The study, published online on March 15, explains how epithelial cells expand as a sheet and migrate to engulf the entire avian egg yolk as it grows. It also reveals the presence of certain molecules during this process that have not been previously reported in other major developmental models, including Xenopus frogs and zebrafish.

"These molecules and mechanisms of early development in the avian embryo may demonstrate evolutionary differences across species in the collective movement of epithelial cells and motivate additional studies of avian embryo development," said Evan Zamir, an assistant professor in the George W. Woodruff School of Mechanical Engineering at Georgia Tech.

Matt Futterman, who worked on the project as a graduate student at Georgia Tech, and mechanical engineering professor Andrés García also contributed to this study. The research was funded by Zamir's new faculty support from Georgia Tech and by a grant to García from the National Institutes of Health.

In the study, the researchers conducted immunofluorescence and high-resolution confocal microscopy experiments to examine the spatial distribution and expression of five proteins -- vimentin, cytokeratin, β-catenin, E-cadherin and laminin -- as cells moved to wrap the yolk sac of quail embryos during development.

The results showed that during this process, four of the proteins -- vimentin, cytokeratin, β-catenin and E-cadherin -- appeared in the cells located at the free edge of the migrating cell sheet. Finding dense interconnected networks of both vimentin and cytokeratin in the edge cells surprised the researchers.

"Since cytokeratin is generally associated with the epithelial phenotype and vimentin is generally associated with the mesenchymal phenotype, it's rare to see them expressed in the same cells, but this does occur in metastasizing tumor cells," said Zamir.

Cells expressing the mesenchymal phenotype are typically found in connective tissues -- such as bone, cartilage, and the lymphatic and circulatory systems -- whereas cells of the epithelial phenotype are found in cavities and glands and on surfaces throughout the body.

This finding provides evidence that epithelial cells normally attached to a membrane surface underwent biochemical changes that enabled them to assume a mesenchymal cell phenotype, which enhanced their migratory capacity. This process, called partial epithelial-to-mesenchymal transition, has many similarities to the initiation of tumor cell metastasis and wound healing.

Since this epithelial and mesenchymal expression pattern in the edge cells has not previously been reported in Xenopus or zebrafish, it may be unique to the avian embryo. This discovery would make the avian embryo a valuable model for studying tumor cell migration and wound healing.

In addition to detailing protein expression in the quail embryo during development, the researchers also determined the origin of the new cells required at the migrating edge to cover the growing yolk. During development, the radius of the quail yolk doubles every day for the first few days, representing a hundreds-fold increase in the egg yolk surface area.

"For each individual cell that has to cover the egg yolk as it grows, the migration around the yolk is extraordinary, because it's such a large territory -- it would be like an ant walking across the earth," explained Zamir.

Looking more closely at the edge cells, the researchers found strong evidence that expansion of the edge cell population was due exclusively to cells relocating from an interior region to the edge as the embryo expanded. The cells located at the free edge generated the bulk of the traction force necessary for expansion and towed the cells within the interior of the epithelium.

"These experiments confirm that edge cell proliferation is not the primary mechanism for expansion of the edge cell population," noted Zamir. "And our observation of epithelial-to-mesenchymal transition in the edge cells explains how these epithelial cells might be changing phenotype to become migratory in this rapidly expanding sheet."

To determine if this study's findings are indeed unique to the avian embryo, Zamir plans to conduct further studies to characterize protein expression and cell migration in Xenopus and zebrafish.

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Scientists at Emory University School of Medicine, Georgia Institute of Technology, and the University of Pennsylvania described the device in an article published this week in the journal Analytical Chemistry.

What a patient with a tumor wants to know after surgery can be expressed succinctly: "Did you get everything?" Statistics indicate that complete removal, or resection, is the single most important predictor of patient survival for most solid tumors.

"This technology could allow a surgeon to directly visualize where the tumors are, in real time. In addition, a post-surgery scan could check tumor margins," said Shuming Nie, a professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University. "A major challenge is to completely remove the tumor as well as identify lymph nodes that may be involved."

The SpectroPen can be used to detect fluorescent dyes, and also scattered light from tiny gold particles, a technology that Nie and his colleagues have been refining.

The particles consist of polymer-coated gold, coupled to a reporter dye and an antibody that sticks to molecules on the outsides of tumor cells more than it sticks to normal cells. Through an effect called surface-enhanced Raman scattering, the gold in the particle greatly amplifies the signal from the reporter dye. Nie and his team have been able to show that the particles can detect tumors smaller than one millimeter grafted into rodents.

The SpectroPen combines a near-infrared laser and a detector for fluorescence or scattered light. It is connected by a fiber optic cable to a spectrometer that can record fluorescence and Raman signals.

In the Analytical Chemistry paper, the researchers used the pen to detect the dye indocyanine green, infused intravenously into mice with implanted human breast cancer cells. The dye accumulates at a higher rate in tumor cells because of the leaky blood vessels and membranes surrounding tumors. The SpectroPen’s signal from the tumor is ten times higher than from normal tissue. Indocyanine green has been approved by the FDA for purposes such as measuring cardiac output and liver function.

The cancer cells had a gene from fireflies added, so that tumors glow after the mice are given a "luciferin" solution. This allowed the scientists to check that the outline of the tumor seen through the SpectroPen matched the glow.

"Our in vivo studies demonstrate that the tumor borders can be precisely detected preoperatively and intraoperatively, and that the contrast signals are strongly correlated with tumor bioluminescence," Nie said.

In the laboratory, the fluorescence and Raman signals are resolvable when the nanoparticles are buried 5-10 mm deep in fresh animal tissues. However, the gold nanoparticles are 40 to 50 times more sensitive than fluorescent dyes.

Future plans include in vivo tests of the nanoparticle contrast agents, along with the SpectroPen.

The research was carried out by an interdisciplinary team of senior investigators including May Wang, Coulter Department at Georgia Tech and Emory University; Sunil Singhal, University of Pennsylvania; and James Provenzale and Brian Leyland-Jones, Emory University. They are developing an integrated spectroscopic and wide-field color imaging system for image-guided surgery and cancer detection during surgery using animal models.

Provenzale and surgeons at the University of Georgia College of Veterinary Medicine are currently using this device to operate on dogs with naturally occurring tumors. Singhal, who is director of the Thoracic Surgery Research Laboratory at the University of Pennsylvania School of Medicine, is applying to conduct clinical trials involving patients with lung cancer.

The research was supported by a Grand Opportunities (GO) grant from the National Cancer Institute (NCI) and the NIH Director’s Office, and by the NCI Centers of Cancer Nanotechnology Excellence (CCNE) at Emory and Georgia Tech.

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The research team plans to develop a system that will excavate brain tumor cells by directing them away from their location in the interior of the brain to a more external location where they can be removed or killed. Nanofiber-based polymer thin films coated with biochemical cues will be aligned in the brain to provide a corridor for tumor cells to follow to a gel-based ‘sink’ where they will be captured and safely removed or encouraged to die through chemical signaling.

“We believe this is the first attempt to exploit the invasive, migrating properties of brain tumors by engineering a path for the tumors to move away from the primary site to a location where treatment can occur,” said lead investigator Ravi Bellamkonda, a professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University.

Collaborating with Bellamkonda on this project are Tobey MacDonald, director of the pediatric neuro-oncology program at the Aflac Cancer Center and Blood Disorders Service of Children’s Healthcare of Atlanta and an associate professor of pediatrics at the Emory University School of Medicine; and Barun Brahma, a pediatric neurosurgeon at Children’s Healthcare of Atlanta. The initial partnership between the researchers began with seed funding from the Georgia Cancer Coalition and Ian’s Friends Foundation.

The National Cancer Institute is providing more than $1 million for the EUREKA grant. For the project, Bellamkonda, MacDonald and Brahma are focusing on treating medulloblastomas -- highly malignant brain tumors that account for more than 20 percent of pediatric brain tumors.

“Medulloblastoma is the most common malignant brain tumor we see in children, but unfortunately the five-year survival rates for children with this cancer only range from 50 to 70 percent and the majority of survivors have a significantly reduced quality of life as a result of treatment-related toxicities,” said MacDonald, who is also a Georgia Cancer Coalition Distinguished Scholar. “An increasing number of survivors are also at risk for developing secondary malignancies as a result of the treatment we now administer. Clearly we have to do a much better job at treating these tumors; however, improving survival while reducing the toxic effects of treatment will require a highly innovative approach.”

Medulloblastoma treatment currently involves surgery followed by radiation therapy to the entire brain and spine and up to one year of intensive intravenous chemotherapy. However, radiation is often delayed or omitted altogether in young children due to its debilitating long-term side effects on the developing central nervous system.

These changes to the timing of radiation administration can adversely impact survival. And while surgery is a mainstay of treatment, it too can cause a significant loss of cognitive and neurological function due to the critical areas of the brain that may be involved by the tumor’s spread but require an extensive surgical area to remove as much of the tumor as possible.

This EUREKA grant aims to address the urgent need to develop therapies to safely treat invasive medulloblastomas in children.

“Our plan is to deliver the tumor to the drug -- by directing tumor cells to a specially engineered gel that can be removed or designed to kill the cells -- rather than the current strategy of delivering the drug to the tumor, which is problematic due to the irregular vasculature and poor diffusivity of the tumor tissue,” explained Bellamkonda, who is also a Georgia Cancer Coalition Distinguished Scholar.

The researchers plan to design a polymer thin film system that will include topographical and biochemical cues similar to those that guide the initial brain tumor invasion. The thin films will be rolled up and deployed with minimally invasive catheters. Because neural tissue will not be suctioned and the films are very thin, there should be minimal tissue and tumor disruption.

The films will also be non-toxic to the patient because they will be engineered with biocompatible, stable polymers. In previous studies, the polymers have been implanted in the nervous systems of small animals for more than 16 weeks with no adverse tissue reactions.

“This research represents a radical approach to treating invasive tumors that is based on the universal properties and mechanics of cell motility and the migration characteristic of metastasis, regardless of the molecular and genetic origins of the tumor,” added Bellamkonda.

If successful, this approach would identify a new and innovative way to treat pediatric medulloblastomas and has the potential to open a new avenue for the treatment of other invasive solid tumors, such as brain stem tumors. These cancers are incurable because they are located in an inoperable region and/or they are resistant or inaccessible to the delivery of chemotherapy agents.

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"This assay is important because researchers and pharmaceutical companies need a dependable method for sensitively detecting a small amount of cathepsin K and quantifying its activity to develop inhibitors to the enzyme that can fight the diseases while minimizing side effects," said Manu Platt, an assistant professor in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory University.

Cathepsin K is required to maintain adequate calcium levels in the body, but it can be highly destructive because it has the ability to break down bone by degrading collagen and elastin.

Platt described the cathepsin K detection protocol in the June issue of the journal Analytical Biochemistry. This research was funded by new faculty support from Georgia Tech, and the Facilitating Academic Careers in Engineering and Science Scholars (FACES) and Summer Undergraduate Research in Engineering (SURE) programs at Georgia Tech.

The benefits of this assay over existing techniques are numerous, according to Platt. The major advantage of this protocol, he said, is the definitive knowledge that mature cathepsin K is being detected in cells and tissues -- and not its immature form or one of the other 10 cathepsin varieties: B, H, L, S, C, O, F, V, X or W.

Another advantage of this technique is that it is more sensitive and less expensive than current, less reliable techniques. The new assay allows cathepsin K to be detected in quantities as small as a few femtomoles and does not require antibodies, which can be expensive and cannot be used across different species.

"In our experiments we were able to detect mature cathepsin K activity in quantities as small as 3.45 femtomoles with zymography, which was 10 to 50 times more sensitive at detecting the enzyme than conventional Western blotting," noted Platt, who is also a Georgia Cancer Coalition Distinguished Cancer Scholar.

In addition, zymography allowed the researchers to measure the activity of the enzyme, whereas Western blotting just measured its presence.

To detect mature cathepsin K with gelatin zymography, Platt and Georgia Tech undergraduate student Weiwei Li first separated the enzymes present in cells by their molecular weights. This allowed them to distinguish the mature form of cathepsin K from the immature form and other cathepsin varieties.

Then, to verify the identity and presence of mature cathepsin K, the team activated the enzymes in the gel. They created the perfect acidic environment for cathepsin K to thrive and added inhibitors to block the activity of all enzymes except mature cathepsin K.

To validate the cathepsin K activity detected in the laboratory experiments, Platt and Georgia Tech undergraduate student Zachary Barry developed a computational kinetic model of the enzyme's activity. By solving a system of differential equations, they were able to calculate the concentrations of immature, mature and inactive cathepsin K present at all times during the experimental procedure.

"It is more challenging to work with enzymes than proteins because enzymes have to be functional, which means they have to be folded correctly to be active," explained Platt. "The model suggested that even after the slight denaturation and refolding required by our assay, the cathepsin K activity determined by zymography reflected what happens in nature and was not an artifact of the experimental procedure."

The model also predicted something unexpected -- the inactive form of cathepsin K commonly purchased from supply houses contained 20 percent mature enzyme.

"Cathepsins are implicated in many different diseases and the value of this assay is that it enables the measurement of previously undeterminable cathepsin activity in normal and diseased cells and tissues," noted Platt.

With this assay, Platt’s team is currently investigating whether cathepsin K activity is different in the cells of individuals with metastatic and non-metastatic breast and prostate cancers, and the role of cathepsin K in cardiovascular diseases, such as stroke, in children with sickle cell anemia. They are also examining whether cathepsin K plays a role in the inflammation associated with HIV.

"This research should provide new information on a number of existing pathophysiological conditions where cathepsin K activity had been previously undetectable," added Platt.

Additional contributors to this work included Georgia Tech research technologists Catera Wilder and Philip Keegan; former graduate student Rebecca Deeds; and Joshua Cohen, a summer researcher at Georgia Tech and currently an undergraduate at the Massachusetts Institute of Technology.

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“We are primarily interested in developing an effectivemethod to reduce the spread of ovarian cancer cells to other organs ,” saidJohn McDonald, professor at the the School of Biology at the Georgia Instituteof Technology and chief research scientist at the Ovarian Cancer Institute.

 The idea came to the research team from the work of KenScarberry, then a Ph.D. student at Tech. Scarberry originally conceived of theidea as a means of extracting viruses and virally infected cells. At hisadvisor’s suggestion Scarberry began looking at how the system could work withcancer cells.

 He published his first paper on the subject in the Journalof the American Chemical Society in July 2008. In that paper he andMcDonald showed that by giving the cancer cells of the mice a fluorescent greentag and staining the magnetic nanoparticles red, they were able to apply amagnet and move the green cancer cells to the abdominal region.

 Now McDonald and Scarberry, currently a post-doc in McDonald’slab, has showed that the magnetic technique works with human cancer cells.

 “Often, the lethality of cancersis not attributed to the original tumor but to the establishment of distanttumors by cancer cells that exfoliate from the primary tumor,” said Scarberry.“Circulating tumor cells can implant at distant sites and give rise tosecondary tumors.  Our technique isdesigned to filter the peritoneal fluid or blood and remove these free floatingcancer cells, which should increase longevity by preventing the continuedmetastatic spread of the cancer.”

 In tests, they showed that theirtechnique worked as well with at capturing cancer cells from human patientsamples as it did previously in mice. The next step is to test how well thetechnique can increase survivorship in live animal models. If that goes well,they will then test it with humans.

 “We’ve developed a system that can kill cancer cells by shining light on gold nanoparticles, but what if the cancer is in a place where we can’t shine light on it? To fix that problem, we’ve decorated the gold with a chemical that brings it inside the nucleus of the cancer cell and stops it from dividing,” said Mostafa El-Sayed, Regents professor and director of the Laser Dynamics Laboratory at Georgia Tech.

 Once the cell stops dividing, apoptosis sets in and kills the cell.

 “In cancer, the nucleus divides much faster than that of a normal cell, so if we can stop it from dividing, we can stop the cancer,” said El-Sayed.

 The team tested their hypothesis on cells harvested from cancer of the ear, nose and throat. They decorated the cells with an argininge-glycine-aspartic acide petipde (RGD) to bring the gold nano-particles into the cytoplasm of a cancer cell but not the healthy cells and a nuclear localization signal peptide (NLS) to bring it into the nucleus.

 In previous work they showed that just bringing the gold into the cytoplasm does nothing. In this current study, they found that implanting the gold into the nucleus effectively kills the cell.

 “The cell starts dividing and then it collapses,” said El-Sayed. “Once you have a cell with two nuclei, it dies.” The gold works by interfering with the cells’ DNA, he added. How that works exactly is the subject of a follow-up study.

 “Previously, we’ve shown that we can bring gold nanoparticles into cancer cells and by shining a light on them, can kill the cells. Now we’ve shown that if we direct those gold nanoparticles into the nucleus, we can kill the cancer cells that are in spots we can’t hit with the light,” said El-Sayed.

 Next the team will test how the treatment works in vivo. 

Shafiq Khan, director of Clark Atlanta's CCRTD, said, "The molecular, bioinformatic and clinical expertise necessary to move forward with such a personalized cancer diagnosis and treatment program exists at the collaborating institutions. Establishment of CCGC will complement the existing experimental infrastructure necessary to generate the genomic data required to attain our goals."

John McDonald, director Georgia Tech's ICRC, added, "We are particularly interested in developing algorithms that will allow us to use gene expression and DNA sequence data that we gather from specific patients to generate a customized prognosis and optimal therapeutic treatment program for individual cancer patients."

Under the collaborative agreement, CCRTD will house and operate the CCGC's high-throughput next generation sequencing instruments. The resulting sequence data will be assembled and analyzed at ICRC. Patient samples will be provided by the Ovarian Cancer Institute (OCI) and Saint Joseph's Hospital's Translational Research Initiatives in Oncology for the Management of Personalized Healthcare (TRIOMPH ) program. Clark Atlanta and Georgia Tech scientists will join clinical experts from OCI and TRIOMPH to interpret and evaluate the resulting data. Housed at CAU in the Thomas W. Cole Jr. Research Center for Science and Technology, the CCGC is scheduled to begin operation in the fall of 2009.

Fifty-one researchers submitted proposals for the 2009 awards. Those reviewing the proposals included nationally recognized scientists and clinicians from across the country.

Dawson completed a Postdoctoral Research Fellowship at Massachusetts General Hospital and Harvard Medical School in 2008. She earned her Ph.D. in Chemical and Biomolecular Engineering from The Johns Hopkins University in 2005, where she was awarded a Graduate Research Fellowship from the National Science Foundation.

"Dr. Dawson is an outstanding and highly motivated researcher,

Read more at:
http://whsc.emory.edu/home/news/releases/2009/01/rules-for-silencing-cancer-cells-identified.html

Magnetic nanoparticles coated with a specialized targeting molecule were able to latch on to cancer cells in mice and drag them out of the body. The results are described in a study published online this month in the Journal of the American Chemical Society. The study's authors, researchers at Georgia Institute of Technology, hope that the new technique will one day provide a way to test for--and potentially even treat--metastatic ovarian cancer. (more)