Understanding the mechanics of breast cancer metastasis

Ghajar Lab research reveals why muscle tissue doesn’t get mets, how dormant tumor cells in the brain wake up and proliferate
A dormant pair of disseminated breast tumor cells in the brain (left). Note the astrocytes in cyan (blue/green) bordering the disseminated tumor cell-associated blood vessels (red). It turns out that reflects a functional, tumor-suppressive role. In the center image, you see the the same model, this time with a brain metastasis. Here you can note the astrocytes in cyan again are stripped from the blood vessels (red) within the metastasis and clustered at the edges. The final image (at right) is an intravital image of a brain metastasis (within a live mouse) with associated blood vessels perfused with a red, fluorescent chemical to allow them to be visualized.
A dormant pair of disseminated breast tumor cells in the brain (top). Note the astrocytes in cyan bordering the disseminated tumor cell-associated blood vessels (red). It turns out that reflects a functional, tumor-suppressive role. In the center image, you see the the same model, this time with a brain metastasis. Here you can note the astrocytes in cyan again are stripped from the blood vessels (red) within the metastasis and clustered at the edges. The final image (at bottom) is an intravital image of a brain metastasis (within a live mouse) with associated blood vessels perfused with a red, fluorescent chemical to allow them to be visualized.

Photos courtesy of Ghajar Lab, Fred Hutchinson Cancer Research Center

Breast cancer in the breast doesn’t kill you: It’s breast cancer that’s traveled out of the breast and into a friendly microenvironment within some organ system — your bones or lungs or liver or brain — where it begins the unchecked cell growth that will eventually become a metastatic tumor.

That’s the breast cancer that kills you.

Not all patients develop metastatic breast cancer, or MBC. But research at Fred Hutchinson Cancer Research Center in Seattle found that 20% of early-stage breast cancer patients will develop metastasis, also known as stage 4 or secondary cancer, within 20 years of their original diagnosis.

There are currently many therapies, but no cures for metastatic disease. There’s also no consensus as to why some patients develop “mets” and some don’t, although scientists do know that not all cancer cells that escape a primary cancer site automatically become distant metastatic tumors.

But two new studies from the Hutch’s Ghajar Lab, one published this week in Nature Cell Biology (and another in the January 2022 issue of Nature Cancer), have provided intriguing answers to key questions about metastatic disease.

Why do some dormant cells wake up and start growing tumors in our brains or other organs while other dormant cells stay quiet? Why is the tissue in our lungs friendly to cancer proliferation while other tissue, like that in our muscles, is hostile to metastatic disease?

Read on for new findings. And expect more to come, as translational researcher Dr. Cyrus Ghajar and his team delve into the mechanics — and potentially exploitable vulnerabilities — of breast cancer metastasis, the breast cancer that kills.  

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Photograph of Dr. Sarah Crist
During her graduate work in the Ghajar Lab, Dr. Sarah Crist examined why metastatic tumors rarely form in muscle. Photo by Robert Hood / Fred Hutch News Service

Why don’t muscles get mets?

Metastasis is not a given. Our body’s immune system destroys most of the cancer cells that leave a tumor site. And some tumor cells become so comfy in their tiny microenvironment niches, they stay dormant — harmlessly asleep — and never wake up to start their deadly proliferation.

Autopsies performed on breast cancer survivors who’ve succumbed to something else have revealed slumbering cancer cells in their bone marrow or livers or brains. Autopsies have also revealed single tumor cells in the muscle tissue of MBC patients, but metastatic tumors in muscle tissue are quite rare.

Dr. Sarah Crist, a former graduate student in the Ghajar Lab who is now a postdoctoral fellow at the University of Minnesota, teamed up with Ghajar and others at the Hutch as well as researchers in Colorado and Australia to learn why muscles don’t seem to develop metastatic tumors. They discovered at least one process by which muscle tissue is able to keep disseminated tumor cells from growing into tumors: by completely stressing them out.

“One thing that’s unique about muscle is it’s a highly metabolic organ,” Crist explained. “It contracts all the time and it takes a lot of energy to make it function. It’s incredibly active and because it’s so active, it needs to be constantly building energy.”

Crist and colleagues wondered if the metabolic nature of muscle was somehow related to the harsh environment it presents for metastatic cancer growth.

“Our hypothesis was that the muscle was consuming something, so that something was no longer available for the tumor,” she said.  

What they found was that metastasis was not able to form because the tumor cells were too stressed out by oxidants — reactive molecules that can cause damage in the cell — as part of an essential and intricate cellular process called redox metabolism.

“Muscle deprives tumor cells of antioxidant building blocks while simultaneously putting them under high oxidative stress,” said Ghajar, co-author on the study. “It’s a bad combination.”

'This is the first study to show that dormancy is the rate-limiting step of brain metastasis, which means that we should be focused on dormancy and preventing metastasis by targeting these dormant disseminated tumor cells.'

— Fred Hutch translational researcher Dr. Cyrus Ghajar

Photograph of Dr. Cyrus Ghajar
Fred Hutch translational researcher Dr. Cyrus Ghajar and his team are trying to find therapies that can shut down dormant disseminated tumor cells in the brain before they can wake up and start forming tumors. Fred Hutch file photo

Disrupting the balance

Using mice, organotypic culture models (a recreation of a human tissue in a petri dish) and metabolomic profiling, which captures the unique chemical fingerprints specific cellular processes leave behind, the researchers traced various molecular pathways to discover that skeletal muscle imposes a sustained oxidative stress on disseminated tumor cells which impairs their ability to proliferate.

“We found these tumor cells are immediately stressed upon traveling to the muscle,” Crist said. “It’s like their balance is off in terms of the redox metabolism — they can’t get past the single-cell stage because the environment is so stressful. They basically don’t have the ability to do anything but survive. They’re using all of their resources just to live in this harsh environment.”

The scientists validated their finding in multiple ways, including by giving tumor cells additional antioxidants.

“That’s the only time we saw the disseminated tumor cells grow in muscle tissue,” she said. “We gave them an extra dose, a much higher amount than is normally present.”

Scientists, of course, are not trying to find new places to grow metastatic tumors. The idea is to identify whatever it is that makes muscle tissue unfriendly to cancer, then use that information to make welcoming environments — like the lung, the liver, the brain and other organs — more hostile.

In their studies they were able to do just that, slowing the proliferation of disseminated tumor cells in the lung by disrupting the delicate balance of redox metabolism, subjecting them to the exact amount of oxidative stress that they experience in skeletal muscle.

“When we did this, we showed profound growth suppression in lung tissue,” Ghajar said. “And if they pushed the cancer cells even further, they died, suggesting a new vulnerability of disseminated tumor cells that can be exploited in patients.”

Of mice and men and women

But patients aren’t mice. Can they do this in people?

“That is the question,” said Crist, recent winner of the Hutch’s Harold M. Weintraub Graduate Student Award. “We’re thinking about how we would administer something that would be more oxidative, to make the tumor cells more stressed. But it’s all about finding the sweet spot — that ‘Goldilocks amount’ — not too much, not too little. You need to stress out the cancer cells without stressing out the body.”

And it’s not necessarily outright cell death they’re after: It’s redox imbalance.

“If you can create enough imbalance, you can stop these cells from growing,” she said. “It might be more realistic to make them stable, to keep them dormant. We don’t know at this point what to give to people, but we know this is a vulnerability we can try and target.”

Ghajar said his lab is working toward putting a clinical trial together to test the theory out.

“Our lab is trying to get to the bottom of the tissue-specific mechanisms of dormancy,” he said. “The hope is at some point that even though the players — the cells and the molecules — are different, maybe they converge on a common theme, providing a means by which one can keep these cells asleep throughout our body.”

Photograph of researcher Dr. Jinxiang “David” Dai
Ghajar Lab postdoctoral fellow Dr. Jinxiang “David” Dai examined the biological factors shaping formation of brain metastases. Fred Hutch file photo

How do brain mets get started?

Metastasis in the brain starts like other mets: Single cancer cells or tumor cell clusters travel there, just as they travel to other organs in the body, and if they make it to the brain alive, they soon become dormant, sometimes for months, sometimes for years or even decades.

In a certain segment of people, though, something rouses these sleeping cells and signals them to start dividing.

Approximately 15% of all MBC patients go on to develop brain metastasis, although it’s more common in those with HER2-positive or triple-negative disease (around 30% of metastatic triple-negative breast cancer patients get brain mets). New cancer therapies have helped MBC patients live longer but living longer also means the cancer has time to travel to the brain and set up shop. Incidence of MBC brain metastasis has increased over the last 10 years.

Brain mets are painful, debilitating and often fatal. The blood-brain barrier, a network of blood vessels and tissue made up of closely spaced cells that keep harmful substances (think pathogens) from reaching the brain, makes it difficult to deliver drugs. And MBC tumor cells are adept at outsmarting our body’s immune system. Therapies to either prevent or treat these cancers are desperately needed.

But a deep understanding of the process — the whole metastatic cascade — is also necessary.

Another paper from the Ghajar Lab, published in Nature Cancer in January, provided key insights into how these tumor seeds reawaken and begin to spread in the brain — a process that has largely gone without study.

It also offers hints as to how they might be stopped.

“This is the first study to show that dormancy is the rate-limiting step of brain metastasis,” Ghajar said. “Which means that we should be focused on dormancy and preventing metastasis by targeting these dormant disseminated tumor cells. From this point, it’s a long way to where you have something that will impact patients, but you have to start somewhere.”

A window into the world of brain mets

Led by Dr. Jinxiang “David” Dai, the research team (which included Ghajar, other Hutch scientists and partners from institutions in California, Colorado, New York, Pennsylvania, Utah and Germany) used “serial intravital imaging” — a literal window installed into the brains of living mice — to watch how single cancer cells escape from a dormant state within the brain’s microenvironment and begin to grow and spread as deadly tumors.

“We wanted to know, on a more granular level, what these dormant cells were doing,” Ghajar said. “Are there clusters of cells? Single cells? Are they growing slowly over the course of years? Are they dividing every two weeks, every four weeks, every week? No one had watched the cells that can form brain metastasis as they come into the brain and contrasted them with those that can’t to see where they differ.”

Dai, Ghajar and teammates installed intravital windows into live mice in order to witness the progression — or lack thereof — of metastatic triple-negative breast cancer cells in the brain. Some of the cells were dormant and some were selected for their ability to form brain metastases.

Through the brain windows, they captured images of how these tumor cells migrate to and occupy vascular niches within the brain — sites rich in blood vessels that create microenvironments that affect the behavior of certain cells — discovering quiescent or sleeping cells are drawn to a particular locale: astrocyte endfeet.

Astrocytes are the most common cell in the central nervous system; their endfeet are specialized structures that ensheathe the brain’s microvessels, controlling blood vessels and blood flow.

Through various experiments, the research team discovered that a critical component of the blood-brain barrier called laminin-211 that’s deposited by the astrocytes is what keeps dormant tumor cells dormant.

The researchers found the brain’s normal signaling is “coopted” by disseminated tumor cells in vascular niches containing astrocytic endfeet and this induces signaling that steers them into their quiescent state. As micro-metastases start to proliferate, astrocytes and their endfeet are then stripped from the brain’s vessels.

What triggers this? Ghajar said that’s an unknown for now, calling it a “classic chicken/egg problem.”

“We looked at brain metastasis from 24 different people, including brain mets from patients who’d died. And in each of those metastatic tumors, the astrocytes are gone, the astrocytic protein is gone and there are proliferative tumor cells around it,” Ghajar said. “Does a cell proliferate and as it does it destroys this axis, or does the axis get compromised which allows growth? My guess is either, depending on the situation. We can show the correlation in a human, but not cause. But we could show cause in a mouse.”

A window of opportunity

In addition to pinpointing the cellular mechanics of dormant tumor cells in brain tissue, the researchers also learned that the mechanism is unique.

“One of the major points of the study is that this process is very specific to the brain,” Ghajar said. “The mechanism does not seem to exist anywhere else.”

The bottom line? Now that they’ve located this vulnerability, they can work on ways to target the seeds of brain mets before they have a chance to sprout.

“Tumor cells need to invade, to get into the bloodstream, get out of the bloodstream, to survive in the organ they’re trying to colonize,” Ghajar said. “People have always focused on the ability of these cells to survive. They thought their ability to survive was the rate-limiting step.”

This data, Ghajar said, shows that the bottleneck to brain metastasis is not cells gaining entry to the brain or surviving once there. Instead, the research definitively points to escape from dormancy as the rate-limiting step.

And that presents a window of opportunity.

“What we want to do ultimately is target the dormancy phase,” Ghajar said. “We have real evidence that this is what limits metastasis. If we could keep things in this state, or if we could eliminate them while they’re in the state, that would help prevent metastasis. Now we just have to figure out the precise way to do this. Stay tuned!”

The research described in this story was funded by the W.M. Keck Foundation, the National Cancer Institute, the Department of Defense Breast Cancer Research Program and Fred Hutch, and the research was supported by Fred Hutch Shared Resources.


Diane Mapes is a staff writer at Fred Hutchinson Cancer Center. She has written extensively about health issues for NBC News, TODAY, CNN, MSN, Seattle Magazine and other publications. A breast cancer survivor, she blogs at doublewhammied.com and tweets @double_whammied. Email her at dmapes@fredhutch.org. Just diagnosed and need information and resources? Visit our Patient Care page.

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