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NCI Scientists Develop Angiogenesis Assay That Mimics In Vivo Environment In Vitro

Scientists at the National Institutes of Health’s National Cancer Institute have developed several in vitro angiogenesis assays and a software application that can mimic in vivo angiogenesis.
According to the researchers, the assays and software in combination can be used to investigate the relationships between different cells involved in angiogenesis, develop new combinatorial approaches to boost the efficiency of existing therapies, and facilitate the discovery of potential single or combination therapies.
The assay is based on immortalized cell lines the scientists developed for high-throughput angiogenesis and cytotoxicity screening. These cell lines, which constitutively express different fluorescent proteins, were selected to represent the different types of cells that participate in angiogenesis in vivo.
The NCI team has also developed a cytotoxicity assay using these cells that would be appropriate for screening potential therapies.
The cell lines, assays, and software applications are currently available for non-exclusive licensing. A patent application (No. 12/060,752) has also been filed with the US Patent and Trademark Office, the researchers said.  
The assays have several advantages over other currently available assay kits, an NCI official told CBA News this week, including the ability to monitor in real time cellular interaction and activity, shortened and simplified protocols, and no added detection reagents to disrupt assay results.
With most angiogenesis assays, researchers are counting cells, said Frank Cuttitta, director of the NCI’s Angiogenesis Core Facility. These cell counts are time-consuming and involve killing the cells and quantifying the enzymes that are in the cell or associated with the mitochondria.
“But with our assay, you do not kill the cells,” Cuttitta said. “It just looks at fluorescence, so you do not have to stop your assay. Most of the assay systems that are available today have a time point where the assay is terminated, and the number of cells is measured, either indirectly or directly. Our assay does not stop. You can actually look at it over 12 days, if you want to,” he added.
“Let’s suppose I want to know what compounds kill the endothelial cells on day one,” said Cuttitta. “I just pull the plate out of the incubator, add the drug, measure the fluorescence, and then put it back into the incubator.”
Investigators can also determine rates of cell growth inhibition or stimulation because they get slopes of curves of the cells are being suppressed. “You cannot do that routinely in the currently used assay systems, unless you do multiple assays for each time point,” Cuttitta said.
Enrique Zudaire, a staff scientist at the Angiogenesis Core Facility, agreed with Cuttitta that one of the major advantages of using these cell lines is that the assay can be followed in real time. In other commercially available systems, a chemical will be added that will be indirectly related to the number of cells in the well, and there is a mathematical relationship between the intensity of the particular dye used and the cell count.
“Your assay stops there, however,” Zudaire said. “In our system, you do not disrupt the environment of the cells.” No chemicals need to be added, because the cells are already fluorescent.

“We have induced different dynamics in the growth of the cells with different drugs.”

Cuttitta explained that when using the assay, “you set up one number of cells and continue the assay for five days or six days. Although you have not set up new assay systems, you still get the critical data.”
Cuttitta, Zudaire, and colleagues have done high-throughput screening on a variety of pharmaceuticals and NCI’s diverse set of small molecules, Cuttitta said. By just looking at fluorescence intensity, one can rapidly screen 2,000 drugs and identify which of those would suppress endothelial cell growth, he said. 
Zudaire said that the NCI investigators have tried to set up high-throughput screening assays based on their growth assay. “One interesting thing is that we have induced different dynamics in the growth of the cells with different drugs.”
Looking at the cells in real time means that “you can observe the dynamics of growth for that particular cell type in the presence or absence of your drug,” Zudaire said. And different drugs induce different cellular dynamics, which “gives us a lot of information about the drugs’ mechanism of action on those cells.”
In addition, Cuttitta said that a driving force behind the development of this assay was that “we wanted to develop an assay where if you are a lab that had, say, only $40,000 worth of grant money, as compared to a lab that had $2 million in grants, both labs could run these assays with no problem whatsoever.”
A lab does not need to spend $1 million to acquire the instrumentation to run this assay. “You just look at fluorescence, which most labs have, and it becomes much cheaper than using routine cell-growth assays that are currently available,” said Cuttitta.
Although instruments that will do real-time analysis are available, “they will run you $60,000. We wanted all labs to be on an even keel.” 
Under the TARP
The Angiogenesis Core Facility came out of a consortium at the NIH called the Transinstitute Angiogenesis Research Program, or TARP, that began in 2003. Multiple institutes are involved in TARP, including the National Cancer Institute; the National Heart, Lung, and Blood Institute; the National Institute of Diabetes and Digestive and Kidney Diseases; the National Institute of Neurological Disorders and Stroke; the National Institute of Child Health and Human Development; and the National Eye Institute.
TARP then formed a steering committee including Judah Folkman, who passed away recently, and Donald McDonald of UCSF, who told TARP that the biggest problem in the field was the lack of standardization, Cuttitta said.
“People were just doing angiogenesis assays helter skelter, and it made it really difficult, if you were in a lab on the West Coast, to compare data with a lab on the East Coast, for example,” he said.
The TARP Angiogenesis Core Facility came out of that mandate. “The Center for Cancer Research, which is basically the center that funds me, and is part of the NCI, said, ‘We need to formulate an NCI angiogenesis research facility to standardize angiogenesis assays and standardize reagents, et cetera,’” Cuttitta said.  
That is where the group’s fluorescent cell-assay system evolved from, said Cuttitta. “One of the biggest problems was to find, in the field, a standard endothelial cell that everyone could use,” he said. “And to be honest with you, that did not exist. There was a huge variation among commercial sources of immortalized endothelial cell lines and primary endothelial cell lines.”
Zudaire headed the entire project on transfecting existing immortalized endothelial cells with different fluorescent dyes, said Cuttitta. He has established endothelial cell lines that have different colors and human tumor cells that have different colors.
Zudaire had shown that, as the population of a given fluorescent color endothelial cell increased, so did the intensity of that fluorescence, so there was a direct correlation between intensity of fluorescence and cell number, Cuttitta said. 
One of the assays routinely used in angiogenesis is the tube-formation assay, which is probably one of the better assays to use, because multiple processes are involved.
However, one of the problems with the tube-formation assay is how one would quantitate it. Zudaire took this problem to a variety of sources outside, and found out that nobody had a user-friendly software program that could quantitate tube formation, Cuttitta said.
Cuttitta went on to explain that Zudaire has a very strong background in areas such as image analysis, software development, and computer interaction, “so he developed his own software program to quantitate tube-formation assays.”
Unfortunately, evaluating the effects of compounds on cell growth and tube formation is only half of the story, “because you can have a drug that can suppress the growth of endothelial cells but that is also cytotoxic,” said Cuttitta.  
He added that Zudaire also developed a method to determine which compounds are cytotoxic. “You can quickly look at the amount of fluorescence in and around the cells, and determine if the compound is cytotoxic or not,” Cuttitta said. 
When faced with a cytotoxic compound, it takes around 30 minutes for the fluorescent compound to lyse the cells and spill out into their environment, Zudaire said. 
Comparisons to commercially available cytotoxicity assays have been made. Those assays are usually based on an indirect relationship to an enzyme that exists in the cytoplasm. These currently used assays usually take about five to six hours to complete, “while our assay is very appropriate for high-throughput screening, because it can be done within 30 minutes,” Zudaire explained.
The actual assay is done in a minute, he added. “I am including … the time required to incubate the cells with your particular drug. The assay itself is a matter of looking at the fluorescence in the liquid where the cells are being cultured.”
A number of companies sell cytotoxicity assays based on lactate dehydrogenase. Most of those assays are based on the fact that LDH produces certain biological byproducts, and you can immunologically detect those byproducts, and thus, indirectly, the enzyme.
However, that involves an antibody that has to bind to the enzyme. The antibody is typically labeled with a peroxidase, for example, and later steps can be added to develop the enzymatic activity of the peroxidase.
These assays involve multiple steps that, in the end, “we believe are more time consuming, more expensive, and are indirect systems, because they are amplified systems,” said Zudaire. “In our system, what you see is a direct measurement of the fluorescence that was in the cell, and because the cell membrane was disrupted, that fluorescence is now in the culture media, and therefore is a direct reading of a component that is being produced by the cell.”
Zudaire said that a number of companies have already expressed interest in this product, or are in the process of licensing part of or the whole package.  
“Obviously, a lot of things can be done” to further develop these assays, said Zudaire. At the NCI Angiogenesis Core Facility, researchers try to develop complex assays that include multiple cell types that are as close as possible to the real angiogenesis environments found in vivo.  
“The only way that we feel that we can do that is to consider the multiple cell types that participate in the angiogenesis process,” Zudaire added. These cell types would include tumor cells, inflammatory cells, and other auxiliary cells, such as fibroblasts.
It would be a “huge advantage,” Zudaire said, to be able to label these cells with fluorescent proteins in such a way that would render additional processing of the assay to get a readout unnecessary.

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