Luminous With Promise

Almost 12 years ago, the British merchant vessel S.S. Lima was cruising under a clear night sky along a southwesterly course in the Indian Ocean about 150 miles off the coast of Somalia. "A whitish glow was observed on the horizon," a crew member later reported. "After 15 minutes of steaming, the ship was completely surrounded by a sea of milky white color with a fairly uniform luminescence. ... It appeared as though the ship was sailing over a field of snow or gliding over clouds." Overhead, in the dark silence of space, a U.S. Department of Defense meteorological mapping satellite was zooming by, looking down with an uncommonly sensitive light detector.

A decade later, with the "milky sea" report from the Lima as the clue, bioluminescence researchers retrieved the satellite's archived imagery. In images captured on Jan. 25-27, 1995, Steven H. D. Haddock of the Monterey Bay Aquarium Research Institute, Steven D. Miller of the Naval Research Laboratory, and their colleagues could make out a Connecticut-sized patch of slightly bright pixels right where the Lima had been cruising.

Using image enhancement techniques, the scientists discerned a living phenomenon that no one had seen from space before. In their write-up of the findings, the scientists proudly stated that they had bagged "the first satellite measurements of bioluminescence from a milky sea" (Proc. Natl. Acad. Sci. USA 2005, 102, 14181). Generated most likely by the bacterium Vibrio harveyi, this awesome display of flamboyant biological chemistry happened on a vast scale: The researchers estimate that it took a bloom of 40 billion trillion (4 X 10²²) bioluminescent cells to generate the milky sea that the Lima had encountered.

This October at the 14th International Conference on Bioluminescence & Chemiluminescence in San Diego, Haddock's recounting of this brilliant moment in the annals of microbial chemistry was all the more striking in terms of scale. Other scientists at the meeting detailed how angstrom-scale displacements of specific amino acids influence the function of the luciferase enzymes that perpetrate much of the world's bioluminescence, including that of V. harveyi. Bioluminescence, it appears, is a phenomenon that spans 14 orders of spatial magnitude.

The collective message from the 100 or so researchers at the conference was that luminescent chemistry, whose expressions in the biological world span from fungi and fireflies to plankton and jellyfish, continues to diffuse into and energize some of the hottest research arenas. Gene expression analysis, pathogen detection, and high-throughput screening and assays for pharmaceutical research are just some of these areas.

Evidence for the diffusion of bioluminescence shines everywhere in the molecular, cellular, and biomedical sciences. "If you look at any journal today, you will see all of these beautiful, colorful images," observed Aladar A. Szalay, president of the International Society for Bioluminescence & Chemiluminescence (ISBC) and chairman of the San Diego symposium. "All of it is coming as a result of this research community," claimed Szalay, who is also president and chief executive officer of Redland, Calif.-based Genelux, a company that is developing bioluminescence-based agents and tools for biomedical applications.

"These are examples of how basic science turns into things you would never expect," said Keith Wood, a longtime bioluminescence researcher at Madison, Wis.-based Promega. Luminescent chemistry derived from fireflies, click beetles, and sea pansies accounts for a significant portion of the $175 million in revenues his company earned in 2006 from the sales of analytic and diagnostic kits, Wood said.

Participants convened at the San Diego conference from 17 countries to share a passion, in this case a passion for understanding, exploiting, and even just playing with luminescent chemistry. Patrick Hickey, for example, came armed with vials of luminescent powders, and he was as ready to turn a glass of water into a crowd-pleasing glowing liquid as to expound upon encounters with luminescent mushrooms.

Hickey is the founder and director of LUX Biotechnology in Edinburgh, Scotland, a small company that sells calibration standards, light detectors, and other laboratory accoutrements useful to his fellow luminescence aficionados. He has a knack for fanning interest in the field, and he recently partnered with a theater troupe in Glasgow to create bioluminescent stage props as well as images and movies of bioluminescent creatures for a "staged cantata" called "The Paper Nautilus," which ran three performances in early November. The goal of the collaboration, according to a promotional flyer, was to "transfer 'living light' from the laboratory to the stage, producing a magical biochemical display."

To be sure, bioluminescence has a way of prodding those who witness and study it to come up with wild ideas. Perhaps that's because of its inherent outlandishness: Consider, for instance, those flying beetles that light up summer evenings with built-in blinking lights made of biological tissue. And then consider that this same sort of chemistry is finding its way into potential medical innovations.

The biggest buzz at the meeting came from the work of biomedical researchers, who described how bacteria and viruses engineered to glow are cranking up momentum in an emerging field called oncolytics. Building luminescent properties into oncolytic—cancer-busting—infective agents has been opening up surprising new approaches to tracking and treating cancer, even the small, distributed, and hard-to-detect specks of diseased tissue that so often are the ones that ultimately kill.

"This is one of the most exciting things I have heard," commented Bruce Branchini, a chemist at Connecticut College, New London, and president-elect of ISBC.

As the word implies, chemiluminescence is the phenomenon by which a chemical vents some energy by emitting light. Bioluminescence is a version of chemiluminescence that takes place in living organisms. Most bioluminescence depends on enzymes known as luciferases. Luciferases catalyze the oxidation of small-molecule substrates known as luciferins in the presence of one or more cofactors, among them oxygen, adenosine triphosphate, and metal ions. A product of this reaction is light, exemplified by the green glow from a firefly's lantern. Unlike the chemically generated light of bioluminescence, the light generated by fluorescent proteins—for example, green fluorescent protein (GFP), which is used extensively in cellular and biomolecular studies—is the result of excitation from another light source. Produced by certain jellyfish, GFP fluorescence is stimulated by bluish light from a separate but nearby bioluminescent reaction.

The earliest written records of bioluminescence date as far back as 2,500 years, but scientists continue to uncover new details about the phenomenon.

Consider Kazuki Niwa of the National Institute of Advanced Industrial Science & Technology, Osaka, Japan, who investigated a mystery in the firefly's biosynthesis of luciferin. In bench studies, Niwa found that only the D-isomer of synthetic firefly luciferin could be made to bioluminesce. Scientists have long thought that the firefly makes only the L-isomer because its precursor, the amino acid cysteine, is available only in the L-form.

Niwa discovered a potential way out of this enantiomeric paradox by showing that a firefly's luciferase enzyme, in addition to its role in eking light out of luciferin, can also convert L-luciferin into D-luciferin, the apparent bioluminescent enantiomer.

Conference organizer Szalay, who sat in the front row for every talk and nervously oversaw the meeting like it was his own wedding, is a champion of the role that basic studies like Niwa's play in the development of light-emitting technologies that can transform lives and societies. He and his colleagues at Genelux are convinced they are pursuing a cancer diagnosis and treatment lead that has this kind of potential.

Genelux' Tony Yu, for example, described how he and coworkers used bacteria, into which they had inserted a light-generating luciferase gene, to help visualize tumors and even small metastatic offshoots of primary tumors in live mice. After intravenously injecting the bacteria, the researchers tracked the distribution of the microbes in anesthetized mice placed in a chamber fitted with a low-light camera capable of picking up luminescent signals through the animal's intact skin. In imagery taken over a period of days, the signals first are visible throughout the animal before localizing in the liver and then disappearing as the animal's immune system clears the microbe.

The researchers repeated the experiment in nude mice deliberately made ill with a leg-based glioma tumor. Over a period of days, Yu said, "the bacteria clear from the blood and concentrate in the tumor." This finding gels with previous data indicating that tumors serve as "immunological safe havens" for some bacteria and viruses. The researchers have seen similar results with a variety of glowing bacteria in several cancer models, including ones for breast, lung, and bladder cancer. "The bacteria even find metastatic tissue," which has spread from the original tumor and often is too small or otherwise unapparent for surgeons to find during operations, Yu noted.

Yanghee Woo of Memorial Sloan-Kettering Cancer Center, in New York City, reported that she and her coworkers, including lab director Yuman Fong, have been testing a fluorescent version of the herpes simplex virus (HSV-1) for its value in detecting and shrinking tumors. Recently, researchers have been finding that HSV-1 selectively replicates in a number of different cancers, including lung, esophageal, gastric, pancreatic, and colorectal cancers.

"We were struck by just how specific the oncolytic virus is for cancer," Woo said during a presentation that portrayed the potential medical versatility of the fluorescently labeled HSV-1 virus designated NV1066. The virally infected cells and tissues glow green when bathed by a beam of light.

A primary goal of Woo's group is to develop new tools for cancer surgeons. The idea is to inject patients with a fluorescent agent like NV1066 that would enable the surgeons to literally see virally infected and therefore presumably cancerous cells and tissues. "What is exciting is that the viruses showed small areas of cancer that we couldn't see otherwise," Woo said, referring in this case to small crumb-sized nodules of cancerous stomach tissue in a mouse model her team was studying. Such reagents could eventually enable surgeons, perhaps using minimally invasive, camera-equipped laparoscopes and endoscopes, to know with more precision what tissue they need to excise.

In an extensive investigation of the technique's potential utility, a Sloan-Kettering team led by Fong reported earlier this year that a single dose of NV1066, administered either locally or systemically, zeroes in on "110 types of cancer in 16 different primary organs" (FASEB J. 2006, 20, 726).

For Christopher Contag of Stanford University, the ability to render recombinant vaccinia virus visible with a luciferase gene has helped him push forward a cancer-attacking strategy that he and colleagues have been pursuing for years. In their tack, they begin with so-called cytokine-induced killer (CIK) cells, a type of immune cell known to home in on tumors and to sometimes put a drag on the cancer's growth. Contag's group preloads the CIKs with the luciferase-expressing vaccinia virus particles, which the CIKs then smuggle directly into tumor tissue. In experiments reported earlier this year in Science (2006, 311, 1780) and recounted by Contag at the meeting, the researchers gave an intravenous dose of these virus-infected CIK cells to mice with tumors. They found that a single dose of this oncolytic preparation cleared the disease from up to 75% of the mice.

Other scientists are heartened by this result but caution that efforts to translate the technology to people have only just begun. A key concern is whether the initial treatment primes the immune system to take on and neutralize the virus in second and subsequent treatments. Contag has shown, however, that virus cloaked within CIK cells appears not to elicit the immune system's ire. This opens the possibility for effective delivery, even to solid tumors, which has not been demonstrated before with this virus, Contag said.

"Imaging enables the in vivo study of cell biology and can reveal the nuances of disease mechanism," Contag said, referring to the use of both fluorescent and bioluminescent tags. If all goes well with the CIK-cloaked oncolytic virus technique, he added, "it may become possible to eliminate the last tumor cell in cancer patients and prevent relapses." Toward that end, Contag and several partners founded Xenogen, a company that was recently acquired by Caliper Life Sciences, to commercialize luminescence-based tools for studying biological processes in vivo and accelerating drug development.

Curing cancer. That would be quite a windfall from what started as mere curiosity about the luminosity of blinking fireflies and jellyfish.