Fluorescent Probes May Permit Real-Time Monitoring of Chemotherapy Effectiveness against Tumors, Stanford Study Shows

STANFORD, Calif.--(Business Wire)--
Going out like a brilliant flame is one way to get attention. If physicians
could watch tumor cells committing a form of programmed suicide called
apoptosis, a desired effect of workhorse cancer treatments such as chemotherapy
and radiotherapy, they could more quickly pick the most effective treatment. Now
scientists at the Stanford University School of Medicine have found a way to do
just that, by lighting up cells as they die.

Apoptosis is a carefully orchestrated sequence of intracellular events that
leads to the cell`s death. "The cell takes itself apart in a finite series of
steps," said Matthew Bogyo, PhD, associate professor of pathology and of
microbiology and immunology, and a member of the Stanford Cancer Center.

Bogyo is senior author of a study to be published online July 13 in Nature
Medicine in which he and his Stanford colleagues demonstrated in mice that it is
possible to noninvasively image the degree of apoptosis occurring in living
animals` tumors, and thereby to gauge the effectiveness of apoptosis-inducing
treatments. Several steps still remain before it can be determined whether this
diagnostic method is safe for use in humans.

Apoptosis occurs all the time in healthy bodies. Cells have this suicide system
in place to deal with viral infections, or just to complete their normal life
cycle. Cells lining the gut, for example, or immune cells in the spleen and
thymus are meant to live only a couple of days. "You lose three-quarters of a
million cells per second in your body due to apoptosis," noted Guy Salvesen,
PhD, director of the program on apoptosis and cell-death management at the
Burnham Institute for Medical Research, in La Jolla, Calif.

But apoptosis is also a check against unwanted cell division, as occurs during
tumor growth, said Salvesen, who collaborates with Bogyo on various research
projects but did not participate in this study. "Cancer cells have to learn to
do two things," Salvesen said. "First, they`ve got to learn to start dividing
rapidly. But once they do that, they become very vulnerable to apoptosis. So
they`ve got to learn to switch off this death mechanism. Chemotherapy and
radiotherapy aim to turn it back on."

One way of determining whether they`ve succeeded is by monitoring key early
players in apoptosis called caspases, a family of usually quiescent enzymes
found inside every mammalian cell. Activated by various biochemical cues from
within or outside of the cell, caspases commence a cascade of molecular steps
that steer the cell to a clean, quiet, orderly death.

"Caspases have to be very tightly controlled, since they are regulating cell
death. If they get turned on, the cell dies," said Bogyo. His team created
probes by affixing fluorescent "tags" to small molecules that were engineered to
bind-and stay bound-almost exclusively to caspases, and only when the caspases
are in an active state. The resulting probes are excited by certain wavelengths
of light that travel through skin without being absorbed. The probes respond by
giving off their own light, which can be imaged by a special detector.

"Our probe can`t bind to inactive caspases," Bogyo said. "It can go into cells,
but it doesn`t get stuck-it just circulates back out. So the only cells that
fluoresce are the ones approaching death."

In an early test of the probe, Bogyo and his colleagues gave mice a drug called
dexamethosone, which preferentially induces apoptosis in certain immature immune
cells residing primarily in the thymus. After systemically injecting a solution
containing the probes, the investigators observed fluorescence in the thymus, as
predicted. They confirmed by chemical methods that the fluorescent probes were
indeed binding to caspases.

Next, the team performed experiments with a new, experimental monoclonal
antibody that activates caspases, by a mechanism different from that of
dexamethasone, and initiates apoptosis particularly in rapidly dividing cells
such as those in tumors. In one such test, the researchers administered this
antibody to mice onto which human tumors had been engrafted. After injecting
these live mice at various time points with the fluorescent probes, Bogyo and
his colleagues again saw the many tumor cells undergoing apoptosis light up, but
not the healthy surrounding tissues.

The potential for practical payoffs is significant, Bogyo said. Radiotherapy and
many chemotherapeutic selectively damage DNA in rapidly replicating cells,
dramatically boosting the amount of apoptotic death happening in tumors. Some
experimental models indicate that inducing apoptosis is the main way these
treatments kill cancer cells.

"Different individuals respond differently to a given treatment. The quicker you
can make a decision about whether a drug is working or not, the better," Bogyo
said. Moreover, he said, new-generation drugs, some of them now in clinical
trials, are designed specifically to turn on caspases.

Because caspase activation is a very early event in apoptosis, monitoring it
could speed clinicians` ability to determine whether, how and when these new
drugs work, said Bogyo. He has started a company, Akrotome, to speed the
fluorescent probes` development and commercialization. Stanford has licensed
this technology to Akrotome for a 4 percent ownership stake.

"The entire cancer chemotherapy field is very, very excited about probes like
this," said the Burnham Institute`s Salvesen, who has no financial ties to the
current study or to Akrotome. The approach also holds promise for tracking
unwanted apoptotic damage to tissues in disorders such as macular degeneration
or traumas such as post-ischemic reperfusion injury.

The National Institutes of Health funded the study. Co-authors were graduate
student Laura Edgington and former graduate student Alicia Berger, PhD, and
postdoctoral researchers Galia Blum, PhD; Victoria Albrow, PhD; and Margot
Paulick, PhD, all of the Bogyo lab. (Berger is now with the Boston Consulting
Group.) Another contributor was postdoctoral scholar Neil Lineberry, PhD.

The Stanford University School of Medicine consistently ranks among the nation`s
top 10 medical schools, integrating research, medical education, patient care
and community service. For more news about the school, please visit
http://mednews.stanford.edu. The medical school is part of Stanford Medicine,
which includes Stanford Hospital & Clinics and Lucile Packard Children`s
Hospital. For information about all three, please visit
http://stanfordmedicine.org/about/news.html.

Stanford University School of Medicine
Bruce Goldman, 650-725-2106 (Print Media)
goldmanb@stanford.edu
M.A. Malone, 650-723-6912 (Broadcast Media)
mamalone@stanford.edu

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