Frustrated by the limitations of basic imaging research, Dr. Christopher Contag has literally found a solution glowing in the dark.
By harnessing the natural light-emitting properties that allow fireflies to flicker in darkness, he and his colleagues have used bioluminescence imaging to track cancer cells, bacteria, and numerous other processes. Their work is based on the use of a family of enzymes known as luciferases, which are found in organisms that emit a bioluminescent glow.
"The technology arose out of the frustration of standard assay techniques that required biopsy and necropsy tissues to understand where biological processes are taking place in the body," said Contag, an assistant professor of pediatrics at Stanford University. "All of those methods are subject to sampling limitations. You can't sample entire organisms or even an entire organ or tissue. You don't know when to look or where."
Enter bioluminescence imaging as a user-friendly method for detecting biological changes in vivo. By tagging cells with the luciferase gene, the cells can be followed throughout the body by means of a digital camera that employs charge-coupled device detectors.
Bioluminescence imaging overcomes limitations of conventional nuclear medicine techniques. For example, if an immune cell is labeled with indium-111 and followed over time, the signal is lost when the cell divides. The same is true for imaging that uses fluorescent dyes. When a reporter gene is introduced into a cell, however, the gene is replicated with each cell division, and as a result, the signal produced by a luciferase gene is perpetuated over time. The perpetual signal enables researchers to study disease progression, response to therapy, growth of new cells and tissue, and many other processes.
Originally developed as a tool for tracking and monitoring biological processes, bioluminescence imaging has evolved into a potentially more valuable aid for assessing functional properties. Examples include protein-protein interactions with cells, gene regulation at the transcription level, protein degradation over time, enzymatic activity associated with tumor progression, and cell death.
"Modifications to a reporter gene and the protein that it encodes tell you something about the biology of the cell as opposed to just where the cell is and what it is doing," Contag said.
The field of bioluminescence imaging has grown dramatically in a relatively short period of time; hundreds of investigators are evaluating it in laboratories around the world.
"If you look at applications of molecular imaging strategies across the board, bioluminescence imaging has been galloping along at the fastest pace over the last 24 months for preclinical work," said Dr. David Piwnica-Worms, director of the Molecular Imaging Center at Washington University in St. Louis. "If you just look at the number of papers published and the way the techniques are being used-comparing MR, PET, SPECT, radiopharmaceutical, fluorescence, ultrasound, and bioluminescence-in preclinical studies and in basic science studies, bioluminescence imaging seems to be dominating the playing field."
Bioluminescence imaging has many advantages in terms of sensitivity and ability to generate genetically encoded reporters that incorporate luciferases, he said.
It can be applied to all disease processes in all areas of small-animal models, Piwnica-Worms said. With a sufficiently strong reporter gene, bioluminescence can easily be seen arising from deep in the brain, thorax, abdomen, and other in vivo locations.
"Mouse models are of great interest for a wide variety of diseases because of all the genetic models that are coming along. We and others are developing bioluminescence reporter mice that are genetically engineered to express luciferases in a variety of organs and under a variety of control strategies," he said. "Quite frankly, using a reporter strategy, you could study anything at the cellular and mouse level."
Examples of ongoing applications include cancer, inflammatory disease, neurodegenerative disease, gastrointestinal physiology, renal physiology, cell trafficking, stem cell research, transplant science, and muscle physiology.
Contag foresees growth of bioluminescence imaging in several fields of investigation, including regenerative medicine, developmental therapeutics, treatment of residual minimal disease, and the concept of the cancer stem cell. The cancer stem cell has been hypothesized for some time, based on observations that only a fraction of tumor cells in transplanted tumors would grow and replicate in animal models, but it has been difficult to study.
"The hypothesis is that there is a certain category of cell within a tumor that has the self-renewing capability, and those cells need to be the target of therapy," Contag said. "Reducing the bulk of the tumor is not going to get rid of the cancer-generating or cancer-perpetuating cells that hypothetically exist in the tumor."
The major role that bioluminescence imaging has in preclinical models is unlikely to translate into a notable clinical role, at least not using the insect forms of luciferase, said Dr. Sanjiv (Sam) Gambhir, director of the Molecular Imaging Program and head of nuclear medicine at Stanford. Limitations of bioluminescence imaging in clinical applications include the need for different luciferases to monitor different genes or processes, the fact that firefly luciferase uses cellular energy in the form of ATP to generate light, and, perhaps most important, the need to introduce a foreign gene into humans.
In contrast, marine-derived luciferases avoid the problems inherent in clinical use of firefly luciferase. Renilla luciferase can be injected as protein and does not require ATP to generate light, Gambhir said. Moreover, renilla luciferase's substrate, coelenterazine, is found naturally in shrimp and other forms of seafood that humans consume. The toxicity of the substrata for firefly luciferase is unknown at this point. Renilla luciferase is also a smaller protein, which can be split more easily to monitor and measure cellular activities and processes.
Perhaps the greatest challenge to bioluminescence imaging will come from fluorescence imaging techniques, Gambhir said. Because problems related to autofluorescence and background signal have been addressed, fluorescence appears to be more generalizable, compared with bioluminescence imaging.
"With fluorescence imaging you can develop small molecules that fluoresce," Gambhir said. "You can't develop small molecules that bioluminesce. Bioluminescence is more limited to genes and proteins. As soon as you go over to small molecules and small ligand receptor interactions, bioluminescence imaging isn't a player."
Contag counters that labeling with fluorescent dyes suffers from the same limitation as radionuclide imaging; namely, the signal is lost with cell division.
"The advantage of using an inserted reporter gene is that you can engineer it to do other things than simply report where it is," he said.