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Movies of Metastasis Shed Light on How Cells Move in the Body

Rabiya S. Tuma

How tumor cells migrate from one site to another in the body is a major question for cancer biologists, but standard metastasis assays look only at the end points of the problem—where the cells start and where they end up.

Now, Peter Friedl, M.D., Ph.D., from the University of Würzburg, Germany, has created a system that allows him to watch tumor cells as they move through the dermis of a live mouse. From these experiments and companion work in vitro, he can see that the cancer cells have a garage full of transportation alternatives and that if one mechanism is blocked, they simply turn to another.



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Dr. Peter Friedl

 
In the simplest version of these experiments—using time-lapse confocal microscopy to watch individual tumor cells migrate through a collagen matrix in vitro—Friedl and his colleagues see what many others had predicted: Tumor cells, including melanoma and rhabdomyosarcoma cells, move like fibroblasts. The leading edge of the cell grabs onto the collagen matrix via {beta} integrins and drags the rear edge of the cell behind it, in an inchworm-like movement.

GoAlso as predicted, tumor cells do not move innocuously through the matrix. Rather, the integrins seem to recruit metalloproteases to the leading edge of the cell, where the proteases clip collagen strands, opening corridors for the large cells to pass through. Often one leading cell, or guerrilla cell as Friedl refers to it, seems to pave the way for other cells that follow in its wake.



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The image above shows a melanoma cluster in collagen (structure shown in green) stained for {beta}1 integrin. Fiber can be seen pulling at the leading edge of the cluster (shown in red on the left). Several cells work together at the leading edge, and a small matrix defect can be seen at the trailing edge. (Source: Cancer Res 2002 Apr 1;62(7):2125-30) (© 2002 American Association for Cancer Research. Reprinted with permission.)

 
Role of Metalloproteases

Up to this point all of these observations fell in line with the leading models in the field, but what happened next threw Friedl and his colleagues for a loop. To decipher the role the metalloproteases play in tumor cell movement, the team investigated whether inhibiting protease activity could block movement. To their surprise, they found that treating the cells with specific inhibitors of the metalloproteases did not slow the cells’ movement or alter their trajectories at all. In fact, even when the research team treated the cells with a complex mixture, or cocktail, of protease inhibitors, which seemed in biochemical assays to prohibit any cleavage of the collagen fibers, the cells still moved at the same rate through the collagen matrix.

These data did not make sense, said Friedl. The cells were still moving at the same rate, but by both visual and biochemical inspection the collagen matrix appeared to be intact. So what was allowing the large cells to move between densely packed fibers?

"We had been looking at these movies [of the tumor cell movement] for a couple of weeks when suddenly we realized that the [cell] morphology was different," said Friedl at the 93rd annual meeting of the American Association for Cancer Research, held in San Francisco in April, where he presented the work. The cells, said Friedl, did not look spread out like fibroblasts but rather appeared spherical. And they seemed to move in a manner similar to lymphocytes, which are much smaller cells. That is, somehow these large tumor cells were squeezing between the matrix fibers rather than cutting a path through them.

At higher magnification, Friedl’s time-lapse movies showed that the cells narrow at one end and then push themselves through an existing gap in the randomly crisscrossing collagen fibers. For the cells to sneak through these too-small spaces, they constantly change shape, compressing first one part and then another.

This type of movement, which Friedl called amoeboid movement, resembles the mode of motility used by single-celled organisms, but it has not been seen previously in these large, fibroblast-like tumor cells.

"We are convinced that this is a non-proteolytic means of moving through the collagen matrix," said Friedl.

The results have taken some researchers by surprise. "People always assume that cancer cells only have one way to move, wandering around like little fibroblasts," said Ann Chambers, Ph.D., who studies metastasis at London Regional Cancer Centre in London, Ontario. "[Friedl’s] stuff so elegantly shows that they can wander around like fibroblasts, wander around in a clump of cells that move together, or they can do this amoeboid thing, which I haven’t seen before, when you block off all the proteases." Chambers acknowledged that this might not be the best news for scientists trying to inhibit metastasis in patients.

Watching Metastasis in Mice

But even the best in vitro data cannot always predict what will happen in vivo. With this in mind, Friedl initiated a collaboration with Ulrich von Andrian, M.D., Ph.D., and Harry Leung at Harvard Medical School in Boston, to use their two-photon confocal microscope to perform similar experiments in live mice. This new confocal technology, said Friedl, enables researchers to peer up to 800 microns into the dermis of a mouse.

To simultaneously watch the movement in the dermis of both cells treated with the protease cocktail and untreated control cells, the researchers labeled the protease-treated cells with red fluorescent die and the control cells with a green dye. They then anesthetized the mouse, removed a patch of epidermis, and injected the cells into the dermis. After 3 hours, the researchers could see clearly that both the protease-treated and control cells had moved from the site of injection.

Because the mouse was always moving slightly, the researchers could not follow individual cells. Instead, they used high-resolution pictures to plot the location and color of each cell at 3 hours after injection and then compared the picture with an image taken 8 hours after injection.

The results were dramatic and convincing: Both types of cells moved, and they moved similar distances in the 5-hour period.

But the researchers did see a difference in the shapes of the two cell types. The protease-treated cells appeared spherical, whereas the untreated ones did not, which was consistent with the treated cells using an amoeboid type of movement.

Mechanism of Migration

"Taking in vivo and in vitro data together we certainly have a default mechanism of migration," concluded Friedl. "Take away the proteases and we are left with amoeboid movement, a salvage strategy to get through the matrix. So a path-generating mode is converted to a path-finding mode."

Lynn Matrisian, Ph.D., chair of the cancer biology department at Vanderbilt University in Nashville, Tenn., who organized the AACR session, says the system Friedl developed will open new doors in the field of metastasis biology.



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Dr. Lynn Matrisian

 
"We have these assays that we’ve been using for years and years," said Matrisian, referring to experiments in which scientists inject tumor cells into the tail vein of mice and then look to see where they end up.

"We’ve made assumptions about the steps in between and where drugs and molecules act during those steps, but until we develop techniques to actually watch where they work, we’ll often be wrong.

"He has the system now to analyze the contributions of any number of molecules and their inhibitors, so we can see how they work and how they affect the system," said Matrisian.

Already this system of visual assays is paying off. In addition to the in vivo work, Friedl described what happens to rhabdomyosarcoma cells that have been freshly excised from a tumor, placed in a culture dish with a collagen matrix, and then exposed to a {beta}1 integrin inhibitor.

In the absence of the inhibitor, the cells move as a cohesive "social" unit, with only the cells on the leading edge expressing significant levels of the {beta} integrin or metalloproteases. But in the presence of the inhibitor, the organized group falls into disarray. Cells throughout the group start expressing the integrin and, more significantly for cancer biologists, the cell group disperses, with the cells migrating individually or in smaller groups.

"I think often people focus on the molecules and forget the context in which they have to work," said Chambers. But she likens Friedl’s work to a "whack on the side of the head" that forcefully reminds people—and enables them to see—that molecules work only in the context of the cell and the organism.



             
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