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The Assay

Quigley uses an assay that has the advantages of being inexpensive, fast, simple, not requiring complicated surgeries, animal protocols, and large spaces.

The assay itself uses chicken eggs with their shells removed that are placed in an incubator to develop for a few days. Because the eggs are only a few days old, they are immunologically naïve and will tolerate human tumor cells.

After ten days, a tumor is placed on the soft chorioallantoic membrane on the inside of the shell and an antibody against the tumor cells is injected into the egg. Quigley uses an aggressive tumor cell that will grow and form metastases in under a week. An antibody of interest can be injected into the egg and tested for the ability to block metastasis. Another advantage of this model is that the volume is small and dilution of antibody over the course of the assay is minimal. "It stays in and does its job," says Quigley.

After a week with the tumor, metastatic cells can be detected by looking for evidence of human (tumor) DNA in a part of the egg that is distinct from where the tumor was implanted a week before. Human DNA has thousands of copies of 30 to 50 base "alu" repeats spread throughout its genome, and finding copies of these alu repeats is analogous to finding a visible tumor. The DNA can be extracted from the sample using a simple prep and amplified through the polymerase chain reaction with alu-specific primers.

Then the amount of DNA extracted from the sample can be quantified by comparing the signal to a standard curve. The number of copies of alu repeats progresses linearly with the number of tumor cells. "We can detect anywhere from 100 to 10,000 cells per sample," says Quigley. "And 100 cells is really seeing micrometastasis—cells that you might never see [looking at sections under a microscope]."

By testing different antibodies against a control, those which alter the metastatic phenotype of the tumor cells can be easily identified. Once an antibody is found that modulates metastasis, its antigen must then be identified by using the same antibody to isolate the correct protein, which then gets sequenced.

Occasionally a protein is found that, when blocked, stops metastasis. Quigley's group has found several of these thus far. Some were to be expected, such as a membrane-spanning integrin-associated protein they identified that is necessary for helping the integrins loose their grip on other cells and on the extracellular matrix, an important first step in metastasis.

Others, however, have turned out to be a surprise. One antigen that was identified was that of an novel protein whose cDNA has been sequenced in an expression database but not yet annotated.

"We have no idea what it is," Quigley says. "But we have cloned it, and we are trying to find out how it works."

Metastasis and Angiogenesis—The Complete Picture

In addition to metastasis, Quigley's laboratory studies angiogenesis, the process where blood vessels are formed and differentiated. The goal of both the metastasis and the angiogenesis work is to identify molecules that could become targets for intervention, and he employs the same basic techniques in both, using subtractive immunization and chicken egg in vivo models to study them.

In the angiogenic models, blood vessels can be easily counted and observed under a microscope, and this forms a basic assay that can then be used to screen for compounds that inhibit, stimulate, or otherwise modulate the angiogenic process.

In fact, the laboratory's first major paper in the field, recently published in Blood, was about growth factor induced angiogenesis. After implanting native collagen onto a chick embryo, they injected angiogenesis growth factors into the collagen, and then studied the effect of new vessel growth on the surrounding area to uncover the active molecules that contributed to the remodeling of the new tissue. Some of the active molecules turned out to be enzymes that break down proteins, the same proteolytic enzymes that Quigley had been studying for years as part of his research into the tumor invasion process. Now his research has turned up the fact that the same enzymes are also involved in the formation of new blood vessels, a gratifying breakthrough.

In other instances, as well, these two problems merge. For instance, tumor angiogenesis—the process whereby tumors will cause a proliferation of blood vessel growth—is an important first step in metastasis. A tumor placed on a membrane will cause new vessels to grow into a drop of collagen that is laid on top of the tumor, and this process can be studied.

"In some cases," he notes, "all the areas in the laboratory merge."

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Basis for quantifying new blood vessel growth (angiogenesis) involves implanting a gridded nylon mesh surrounded by collagen onto the chorioallantoic membrane of a chick embryo (A and B). When the collagen contain specific growth factors, bFGF and VEGF, enhanced appearance of new blood vessels occur in the upper grid of the nylon mesh (C vs. D), which can be easily quantified.