Tuesday, December 14, 2004

In vivo molecular imaging

Molecular imaging is a field that gained major attention in biology in the past few years, starting with the discovery/isolation of GFP, a protein able to emit (green) fluorescence when excited (for example with a laser). Nowadays, a panoply of fluorescent proteins / chemicals allow for precise tracking of different biological components (proteins, organelles, DNA) when coupled to antibodies or fused with proteins. Pretty covers for science magazines, very useful in the lab, everyones happy! Recent developments in the field include in vivo imaging (in real, live animals) and sequence-specific RNA tracking (with molecular beacons). Both were put to good use by a research team of the Abramson Cancer Center in a study related to cancer.

They were able to track the effectiveness of chemoterapy (Doxorubicin chemo, to be precise) using molecular beacons in mice. Doxorubicin kill tumoral cells by a p53 induced mechanism. p53 is a transcription factor; they tracked the expression of a p53-dependant gene, p21/Waf. Molecular beacons are hairpin DNA structures with a fluorophore on one end and a quencher (a molecule that absorb fluorescence and reemit it at another wavelenght) at the other end. When the hairpin is closed, no fluorescence is emitted. When the beacon anneal to a specific sequence, the fluorophore is too far from the quencher, and fluorescence is emitted. Cells express p21 via a Doxirubicin, p53 induced mechanism and go fluorescent. And you get to see it in the animal while its alive (and getting cured of its cancer!), so you can track the response throught time and space. Wonderful application of biotechnology! Here's the press release :

Molecular Tailoring of Chemotherapy with Novel Imaging Techniques Molecular Beacons, Gene Silencing, and Reporter Genes Studied to Better Predict Response to Chemotherapy
(Philadelphia, PA) – Researchers at the Abramson Cancer Center of the University of Pennsylvania are applying a host of imaging techniques to develop better ways to look noninvasively at the molecular characteristics of tumors. The experiments, now in human cell cultures and mouse models, are aimed at better forecasting early response to chemotherapy so that treatment choices can be adjusted.

"Right now in cancer therapy, with the exception of relatively uncommon examples of cancers for which we have tumor markers, we don't have reliable ways of predicting who is going to respond early on to chemotherapy," says Wafik El-Deiry, MD, PhD Associate Professor, Departments of Medicine, Genetics, and Pharmacology. "Currently cancer patients get their chemo and you can't tell if they're responding for several weeks. We need to have tests that will tell us if patients are going to respond to the chemo or the radiation soon after it's first given, and whether these responses are going to last."

Two recent papers in Cancer Biology & Therapy and Cancer Research describe the work of the El-Deiry laboratory. One approach is to use a molecular beacon, a molecule that can be activated within cells due to a specific context, such as in this case, the response to chemotherapy. The beacon recognizes a characteristic change in chemo-treated tumor cells, physically opens up and fluoresces, which can then be measured. "The beacon goes right into the living cell and if it opens up, emitting fluorescence, we can detect the glow," says El-Deiry.

Human lung-cancer cells were treated with the chemotherapeutic agent doxorubicin (Adriamycin), which causes cellular DNA damage. Doxorubicin works through the tumor suppressor protein p53, which ultimately kills many types of cancer cells. "We engineered a molecular beacon to detect expression of a gene called p21, that is turned on directly by p53 when cells are exposed to Doxorubicin," says El-Deiry.

The cells that were exposed to Doxorubicin activated the p53-responsive molecular-beacon tag and emitted a strong fluorescence. From this El-Deiry and colleagues hope to develop a scan that could detect a patient's likely reaction to certain chemotherapies: Strong fluorescence equals a good response to the chemotherapy. They hope to make what he refers to as a "beacon cocktail" that can predict response by monitoring multiple genes simultaneously as well as additional intracellular events in the process of cell death.

In another study El-Deiry and colleagues combine imaging techniques and a mouse model for colon cancer. "In this research, we're combining two very powerful emerging technologies," says El-Deiry. "This is the first example, to my knowledge, of the use of inducible gene silencing and non-invasive bioluminescence imaging in a mouse model for cancer." Gene silencing is a technique that allows researchers to control the expression of any gene in a given cell by introducing small RNA sequences targeting the gene of interest. Inducible refers to the ability to control whether or not the silencing RNA is expressed in the cell so that investigators can compare gene activity to tumor growth, as El-Deiry did in this study. This approach allows researchers to regulate gene expression by what they feed the mice. In this study, the KILLER/DR5 receptor, another protein that responds to chemotherapy by killing cancer cells, is silenced in colon tumors in the mice.

They also labeled the cells with a reporter gene called firefly luciferase, which gives off light. "The use of a reporter like firefly luciferase marks the tumor cells so we can see them by another imaging technique," explains El-Deiry. "Fireflies that we see in the evening carry out the same chemical reaction with their own luciferase protein to give off the light." The imager detects the light and captures its intensity to provide a measurement of the size of the tumor.

"The bioluminescence imaging technology has provided a breakthrough that allows scientists to examine the size of a tumor in living mice with high sensitivity," says El-Deiry. "Since the reporter gene is always on and only in the tumor cell, it's essentially measuring tumor volume. Using the reporter gene along with the KILLER/DR5 silencer, we show for the first time that when we turn off KILLER/DR5, we get bigger tumors."

While the beacon or beacon cocktails have the potential to be used in the clinic to detect mutations in cancer cells or the activation of genes that predict therapeutic response, the major advance with the bioluminescence imaging is in accelerating preclinical drug development. The gene silencing allows precise molecular characterization of targets that are relevant for therapeutic response while the imaging allows non-invasive assessment of drug activity towards implanted tumors. This approach saves time and money because it is possible to see the effects of drugs in living mice without sacrificing them and it also requires fewer mice in experiments.

Because the KILLER/DR5 receptor is involved in the process of cell death by chemotherapy, El-Deiry is also gaining insight into which drugs use it and which drugs work by other mechanisms. "This is important because to maximize tumor killing and to attempt to bypass or reverse resistance to chemotherapy, we need to harness all the ways cancer cells can be killed," he says.

The KILLER/DR5 receptor is engaged by a therapeutic agent currently being developed called TRAIL (Tumor necrosis factor-Related Apoptosis Inducing Ligand). TRAIL is produced normally by natural killer cells and controls tumor spread by binding to a tumor's death-inducing receptor KILLER/DR5. "However, in cancer patients with suppressed immunity and for reasons we still don't understand, there isn't enough TRAIL being produced or effectively delivered by the natural killer cells at the site of tumors and so tumors are not suppressed," says El-Deiry. "The hope is that if TRAIL is administered to patients alone or in combination with chemotherapy, this may in the clinic lead to some benefit." TRAIL looks promising in animal studies but clinical studies that are due to start in the next year or so will determine how toxic TRAIL is and begin to see whether it really works in cancer patients.

The work was funded by NIH grants including a multi-institutional Network for Translational Research in Optical Imaging imaging grant from the National Cancer Institute. The Network provides support for imaging resources to accelerate translational research on cancer.

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