April 23, 2019
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Research Area: Cancer Biology
The substance that bathes tumors in the body is quite different from the medium used to grow cancer cells in the lab, biologists report.
Anne Trafton | MIT News Office
April 16, 2019
Before being tested in animals or humans, most cancer drugs are evaluated in tumor cells grown in a lab dish. However, in recent years, there has been a growing realization that the environment in which these cells are grown does not accurately mimic the natural environment of a tumor, and that this discrepancy could produce inaccurate results.
In a new study, MIT biologists analyzed the composition of the interstitial fluid that normally surrounds pancreatic tumors, and found that its nutrient composition is different from that of the culture medium normally used to grow cancer cells. It also differs from blood, which feeds the interstitial fluid and removes waste products.
The findings suggest that growing cancer cells in a culture medium more similar to this fluid could help researchers better predict how experimental drugs will affect cancer cells, says Matthew Vander Heiden, an associate professor of biology at MIT and a member of the Koch Institute for Integrative Cancer Research.
“It’s kind of an obvious statement that the tumor environment is important, but I think in cancer research the pendulum had swung so far toward genes, people tended to forget that,” says Vander Heiden, one of the senior authors of the study.
Alex Muir, a former Koch Institute postdoc who is now an assistant professor at the University of Chicago, is also a senior author of the paper, which appears in the April 16 edition of the journal eLife. The lead author of the study is Mark Sullivan, an MIT graduate student.
Environment matters
Scientists have long known that cancer cells metabolize nutrients differently than most other cells. This alternative strategy helps them to generate the building blocks they need to continue growing and dividing, forming new cancer cells. In recent years, scientists have sought to develop drugs that interfere with these metabolic processes, and one such drug was approved to treat leukemia in 2017.
An important step in developing such drugs is to test them in cancer cells grown in a lab dish. The growth medium typically used to grow these cells includes carbon sources (such as glucose), nitrogen, and other nutrients. However, in the past few years, Vander Heiden’s lab has found that cancer cells grown in this medium respond differently to drugs than they do in mouse models of cancer.
David Sabatini, a member of the Whitehead Institute and professor of biology at MIT, has also found that drugs affect cancer cells differently if they are grown in a medium that resembles the nutrient composition of human plasma, instead of the traditional growth medium.
“That work, and similar results from a couple of other groups around the world, suggested that environment matters a lot,” Vander Heiden says. “It really was a wake up call for us that to really know how to find the dependencies of cancer, we have to get the environment right.”
To that end, the MIT team decided to investigate the composition of interstitial fluid, which bathes the tissue and carries nutrients that diffuse from blood flowing through the capillaries. Its composition is not identical to that of blood, and in tumors, it can be very different because tumors often have poor connections to the blood supply.
The researchers chose to focus on pancreatic cancer in part because it is known to be particularly nutrient-deprived. After isolating interstitial fluid from pancreatic tumors in mice, the researchers used mass spectrometry to measure the concentrations of more than 100 different nutrients, and discovered that the composition of the interstitial fluid is different from that of blood (and from that of the culture medium normally used to grow cells). Several of the nutrients that the researchers found to be depleted in tumor interstitial fluid are amino acids that are important for immune cell function, including arginine, tryptophan, and cystine.
Not all nutrients were depleted in the interstitial fluid — some were more plentiful, including the amino acids glycine and glutamate, which are known to be produced by some cancer cells.
Location, location, location
The researchers also compared tumors growing in the pancreas and the lungs and found that the composition of the interstitial fluid can vary based on tumors’ location in the body and at the site where the tumor originated. They also found slight differences between the fluid surrounding tumors that grew in the same location but had different genetic makeup; however, the genetic factors tested did not have as big an impact as the tumor location.
“That probably says that what determines what nutrients are in the environment is heavily driven by interactions between cancer cells and noncancer cells within the tumor,” Vander Heiden says.
Scientists have previously discovered that those noncancer cells, including supportive stromal cells and immune cells, can be recruited by cancer cells to help remake the environment around the tumor to promote cancer survival and spread.
Vander Heiden’s lab and other research groups are now working on developing a culture medium that would more closely mimic the composition of tumor interstitial fluid, so they can explore whether tumor cells grown in this environment could be used to generate more accurate predictions of how cancer drugs will affect cells in the body.
The research was funded by the National Institutes of Health, the Lustgarten Foundation, the MIT Center for Precision Cancer Medicine, Stand Up to Cancer, the Howard Hughes Medical Institute, and the Ludwig Center at MIT.
Acidic environment triggers genes that help cancer cells metastasize.
Anne Trafton | MIT News Office
March 21, 2019
Scientists have long known that tumors have many pockets of high acidity, usually found deep within the tumor where little oxygen is available. However, a new study from MIT researchers has found that tumor surfaces are also highly acidic, and that this acidity helps tumors to become more invasive and metastatic.
The study found that the acidic environment helps tumor cells to produce proteins that make them more aggressive. The researchers also showed that they could reverse this process in mice by making the tumor environment less acidic.
“Our findings reinforce the view that tumor acidification is an important driver of aggressive tumor phenotypes, and it indicates that methods that target this acidity could be of value therapeutically,” says Frank Gertler, an MIT professor of biology, a member of MIT’s Koch Institute for Integrative Cancer Research, and the senior author of the study.
Former MIT postdoc Nazanin Rohani is the lead author of the study, which appears in the journal Cancer Research.
Mapping acidity
Scientists usually attribute a tumor’s high acidity to the lack of oxygen, or hypoxia, that often occurs in tumors because they don’t have an adequate blood supply. However, until now, it has been difficult to precisely map tumor acidity and determine whether it overlaps with hypoxic regions.
In this study, the MIT team used a probe called pH (Low) Insertion Peptide (pHLIP), originally developed by researchers at the University of Rhode Island, to map the acidic regions of breast tumors in mice. This peptide is floppy at normal pH but becomes more stable at low, acidic pH. When this happens, the peptide can insert itself into cell membranes. This allows the researchers to determine which cells have been exposed to acidic conditions, by identifying cells that have been tagged with the peptide.
To their surprise, the researchers found that not only were cells in the oxygen-deprived interior of the tumor acidic, there were also acidic regions at the boundary of the tumor and the structural tissue that surrounds it, known as the stroma.
“There was a great deal of tumor tissue that did not have any hallmarks of hypoxia that was quite clearly exposed to acidosis,” Gertler says. “We started looking at that, and we realized hypoxia probably wouldn’t explain the majority of regions of the tumor that were acidic.”
Further investigation revealed that many of the cells at the tumor surface had shifted to a type of cell metabolism known as aerobic glycolysis. This process generates lactic acid as a byproduct, which could account for the high acidity, Gertler says. The researchers also discovered that in these acidic regions, cells had turned on gene expression programs associated with invasion and metastasis. Nearly 3,000 genes showed pH-dependent changes in activity, and close to 300 displayed changes in how the genes are assembled, or spliced.
“Tumor acidosis gives rise to the expression of molecules involved in cell invasion and migration. This reprogramming, which is an intracellular response to a drop in extracellular pH, gives the cancer cells the ability to survive under low-pH conditions and proliferate,” Rohani says.
Those activated genes include Mena, which codes for a protein that normally plays a key role in embryonic development. Gertler’s lab had previously discovered that in some tumors, Mena is spliced differently, producing an alternative form of the protein known as MenaINV (invasive). This protein helps cells to migrate into blood vessels and spread though the body.
Another key protein that undergoes alternative splicing in acidic conditions is CD44, which also helps tumor cells to become more aggressive and break through the extracellular tissues that normally surround them. This study marks the first time that acidity has been shown to trigger alternative splicing for these two genes.
Reducing acidity
The researchers then decided to study how these genes would respond to decreasing the acidity of the tumor microenvironment. To do that, they added sodium bicarbonate to the mice’s drinking water. This treatment reduced tumor acidity and shifted gene expression closer to the normal state. In other studies, sodium bicarbonate has also been shown to reduce metastasis in mouse models.
Sodium bicarbonate would not be a feasible cancer treatment because it is not well-tolerated by humans, but other approaches that lower acidity could be worth exploring, Gertler says. The expression of new alternative splicing genes in response to the acidic microenvironment of the tumor helps cells survive, so this phenomenon could be exploited to reverse those programs and perturb tumor growth and potentially metastasis.
“Other methods that would more focally target acidification could be of great value,” he says.
The research was funded by the Koch Institute Support (core) Grant from the National Cancer Institute, the Howard Hughes Medical Institute, the National Institutes of Health, the KI Quinquennial Cancer Research Fellowship, and MIT’s Undergraduate Research Opportunities Program.
Other authors of the paper include Liangliang Hao, a former MIT postdoc; Maria Alexis and Konstantin Krismer, MIT graduate students; Brian Joughin, a lead research modeler at the Koch Institute; Mira Moufarrej, a recent graduate of MIT; Anthony Soltis, a recent MIT PhD recipient; Douglas Lauffenburger, head of MIT’s Department of Biological Engineering; Michael Yaffe, a David H. Koch Professor of Science; Christopher Burge, an MIT professor of biology; and Sangeeta Bhatia, the John and Dorothy Wilson Professor of Health Sciences and Technology and Electrical Engineering and Computer Science.
Stefani Spranger is exploring multiple avenues for the next immunotherapy breakthrough
Pamela Ferdinand | Spectrum
March 12, 2019
A YOUNG LAB AT THE FOREFRONT OF IMMUNOTHERAPY DISCOVERIES is an exciting yet challenging place to be. MIT faculty member Stefani Spranger, an expert in cancer biology and immunology, understands that better than most people.
Spranger knows that new labs such as hers, which opened in July 2017 at the Koch Institute for Integrative Cancer Research at MIT, face distinct advantages and disadvantages when it comes to making their mark. While younger labs typically have startup grants, they lack the long-term funding, track record, and name recognition of established researchers; on the other hand, new labs tend to have smaller, close-knit teams open to tackling a wider array of investigative avenues to see what works, what doesn’t work, and where promise lies.
That’s when the funds and recognition of an endowed professorship can make a big difference, says Spranger, an assistant professor of biology who last year was named the Howard S. (1953) and Linda B. Stern Career Development Professor. “Not everything will work, so being able to test multiple approaches accelerates discovery and success,” she says.
Spranger is working to understand the mechanisms underlying interactions between cancer and the immune system—and ultimately, to find ways to activate immune cells to recognize and fight the disease. Cancer immunotherapies (the field in which this past year’s Nobel Prize in Physiology or Medicine was awarded) have revolutionized cancer treatment, leading to a new class of drugs called checkpoint inhibitors and resulting in lasting remissions, albeit for a very limited number of cancer patients. According to Spranger, there won’t be a single therapy, one-size-fits-all solution, but targeted treatments for cancers depending on their characteristics.
To discover new treatments, Spranger’s lab casts a wide net, asking big-picture questions about what influences anti-tumor immune response and disease outcome while also zooming in to investigate, for instance, specifically how cancer-killing T cells are excluded from tumors. In 2015, as a University of Chicago postdoc, Spranger made the novel discovery that malignant melanoma tumors with high beta-catenin protein lack T cells and fail to respond to treatment while tumors with normal beta-catenin do.
Her lab focuses on understanding lung and pancreatic cancers, employing a multidisciplinary research team with expertise ranging from immunology and biology to math and computation. One of her graduate students is using linear algebra to develop a mathematical model for translating mouse data into more accurate predictions about key signaling pathways in humans.
Another project involves exploring the relationship between homogenous tumors and immune response. Not every cancer cell is identical, nor does it have the same molecules on its surface that can be recognized by an immune cell; cancer patients with a more homogenous expression of those cells do better with immunotherapy. To investigate whether that homogeneity is due to the tumor or to the immune response to the tumor, Spranger is seeking to build a model system. The research involves a lot of costly sequencing—up to $3,000 per attempt, which is fairly expensive for a young lab—and each try has an element of what Spranger half-jokingly describes as “close your eyes and hope it worked.”
“Being able to generate preliminary proof of concept data for high-risk projects is of outstanding importance for any principal investigator,” she says. “However, it is particularly important to have freedom and flexibility early on.”
Boosting potential
Advancing cancer research and supporting the careers of promising faculty were the intentions of Linda Stern and her late husband Howard Stern ’53, SM ’54, whose gift has supported a series of biology professors since 1993. The first appointee to the chair was Tyler Jacks, now director of the Koch Institute.
Linda Stern says her husband, the cofounder and chairman of E-Z-EM, Inc., and a pioneer in the field of medical imaging, gave thoughtfully to many charitable causes. Yet MIT, where he earned undergraduate and graduate degrees in chemical engineering, had a special place in his heart.
“He was very involved and loved MIT,” says Stern, whose own career path included working as a private detective for 28 years. “He made wonderful contacts and got a wonderful education. He was a real heavy hitter when it came to defending the university.”
MIT’s continued excellence in a competitive environment depends on its ability to recognize and retain faculty, nurture careers, support students, and allow for the pursuit of novel ideas. Like the full professorships awarded to tenured faculty members, career development professorships such as the one endowed by the Sterns fund salary, benefits, and a scholarly allowance. These shorter-term (typically three-year) appointments, however, are specifically meant to accelerate the research and career progression of junior professors with exceptional potential.
“The professorship showed me that MIT as a community is invested and interested in fostering my career,” says Spranger. The discretionary funds she receives from the chair can cover, without need for an approval process, expenses that are not paid for by grants or that suddenly arise from a new idea or opportunity. They can keep projects running in tough times, fund travel to conferences, and purchase equipment. “It gives you a little more traction,” Spranger says. “It’s probably the best invested money because you have a lot of ideas you want to test, and at the same time, you are still checking the pulse of where the field might go and where you want to build your niche.”
Antibiotics or anti-inflammatory drugs may help combat lung cancer.
Anne Trafton | MIT News Office
January 31, 2019
MIT cancer biologists have discovered a new mechanism that lung tumors exploit to promote their own survival: These tumors alter bacterial populations within the lung, provoking the immune system to create an inflammatory environment that in turn helps the tumor cells to thrive.
In mice that were genetically programmed to develop lung cancer, those raised in a bacteria-free environment developed much smaller tumors than mice raised under normal conditions, the researchers found. Furthermore, the researchers were able to greatly reduce the number and size of the lung tumors by treating the mice with antibiotics or blocking the immune cells stimulated by the bacteria.
The findings suggest several possible strategies for developing new lung cancer treatments, the researchers say.
“This research directly links bacterial burden in the lung to lung cancer development and opens up multiple potential avenues toward lung cancer interception and treatment,” says Tyler Jacks, director of MIT’s Koch Institute for Integrative Cancer Research and the senior author of the paper.
Chengcheng Jin, a Koch Institute postdoc, is the lead author of the study, which appears in the Jan. 31 online edition of Cell.
Linking bacteria and cancer
Lung cancer, the leading cause of cancer-related deaths, kills more than 1 million people worldwide per year. Up to 70 percent of lung cancer patients also suffer complications from bacterial infections of the lung. In this study, the MIT team wanted to see whether there was any link between the bacterial populations found in the lungs and the development of lung tumors.
To explore this potential link, the researchers studied genetically engineered mice that express the oncogene Kras and lack the tumor suppressor gene p53. These mice usually develop a type of lung cancer called adenocarcinoma within several weeks.
Mice (and humans) typically have many harmless bacteria growing in their lungs. However, the MIT team found that in the mice engineered to develop lung tumors, the bacterial populations in their lungs changed dramatically. The overall population grew significantly, but the number of different bacterial species went down. The researchers are not sure exactly how the lung cancers bring about these changes, but they suspect one possibility is that tumors may obstruct the airway and prevent bacteria from being cleared from the lungs.
This bacterial population expansion induced immune cells called gamma delta T cells to proliferate and begin secreting inflammatory molecules called cytokines. These molecules, especially IL-17 and IL-22, create a progrowth, prosurvival environment for the tumor cells. They also stimulate activation of neutrophils, another kind of immune cell that releases proinflammatory chemicals, further enhancing the favorable environment for the tumors.
“You can think of it as a feed-forward loop that forms a vicious cycle to further promote tumor growth,” Jin says. “The developing tumors hijack existing immune cells in the lungs, using them to their own advantage through a mechanism that’s dependent on local bacteria.”
However, in mice that were born and raised in a germ-free environment, this immune reaction did not occur and the tumors the mice developed were much smaller.
Blocking tumor growth
The researchers found that when they treated the mice with antibiotics either two or seven weeks after the tumors began to grow, the tumors shrank by about 50 percent. The tumors also shrank if the researchers gave the mice drugs that block gamma delta T cells or that block IL-17.
The researchers believe that such drugs may be worth testing in humans, because when they analyzed human lung tumors, they found altered bacterial signals similar to those seen in the mice that developed cancer. The human lung tumor samples also had unusually high numbers of gamma delta T cells.
“If we can come up with ways to selectively block the bacteria that are causing all of these effects, or if we can block the cytokines that activate the gamma delta T cells or neutralize their downstream pathogenic factors, these could all be potential new ways to treat lung cancer,” Jin says.
Many such drugs already exist, and the researchers are testing some of them in their mouse model in hopes of eventually testing them in humans. The researchers are also working on determining which strains of bacteria are elevated in lung tumors, so they can try to find antibiotics that would selectively kill those bacteria.
The research was funded, in part, by a Lung Cancer Concept Award from the Department of Defense, a Cancer Center Support (core) grant from the National Cancer Institute, the Howard Hughes Medical Institute, and a Margaret A. Cunningham Immune Mechanisms in Cancer Research Fellowship Award.
New platform enables longitudinal studies of circulating tumor cells in mouse models of cancer.
Bendta Schroeder | Koch Institute
January 23, 2019
Circulating tumor cells (CTCs) — an intermediate form of cancer cell between a primary and metastatic tumor cell — carry a treasure trove of information that is critical to treating cancer. Numerous engineering advancements over the years have made it possible to extract cells via liquid biopsy and analyze them to monitor an individual patient’s response to treatment and predict relapse.
Thanks to significant progress toward creating genetically engineered mouse models, liquid biopsies hold great promise for the lab as well. These mouse models mimic many aspects of human tumor development and have enabled informative studies that cannot be performed in patients. For example, these models can be used to trace the evolution of cells from initial mutation to eventual metastasis, a process in which CTCs play a critical role. But since it has not been possible to monitor CTCs over time in mice, scientists’ ability to study important features of metastasis has been limited.
The challenge lies in capturing enough cells to conduct such longitudinal studies. Although primary tumors shed CTCs constantly, the density of CTCs in blood is very low — fewer than 100 CTCs per milliliter. For human patients undergoing liquid biopsy, this does not present a problem. Clinicians can withdraw enough blood to guarantee a sufficient sample of CTCs, just a few milliliters out of five or so liters on average, with minimal impact to the patient.
A mouse, on the other hand, only has about 1.5 milliliters of blood in total. If researchers want to study CTCs over time, they may safely withdraw no more than a few microliters of blood from a mouse each day — nowhere near enough to ensure that many (or any) CTCs are collected.
But with a new approach developed by researchers at the Koch Institute for Integrative Cancer Research, it is now possible to collect CTCs from mice over days and even weeks, and analyze them as the disease progresses. The system, described in the Proceedings of the National Academy of Sciences the week of Jan. 21, diverts blood to a microfluidic cell-sorting chip that extracts individual CTCs before returning the blood back to an awake mouse.
A menu of sorts
The inspiration for the project was cooked up, not in the lab, but during a chance encounter in the Koch Café between Tyler Jacks, director of the Koch Institute and the David H. Koch Professor of Biology, and Scott Manalis, the Andrew and Erna Viterbi Professor in the departments of Biological Engineering and Mechanical Engineering and a member of the Koch Institute.
As luck and lunch lines would have it, the pair would discuss thesis work being done by then-graduate student Shawn Davidson, who was using a dialysis-like system to track metabolites in the bloodstream of mice in the laboratory of Matthew Vander Heiden, an associate professor of biology. Jacks and Manalis were inspired: Could a similar approach could be used to study rare CTCs in real time?
Along with their Koch Institute colleague Alex K. Shalek, the Pfizer-Laubach Career Development Assistant Professor of Chemistry and a core member of the Institute for Medical Engineering and Science (IMES) at MIT, it would take Jacks and Manalis more than five years to put all the pieces of the system together, drawing from different areas of expertise around the Koch Institute. The Jacks lab supplied its fluorescent small cell lung cancer model, the Manalis lab developed the real-time CTC isolation platform, and the Shalek lab provided genomic profiles of the collected CTCs using single-cell RNA sequencing.
“This is a project that could not have succeeded without a sustained effort from several labs with very different sets of expertise. For my lab, which primarily consists of engineers, the opportunity to participate in this type of research has been incredibly exciting and is the reason why we are in the Koch Institute,” Manalis says.
The CTC sorter uses laser excitation to identify tumor cells expressing a fluorescent marker that is incorporated in the mouse model. The system draws blood from the mouse and passes it through a microfluidic chip to detect and extract the fluorescing CTCs before returning the blood back to the mouse. A minute amount of blood — approximately 100 nanoliters — is diverted with every detected CTC into a collection tube, which then is purified further to extract individual CTCs from the thousands of other blood cells.
“The real-time detection of CTCs happens at a flow rate of approximately 2 milliliters per hour which allows us to scan nearly the entire blood volume of an awake and moving mouse within an hour,” says Bashar Hamza, a graduate student in the Manalis lab and one of the lead authors on the paper.
Biology in their blood
With the development of a real-time cell sorter, the researchers could now, for the first time, longitudinally collect CTCs from the same mouse.
Previously, the low blood volume of mice and the rarity of CTCs meant that groups of mice had to be sacrificed at successive times so that their CTCs could be pooled. However, CTCs from different mice often have significantly different gene expression profiles that can obscure subtle changes that occur from the evolution of the tumor or a perturbation such as a drug.
To demonstrate that their cell-sorter could capture these differences, the researchers treated mice with a compound called JQ1, which is known to inhibit the proliferation of cancer cells and perturb gene expression. CTCs were collected and profiled with single-cell RNA sequencing for two hours prior to the treatment, and then every 24 hours after the initial treatment for four days.
When the researchers pooled data for all mice that had been treated with JQ1, they found that the data clustered based on individual mice, offering no confirmation that the drug affects CTC gene expression over time. However, when the researchers analyzed single-mouse data, they observed gene expression shift with time.
“What’s so exciting about this platform and our approach is that we finally have the opportunity to comprehensively study longitudinal CTC responses without worrying about the potentially confounding effects of mouse-to-mouse variability. I, for one, can’t wait to see what we will be able to learn as we profile more CTCs, and their matched primary and metastatic tumors,” says Shalek.
Researchers believe their approach, which they intend to use in additional cancer types including non-small cell lung, pancreatic, and breast cancer, could open new avenues of inquiry in the study of CTCs, such as studying long-term drug responses, characterizing their relationship to metastatic tumors, and measuring their rate of production in short timeframes — and the entire metastatic cascade. In future work, researchers also plan to use their approach for profiling rare immune cells and monitoring cells in dynamic contexts such as wound healing and tumor formation.
“The ability to study CTCs as well other rare cells in the blood longitudinally gives us a powerful view into cancer development. This sorter represents a real breakthrough for the field and it is a great example of the Koch Institute in action,” says Jacks.
The paper’s other co-lead authors are graduate students Sheng Rong Ng from the Jacks lab and Sanjay Prakadan from the Shalek lab. The research is supported, in part, by the Ludwig Center at MIT, the National Cancer Institute, the National Institutes of Health and the Searle Scholars Program.
A new study linking RNA processing to DNA repair may open new avenues to cancer therapy.
Bendta Schroeder | Koch Institute
December 3, 2018
Mutations in the BRCA1 and BRCA2 genes pose a serious risk for breast and ovarian cancer because they endanger the genomic stability of a cell by interfering with homologous recombination repair (HR), a key mechanism for accurately repairing harmful double-stranded breaks in DNA. Without the ability to use HR to fix double-stranded breaks, the cell is forced to resort to more error-prone — and thus more cancer-prone — forms of DNA repair.
The BRCA1 and BRCA2 genes are not the only genes whose mutations foster tumorigenesis by causing an inability to repair DNA double strand breaks by HR. Mutations in twenty-two genes are known to disrupt HR, giving rise to tumors with what researchers call “BRCAness” characteristics. All but one of these BRCAness genes are known to be directly involved in the HR pathway.
The one exception, CDK12, is thought to facilitate a set of different processes altogether, involving how RNA transcripts are elongated, spliced and cleaved into their mature forms. While the connection between this RNA-modulating gene to DNA repair remained poorly understood, the identification of CDK12 as a BRCAness gene piqued significant clinical interest.
The researchers who pinpointed this connection, Sara Dubbury and Paul Boutz, both work in the laboratory of Phillip Sharp, Institute Professor, professor of biology, and member of the Koch Institute for Integrative Cancer Research. In a study appearing online in Nature on Nov. 28, they describe how they discovered a previously unknown mechanism by which CDK12 enables the production of full-length RNA transcripts and that this mechanism was especially critical to maintain functional expression of the other BRCAness genes.
When the researchers knocked out expression of CDK12, mouse stem cells showed many signs of accumulating DNA damage that prevented DNA replication from going forward, classic indications of a BRCAness phenotype. To identify what roles CDK12 may play in regulating gene expression, the researchers turned to RNA sequencing to determine which genes had increased or decreased their overall expression.
To their surprise, only genes activated by p53 and early differentiation (side effects of accumulating unrepaired DNA damage and BRCAness in mouse stem cells) accounted for the lion’s share of changes to RNA transcription. However, when the researchers instead focused on the types of RNAs transcribed, they found that many genes produced unusually short transcripts when CDK12 was absent.
Not every stretch of DNA in a gene makes it into the final RNA transcript. The initial RNA from a gene often includes sections, which researchers call “introns,” that are cut out of transcript, the discovery that earned Sharp the 1993 Nobel Prize in Physiology or Medicine and the remaining sections. “Exons,” are spliced together to form a mature transcript (mRNA). Alternately, an intronic polyadenylation (IPA) site may be activated to cleave away the RNA sequence that follows it preventing intron removal and generating a prematurely shortened transcript. These processes allow the same gene to produce alternate forms of messenger RNA (mRNA), and thus be translated into different protein sequences.
Surprisingly CDK12 knockout cells produced significantly more IPA-truncated transcripts genome-wide, while full-length transcripts for the same genes were reduced. These shortened mRNAs can vary greatly in their stability, their ability to be translated into protein, and their protein function. Thus, even while a gene may be actively transcribed, its translation into functional proteins can be radically altered or depleted by IPA activation.
While this observation began to illuminate CDK12’s role in regulating mRNA processing, what remained puzzling was why CDK12 loss affected the HR pathway so disproportionately. In investigating this question, Dubbury and Boutz found that BRCAness genes were overrepresented as a group among those genes that have increased IPA activity upon CDK12 loss.
Additionally, while CDK12 suppresses IPA activity genome-wide, 13 of the other 21 BRCAness genes were found to be particularly vulnerable to CDK12 loss, in part, because they possess multiple high-sensitivity IPA sites, which have a compound effect in decreasing the total amount of full-length transcripts. Moreover, because multiple CDK12-senstive BRCAness genes operate in the same HR pathway, the researchers believe that the disruption to HR repair of double-stranded DNA breaks is amplified.
CDK12 mutations are found recurrently in prostate and ovarian cancer patients, making them an attractive diagnostic and therapeutic target for cancer. However, not enough is known about CDK12 to distinguish between true loss-of-function mutations and so-called “passenger mutations” with no functional consequence.
“The ability to identify patients with true loss-of-function mutations in CDK12 would enable clinicians to label a new cohort of patients with bona fide BRCAness tumors that could benefit from certain highly effective and targeted chemotherapeutics against BRCAness, such as PARP1 inhibitors,” says Dubbury, a former David H. Koch Fellow.
Dubbury and Boutz were able to confirm that IPA sites in key BRCAness genes were also used more frequently upon CDK12 loss in human tumor cells using RNA sequencing data from prostate and ovarian tumor patients with CDK12 mutations and by treating human prostate adenocarcinoma and ovarian carcinoma cells with a CDK12 inhibitor. This result suggests that the CDK12 mechanism observed in mouse cell lines is conserved in humans and that CDK12 mutations in human ovarian and prostate tumors may promote tumorigenesis by increasing IPA activity and thus functionally attenuating HR repair.
“These results not only give us a better understanding how CDK12 contributes to BRCAness, they also may have exciting potential impact in the clinic,” Dubbury says. “Currently available diagnostic techniques could be used to probe the usage of IPA sites found in this study to rapidly screen for patients with true loss-of-function CDK12 mutations, who would respond to BRCAness-targeted treatments.”
Paul Boutz, a research scientist in the Sharp Lab, is co-first author of the study, and has plans to follow-up many of these implications for ovarian and prostate cancer his lab at the University of Rochester School of Medicine and Dentistry.
“CDK12 provides a remarkable example of how factors that control the processing of RNA molecules can function as master regulators of gene networks, and thereby profoundly affect the physiology of both normal and cancerous cells,” he says.
Phil Sharp, the senior author on the work, says “Sara’s and Paul’s surprising discovery that CDK12 suppresses intronic polyadenylation has implications for fundamental new insights into gene structure as well as for control of cancer.”
Matthew Vander Heiden seeks new cancer treatments that exploit tumor cells’ abnormal metabolism.
Anne Trafton | MIT News Office
August 28, 2018
Nearly 100 years ago, the German chemist Otto Warburg discovered that cancer cells metabolize nutrients differently than most normal cells. His discovery launched the field of cancer metabolism research, but interest in this area waned; by the 1970s most cancer scientists had shifted their focus to the genetic mutations that drive cancer development.
In the past decade or so, interest in cancer metabolism has resurged, and the first drugs that target cancer cells’ abnormal metabolism were approved to treat leukemia in 2017.
“Cancer metabolism is a very sophisticated field at this point,” says Matthew Vander Heiden, an associate professor of biology at MIT. “We have a lot better understanding of what nutrients cancer cells use and what determines how those nutrients are used. This has led to different ways to think about drugs.”
Vander Heiden, who is also a member of MIT’s Koch Institute for Integrative Cancer Research, is one of the people responsible for the recent surge in cancer metabolism research. As a graduate student and postdoc, he published some of the first studies of how cancer cells alter their metabolism, and now his lab at MIT is devoted to the topic.
“All of the time that I was in grad school and working as a postdoc, I was never working in a lab that was dedicated to studying metabolism. So my vision, if someone gave me a job, was to set up a lab that could really be built in a way that would allow us to ask questions about metabolism,” he says.
Metabolism and cancer
Vander Heiden grew up in a small town in Wisconsin, and unlike most of his high school classmates, he headed out of state for college, to the University of Chicago. He was interested in science, so decided on a pre-med track. A work-study job in a plant biology lab led him to discover that he also enjoyed doing research.
“At that point I already had this idea I was going to go to medical school, but then the idea of MD/PhD came up, and I ended up going down that path,” Vander Heiden says.
While in the MD/PhD program at the University of Chicago Medical School, he worked in the lab of Craig Thompson, now president of Memorial Sloan Kettering Cancer Center. At that time, Thompson was studying the biochemical regulation of apoptosis, the programmed cell death pathway. For his PhD thesis, Vander Heiden investigated the function of a protein called Bcl-x, which is a regulator of apoptosis found in the membranes of mitochondria — cell organelles responsible for generating energy.
“That project really got me thinking about how the mitochondria work and how metabolism works,” Vander Heiden recalls. “At the time, I came to the realization that we don’t understand cell metabolism anywhere near as well as we thought we did, and someone should really study this.”
After finishing his degrees, he spent five years doing clinical training, then decided to pursue research in cancer metabolism.
“Altered metabolism has been known about in cancer for 100 years, but few people were studying it,” Vander Heiden says. “The challenge was finding a lab that would allow me to study metabolism and cancer, which in 2004-2005 was not such an obvious thing to do.”
He ended up going to Harvard Medical School to work with Lewis Cantley, who studies signaling pathways in cells and was receptive to the idea of exploring cancer metabolism. There, Vander Heiden began studying an enzyme called pyruvate kinase M2 (PKM2), which is involved in regulation of glycolysis, a biochemical process that cells use to break down sugar for energy.
In 2008, Vander Heiden, Cantley, and others at Harvard Medical School reported that when cells shift between normal and Warburg (cancer-associated) metabolism, they start using PKM2 instead of PKM1, the enzyme that adult cells normally use for glycolysis. Cantley and Craig Thompson have since founded a company, Agios Pharmaceuticals, that is developing potential drugs that target PKM2, as well as other molecules involved in cancer metabolism.
While at Harvard, Vander Heiden also worked on a paper that contributed to the eventual development of drugs that target cancer cells with a mutation in the IDH gene. These drugs, the first modern FDA-approved cancer drugs that target metabolism, shut off an alternative pathway used by cancer cells with the IDH mutation.
New drug targets
In 2010, Vander Heiden became one of the first new faculty members hired after the creation of MIT’s Koch Institute, where he set up a lab focused on metabolism, particularly cancer metabolism.
His research has yielded many insights into the abnormal metabolism of cancer cells. In one study, together with other MIT researchers, he found that tumor cells turn on an alternative pathway that allows them to build lipids from the amino acid glutamine instead of the glucose that healthy cells normally use. He also found that altering the behavior of PKM2 to make it act more like PKM1 could stop tumor cell growth.
Studies such as these can offer insights that may help researchers to develop drugs that starve tumor cells of the nutrients they need, offering a new way to fight cancer, Vander Heiden says.
“If one wants to develop drugs that target metabolism, one really needs to focus on the context in which it’s happening, which is the environment of the cell plus the genetics of the cell,” he says. “That is what defines the sensitivity to drugs.”
Aviv Regev helped pioneer single-cell genomics. Now she’s cochairing a massive effort to map the trillions of cells in the human body. Biology will never be the same.
Sam Apple | MIT Technology Review
August 23, 2018
Last October, Aviv Regev spoke to a gathering of international scientists at Israel’s Weizmann Institute of Science. For Regev, a computational and systems biologist at the Broad Institute of MIT and Harvard, the gathering was also a homecoming of sorts. Regev earned her PhD from nearby Tel Aviv University in 2002. Now, 15 years later, she was back to discuss one of the most ambitious projects in the history of biology.
The project, the Human Cell Atlas, aims to create a reference map that categorizes all the approximately 37 trillion cells that make up a human. The Human Cell Atlas is often compared to the Human Genome Project, the monumental scientific collaboration that gave us a complete readout of human DNA, or what might be considered the unabridged cookbook for human life. In a sense, the atlas is a continuation of that project’s work. But while the same DNA cookbook is found in every cell, each cell type reads only some of the recipes—that is, it expresses only certain genes, following their DNA instructions to produce the proteins that carry out a cell’s activities. The promise of the Human Cell Atlas is to reveal which specific genes are expressed in every cell type, and where the cells expressing those genes can be found.
Speaking to her colleagues at the meeting in Israel, Regev, who is cochairing the Human Cell Atlas Organizing Committee with Sarah Teichmann of the Wellcome Trust Sanger Institute, displayed the no-nonsense demeanor you might expect of someone at the helm of a massive scientific undertaking. The project had been under way for a year, and Regev, an MIT biology professor who is also chair of the faculty of the Broad and director of its Klarman Cell Observatory and Cell Circuits Program, was reviewing a newly published white paper detailing how the Human Cell Atlas is expected to change the way we diagnose, monitor, and treat disease.
As Regev made her way through the white paper, the possibilities began to seem almost endless. At the most basic level, as a reference map detailing the genes expressed by each different type of healthy cell, the Human Cell Atlas will make it easier to identify how gene expression and signaling go awry in the case of disease. The same map could also help drug developers avoid toxic side effects: researchers targeting a gene that’s harmful in one part of the body would know if the same gene is playing a vital role in another. And because the atlas is expected to reveal many new types of cells, it could also add much more sensitivity to a type of standard blood test, which simply counts different subsets of immune cells. Likewise, looking at individual intestinal cells might provide new insights into the specific cells responsible for inflammation and food allergies. And a better understanding of types of neurons could have far-reaching implications for brain science.
The final product, Regev says, will amount to nothing less than a “periodic table of our cells,” a tool that is designed not to answer one specific question but to make countless new discoveries possible. Eric Lander, the founding director and president of the Broad Institute and a member of the Human Cell Atlas Organizing Committee, likens it to genomics. “People thought at the beginning they might use genomics for this application or that application,” he says. “Nothing has failed to be transformed by genomics, and nothing will fail to be transformed by having a cell atlas.”
Cellular circuits
Regev’s interest in cells began at Tel Aviv University, where she was one of just 15 or so entering students in a highly selective program that gave them the freedom to take high-level courses in any subject. “You could go your first day as a freshman and decide to take a graduate class in political science,” she says.
Regev took a genetics class her first semester and got hooked on the computational challenge of finding order in the complex, interconnected networks of proteins and genes within each cell. She pursued that topic for her doctoral work, characterizing living systems in a mathematical language that had been designed to describe computer processes. As she finished her doctorate in 2002, she was accepted into a program at Harvard’s Bauer Center for Genomics Research that allowed her to start her own lab without first training as a postdoc.
Not long after, Lander, who’d begun his own career as a mathematician after studying algebraic coding theory and combinatorial mathematics at Oxford, was searching for star talent for the newly created Broad Institute, whose mission is to use genomics to study human disease and help advance its treatment. He first met Regev at a lunch at the Bauer Center during which the fellows took turns speaking about their research for five to 10 minutes. “By the time we got all the way around the table I had written down ‘Hire Aviv Regev,’” he recalls.
Convinced by Lander to join the Broad after “many cups of tea” at Cafe Algiers in Harvard Square, Regev continued to apply computational approaches to study the mind-bogglingly complicated machinery of the cell. A single cell is made up of millions of molecules that are in constant conversation as they work together to do all the things cells need to do: divide, grow, repair internal damage, and, in the case of immune cells, signal other cells about threats. Inside the nucleus, the DNA is transcribed into RNA. That in turn gives rise to proteins, the molecules that do the work inside a cell. Meanwhile, proteins on the surface of the cell are constantly receiving molecular messages from outside—glucose is available, an invader has arrived. These must be relayed back to proteins in the nucleus, which will respond by transcribing other DNA, giving rise to new proteins and still more signaling networks.
“It’s like a complex computer that is made of these many, many different parts that are interacting with each other and telling each other what to do,” says Regev. The protein signaling networks are like “circuits”—and you can think about the cell “almost like a wiring diagram,” she says. But using computational approaches to understand their activity first requires gathering an enormous amount of data, which Regev has long done through RNA sequencing. Unlike DNA sequencing, she says, it can tell her which genes are actually being expressed, so it offers a far more dynamic picture of a cell in action. But simply sequencing the RNA of the cells she’s studying can tell her only so much. To understand how the circuits change under different circumstances, Regev subjects cells to different stimuli, such as hormones or pathogens, to see how the resulting protein signals change.
Next comes what she calls “the modeling step”—creating algorithms that try to decipher the most likely sequence of molecular events following a stimulus. And just as someone might study a computer by cutting out circuits and seeing how that changes the machine’s operation, Regev tests her model by seeing if it can predict what will happen when she silences specific genes and then exposes the cells to the same stimulus.
In a 2009 study, Regev and her team examined how exposure to molecular components of pathogens like bacteria, viruses, or fungi affected the circuitry of the immune system’s dendritic cells. She turned to a technique known as RNA interference (she now uses CRISPR), which allowed her to systematically shut genes down. Then she looked at which genes were expressed to determine how the cells’ response changed in each case. Her team singled out 100 different genes that were involved in regulating the response to the pathogens—some of which weren’t previously known to be involved in immune function. The study, published in Science, generated headlines. But according to longtime colleague Dana Pe’er, now chair of computational and systems biology at the Sloan Kettering Institute at the Memorial Sloan Kettering Cancer Center and a member of the Human Cell Atlas Organizing Committee, what really sets Regev apart is the elegance of her work. Regev, says Pe’er, “has a rare, innate ability of seeing complex biology and simplifying it and formalizing it into beautiful, abstract, describable principles.”
From smoothies to fruit salad
There are lots of empty coffee mugs in Regev’s office at the Broad Institute, but very little in the way of decoration. She approaches her science with a businesslike efficiency. “There are many brilliant people,” says Lander. “She’s a brilliant person who can get things done.”
In the fast-changing arena of genomics (“2015 in my field is considered ancient history,” she says), she is known for making the most of the latest innovations—and for helping to spur the next ones. For years, she and others in the field struggled with a dirty secret of RNA sequencing: though its promise has always been precision—the power of knowing the exact code—the techniques produced results that were unspecific. Every cell has only a minuscule amount of RNA. For sequencing purposes, the RNA from millions of cells had to be pooled together. Bulk RNA sequencing left researchers with what she likens to a smoothie. Once it’s blended together, there’s no way to distinguish all the fruits—or in this case, the RNA from individual cells—that went into it. What researchers needed was something more like a fruit salad, a way to separate all the blueberries, raspberries, and blackberries.
In 2011, working with Broad Institute colleague Joshua Levin, PhD ’92, and postdocs Alex Shalek, now at MIT’s Institute for Medical Engineering and Science, and Rahul Satija, now at the New York Genome Center, Regev managed to obtain enough RNA from a single cell to sequence it. To test the method, they sequenced 18 individual dendritic cells from the bone marrow of a mouse. The cells were all obtained in the same way and were expected to be the same type. But to the researchers’ amazement, they were expressing different genes and could be classified into two distinct subtypes. It was like finding out the smoothie you’d been drinking for years had ingredients you’d never known about.
Regev and her colleagues weren’t the only ones figuring out how to sequence a single cell with such sensitivity, nor were they the very first to succeed. Other labs were making similar advances at approximately the same time, each using its own technology and algorithms. And they all faced the same problem: isolating and extracting enough RNA from individual cells was time consuming and expensive. Regev and her colleagues had spent many thousands of dollars to sequence only 18 cells. If the body was full of rare, undiscovered cells, it was going to take an extraordinarily long time to find them.
Skip ahead seven years and the cost of single-cell RNA sequencing is down to only pennies per cell. A critical breakthrough was Drop-Seq, a new technology developed by researchers at Harvard and the Broad Institute, including Regev and members of her lab. The device embeds individual cells into distinct oil droplets with a tiny “bar-coded” bead. When the cell is broken apart for sequencing, some of its RNA attaches to the bead in its droplet. This allows researchers to analyze thousands at once without getting their genetic material mixed up.
Cell theory 2.0
When cell theory was first proposed by German scientists some 180 years ago, it was hard to fathom that our tissues are built from “individual elementary units,” as Theodor Schwann, one of the two scientists credited with the theory, described cells. But it soon became a central tenet of biology, and over the decades and centuries, cells began to give up their secrets. Microscopes improved; new staining and sorting techniques became available. With each advance, new distinctions became possible. Muscle cells could be distinguished from neurons, and then categorized again as smooth or skeletal muscle cells. Cells, it became clear, were all fundamentally similar but came in different forms that had different properties.
By the 21st century, 200 to 300 major cell types had been identified. And while biologists have long recognized that the true number of cell types must be higher, the extent of their diversity is only now coming into full focus, thanks in large part to single-cell RNA sequencing. Regev says that the immune system alone can now be divided into more than 200 cell types and that even our retinas have 100 or more distinct types of neurons. She and her colleagues have discovered several of them.
The idea that knowing so much more about our cells could lead to medical breakthroughs is no longer hypothetical. By sequencing the RNA of individual cancer cells in recent years—“Every cell is an experiment now,” she says—she has found remarkable differences between the cells of a single tumor, even when they have the same mutations. (Last year that work led to Memorial Sloan Kettering’s Paul Marks Prize for Cancer Research.) She found that while some cancers are thought to develop resistance to therapy, a subset of melanoma cells were resistant from the start. And she discovered that two types of brain cancer, oligodendroglioma and astrocytoma, harbor the same cancer stem cells, which could have important implications for how they’re treated.
The excitement in the field has become tangible as more new cell types have been found. And yet Regev realized that if the aim was comprehensive knowledge, the approach needed to be coordinated. If each lab were to rely on its own techniques, it would be hard to standardize the computational tools and the resulting data. The new studies were producing “very nice glimmers of light,” Regev says—“a thing here, a thing there.” But she wanted to make sure those findings could be connected.Regev has also been busily mapping cells from the immune system, brain, gut, and elsewhere. She is not alone. Other labs have started their own mapping projects, each tackling a different part of the body. Last year researchers at the University of Washington attempted to classify every cell type in the microscopic worm C. elegans. “Every single field in biology is saying, ‘Of course we have to look at single-cell resolution,’” says Lander. “How did we ever imagine we were going to solve a problem without single-cell resolution?”
Regev began to advocate creating something more unified: a map that would allow researchers to chart gene expression and cell types across the entire body. Sarah Teichmann had been thinking along the same lines. When she reached out to Regev in late 2015 about the possibility of joining forces, Regev immediately said yes.
A Google Maps for our cells
The Human Cell Atlas is a collaboration among hundreds of biologists, technologists, and software engineers across the globe. Results from single-cell RNA sequencing will be combined with other data points to provide a comprehensive catalogue of all human cells.
But the many researchers involved won’t simply be compiling spreadsheets listing different cell types. The atlas will also reveal where the cells are located in the body, how many there are, what forms they can take, even the developmental history of different cell types as they differentiated from stem cells. And all of this will be made accessible through a data coordination platform and a rich visual interface that Regev compares to Google Maps. It will allow users to zoom in to the molecular level of our cells, but zooming out to the level of tissues and organs will be important too. As a 2017 overview of the Human Cell Atlas by the project’s organizing committee noted, an atlas “is a map that aims to show the relationships among its elements.” Just as corresponding coastlines seen in an atlas of Earth offer visual evidence of continental drift, compiling all the data about our cells in one place could reveal relationships among cells, tissues, and organs, including some that are entirely unexpected. And just as the periodic table made it possible to predict the existence of elements yet to be observed, the Human Cell Atlas, Regev says, could help us predict the existence of cells that haven’t been found.
The plan is not to sequence all 37 trillion cells but to sample from every part of the body. As Regev talks about the project, her enthusiasm evident, she digs up a slide to demonstrate how effective sampling can be. The slide, first only an empty frame of white, begins to fill in, pixel by pixel, with specks of blue and yellow. Soon, even though many of the pixels haven’t yet been filled, the image on the screen is unmistakable: it is Van Gogh’s Starry Night. Likewise, Regev explains, the Human Cell Atlas can give a complete picture even if not every single cell has been sequenced.
To do the sequencing, Regev and Teichmann have welcomed and recruited experts in each different tissue type. Though expected to take years, the project is moving ahead rapidly with such backers as NIH, the EU, the Wellcome Trust, the Manton Foundation, and the Chan Zuckerberg Initiative, which pledged to spend $3 billion to battle disease over the next decade; this year alone it will fund 85 Human Cell Atlas grants. Early results are already pouring in. In March, Swedish researchers working on cells related to human development announced they had sequenced 250,000 individual cells. In May, a team at the Broad made a data set of more than 500,000 immune cells available on a preview site. The goal, Regev says, is for researchers everywhere to be able to use the open-source platform of the Human Cell Atlas to perform joint analyses.
Plenty of challenges remain before the atlas can become a reality. New visualization software must be developed. Sequencing and computational approaches will need to be standardized across a huge number of labs. Conceptual issues, such as what distinguishes one cell type from another, have to be worked through. But the community behind the Human Cell Atlas—including more than 800 individuals as of June—has no shortage of motivation.
One of Regev’s own recent studies, published in August in Nature, is perhaps the best example of how the project could change biology. In mapping cells of the lungs, Regev and Jay Rajagopal’s lab at Massachusetts General Hospital found a new, very rare cell type that primarily expresses a gene linked to cystic fibrosis. Regev now thinks that these rare cells probably play a key role in the disease. More surprising yet, researchers had previously thought that a different cell type was expressing the gene.
“Imagine if somebody wanted to do gene therapy,” Regev says. “You have to fix the gene, but you have to fix it in the right cell.” The Human Cell Atlas could help researchers identify the right cell and understand how the gene in question is regulated by that cell’s extraordinarily complicated molecular networks.
For Regev, the importance of the Human Cell Atlas goes beyond its promise to revolutionize biology and medicine. As she once put it, without an atlas of our cells, “we don’t really know what we’re made of.”
Unexpected findings show that the body’s own immune system destroys retinal cells.
Anne Trafton | MIT News Office
August 11, 2018
Glaucoma, a disease that afflicts nearly 70 million people worldwide, is something of a mystery despite its prevalence. Little is known about the origins of the disease, which damages the retina and optic nerve and can lead to blindness.
A new study from MIT and Massachusetts Eye and Ear has found that glaucoma may in fact be an autoimmune disorder. In a study of mice, the researchers showed that the body’s own T cells are responsible for the progressive retinal degeneration seen in glaucoma. Furthermore, these T cells appear to be primed to attack retinal neurons as the result of previous interactions with bacteria that normally live in our body.
The discovery suggests that it could be possible to develop new treatments for glaucoma by blocking this autoimmune activity, the researchers say.
“This opens a new approach to prevent and treat glaucoma,” says Jianzhu Chen, an MIT professor of biology, a member of MIT’s Koch Institute for Integrative Cancer Research, and one of the senior authors of the study, which appears in Nature Communications on Aug. 10.
Dong Feng Chen, an associate professor of ophthalmology at Harvard Medical School and the Schepens Eye Research Institute of Massachusetts Eye and Ear, is also a senior author of the study. The paper’s lead authors are Massachusetts Eye and Ear researchers Huihui Chen, Kin-Sang Cho, and T.H. Khanh Vu.
Genesis of glaucoma
One of the biggest risk factors for glaucoma is elevated pressure in the eye, which often occurs as people age and the ducts that allow fluid to drain from the eye become blocked. The disease often goes undetected at first; patients may not realize they have the disease until half of their retinal ganglion cells have been lost.
Most treatments focus on lowering pressure in the eye (also known as intraocular pressure). However, in many patients, the disease worsens even after intraocular pressure returns to normal. In studies in mice, Dong Feng Chen found the same effect.
“That led us to the thought that this pressure change must be triggering something progressive, and the first thing that came to mind is that it has to be an immune response,” she says.
To test that hypothesis, the researchers looked for immune cells in the retinas of these mice and found that indeed, T cells were there. This is unusual because T cells are normally blocked from entering the retina, by a tight layer of cells called the blood-retina barrier, to suppress inflammation of the eye. The researchers found that when intraocular pressure goes up, T cells are somehow able to get through this barrier and into the retina.
The Mass Eye and Ear team then enlisted Jianzhu Chen, an immunologist, to further investigate what role these T cells might be playing in glaucoma. The researchers generated high intraocular pressure in mice that lack T cells and found that while this pressure induced only a small amount of damage to the retina, the disease did not progress any further after eye pressure returned to normal.
Further studies revealed that the glaucoma-linked T cells target proteins called heat shock proteins, which help cells respond to stress or injury. Normally, T cells should not target proteins produced by the host, but the researchers suspected that these T cells had been previously exposed to bacterial heat shock proteins. Because heat shock proteins from different species are very similar, the resulting T cells can cross-react with mouse and human heat shock proteins.
To test this hypothesis, the team brought in James Fox, a professor in MIT’s Department of Biological Engineering and Division of Comparative Medicine, whose team maintains mice with no bacteria. The researchers found that when they tried to induce glaucoma in these germ-free mice, the mice did not develop the disease.
Human connection
The researchers then turned to human patients with glaucoma and found that these patients had five times the normal level of T cells specific to heat shock proteins, suggesting that the same phenomenon may also contribute to the disease in humans. The researchers’ studies thus far suggest that the effect is not specific to a particular strain of bacteria; rather, exposure to a combination of bacteria can generate T cells that target heat shock proteins.
One question the researchers plan to study further is whether other components of the immune system may be involved in the autoimmune process that gives rise to glaucoma. They are also investigating the possibility that this phenomenon may underlie other neurodegenerative disorders, and looking for ways to treat such disorders by blocking the autoimmune response.
“What we learn from the eye can be applied to the brain diseases, and may eventually help develop new methods of treatment and diagnosis,” Dong Feng Chen says.
The research was funded by the National Institutes of Health, the Lion’s Foundation, the Miriam and Sheldon Adelson Medical Research Foundation, the National Nature Science Foundation of China, the Ivan R. Cottrell Professorship and Research Fund, the Koch Institute Support (core) Grant from the National Cancer Institute, and the National Eye Institute Core Grant for Vision Research.