A Hard Boiled Look at Metastatic Remodeling Molecules

By Jason Socrates Bardi

The very worst kind of tumors are those drifters that pick up and move to another part of the body, an event called metastasis.

When a tumor metastasizes, it does so by expressing proteins that allow its cells to break free of the colony, enter the bloodstream, survive in the circulation, and arrest in the vessels of another organ. Then these founder cells express more proteins that keep them alive, allow them to divide and divide, bring them blood and nutrients, and help them grow into new metastatic tumor colonies.

Professor James Quigley of the Department of Vascular Biology calls the expression changes within a tumor that begins metastasis remodeling.

"If a cell is going to spread into a neighboring tissue," he says, "there is going to be a lot of molecular remodeling... We're after the early events. What are the molecules that determine why a human tumor cell migrates to and survives in a different organ—a different environment with different growth factors, adhesion molecules, hormones, and glandular products nearby?"

If one could identify these molecules, they would yield information about both the basic biology of metastatic cancer and possible therapeutic targets, and that is exactly what Quigley is trying to do.

Under Glass, In Profile, Under Reason

This is easier said then done, though, because trying to observe metastasis in the laboratory is not trivial. First, scientists must study cells in vivo, because metastasis is a complex, three-dimensional cascade event with too many processes involved to be imitated in vitro. "Metastasis cannot be mimicked at all in culture," says Quigley.

Second, some changes may not be morphological or otherwise observable via some easily detected change or signal. In order to detect the tumors with a microscope, a standard method, one would have to wait for the tumors to grow large enough to be seen through exhaustive searches of tissue sections, a time-consuming processes. Using pigmented melanomas to increase the visibility of the tumors helps, but these melanomas do not necessarily have the desired metastatic properties.

Finally, there is the problem of false positives, which arises from the fact that some of the most important molecules that contribute to the phenotype of metastasis may not be the ones that are most widely expressed. The observation that a protein is up or down-regulated in cell metastasis does not mean that protein is necessary for metastasis.

Plus, the metastatic phenotype may be brought about by extensive combinations of subtle up and down regulations of proteins that activate or suppress other proteins that are normally there, in which case the more interesting observation would be how the expression of the activator and suppressor proteins change.

"We want to identify the molecules that are not just associated or correlated with a given process, but functionally involved," Quigley says.

One way to get at these functionally involved molecules is to reason out which proteins might be necessary for metastasis, given everything we know about the basic biology, and study those. Serine and metalloproteases, for instance, are necessary to free a potentially metastatic cell from the collagens and proteoglycans that make up the stromal tissues to which they are bound. Quigley has worked on such proteases for years, and he continues to devote a significant portion of his time to them.

Quigley and his laboratory have also developed another method, which Quigley calls an unbiased approach to identifying the proteins involved in metastasis. This method has the advantage of working without any preconceived notion as to what these proteins are.

The Unbiased Way

This approach involves first generating many monoclonal antibodies raised against "crude" tumor cell antigen populations—whole cells and cell membranes—and then screening for those that block the metastatic ability of the cell. Quigley reasons that any antibody that arrests the metastasis must have as its target some antigen involved in the process.

"[We do this] without having any idea as to what the nature of the target of that antibody is," says Quigley. "Once we screen for a blocker, then we try to identify its target."

The difficulty in using monoclonal antibodies against tumor cells is that most of the antibodies raised will be against immunodominant antigens—all the high-visibility proteins that the immune system recognizes. But these antigens may not be critical for a biological process like metastasis. One would really like to make antibody against only those minor or low-abundance antigens that are critical.

To accomplish this, Quigley uses a trick called "subtractive immunization" that increases the proportion of non-immunodominant antibody raised.

The trick with subtractive immunization is to first tolerize an organism -with a non-metastatic tumor cell, using an immune suppressant chemical like cyclophosphamide to kill off all the immune cells that recognize the immunodominant antigens.

Once this tolerance is achieved, the next step is to challenge the same organism with a second tumor cell. The second cell should be similar in every way to the first except that it is aggressively metastatic. "It will still have those same common immunodominant antigens on the surface, but the tolerized immune system won't mount a defense against them," says Quigley.

Instead, the immune system will produce antibodies against those proteins that are unique to or enriched in the metastatic cell. Some of the proteins may directly contribute to metastatic spread. Then the B cells that make these antibodies can be used to make antibody-producing cell lines, called hybridomas, and the antibodies produced by these hybridomas can be screened in an assay Quigley has developed to look for the phenotype of blocking metastasis. Then the antigens of those antibodies that do block metastasis can be isolated and sequenced.

Quigley calls this isolation "a hard chore," but once done, these antigens should be cell molecules that are essential components for metastasis and, perhaps, eventual targets for therapeutics.


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James Quigley is a professor in the Department of Vascular Biology.