special. These projects are probably the most interesting of my career. Another major cultural difference is that Scripps is a private but nonprofit research organization, while PARC is a commercial research center. An initial brainstorming session resulted in a large list of potential projects. Those projects were investigated from scientific, technical, and marketing perspectives. The primary criterion in the final selection was whether the project could make a significant impact on
Fig. 5 Four arrays of nanocalorimeter detectors can be processed simultaneously. (Reprinted with permission from Torres, F. E., et al., Proc. Natl. Acad. Sci. USA (2004) 101 (26), 9517. 2004 National Academy of Sciences.)

medicine, clinical treatment, or biomedical research, although Bruce notes that limited markets were a factor that led PARC to reject some projects.

a cell biology or chemistry graduate student. Each brings a missioncritical piece to the table, says Kuhn. The difficulty is really knowing the language, explains Bruce. The level of complexity in the biological sciences is absolutely huge. He believes that physics is based on rules and derivations, while biology uses project-specific terminology. Weve had to learn a fair amount of biology so that we could be sure were working on the right problem, Bruce says. I would say weve learned far more biology than the biologists have learned our disciplines. Kuhn agrees. A meeting takes a little bit longer, because theres a lot of translating that has to go on, he says. Thats what makes it so important that were working (a) in areas where there is a true and actual need, and (b) where we believe we can truly address that need in a fairly large market with significant breakthroughs. The key to overcoming these challenges is understanding the different vocabularies and cultures of the different disciplines, says Kuhn. That simply requires a level of commitment that you can get most easily by doing something that excites everybody involved. Biological scientists seek a solution to a problem, believes Kuhn. Their product will have a true and real impact on either biomedical

These projects are probably the most interesting of my career

As a result, the Scripps-PARC Institute believes they have developed technologies that could bring large commercial rewards. The FAST cytometer, with its potential for commercial and hospital laboratories, would be entering a multibillion dollar market, while there is a multimillion dollar drug-design market for the enthalpy array and mass spectrometry technology. These estimates are based on the potential in the US alone. The two institutions will share the commercial benefits, and they have signed an agreement that covers intellectual property rights. That effectively has made it as if we were working within the same institution, explains Bruce.

Into the future

The next steps involve taking the various projects from their testing stages to eventual commercial production. Were going to move these projects on to fruition, says Bruce. The scientists will also use the lessons learned so far to determine which new projects will be worked on by the Scripps-PARC partnership.

FURTHER INFORMATION
Scripps-PARC Institute for Advanced Biomedical Sciences: www.parc.com/research/scripps-parc/ Palo Alto Research Center: www.parc.com The Scripps Research Institute: www.scripps.edu

Cancer cell screening: Krivacic, R. T., et al., Proc. Natl. Acad. Sci. USA (2004) 101 (29), 10501 Enthalpy array: Torres, F. E., et al., Proc. Natl. Acad. Sci. USA (2004) 101 (26), 9517 Kuhn Laboratory at Scripps: http://scrippsparc.scripps.edu Richard Bruce: www.parc.com/about/management/bruce.html

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