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Pancreatic Cancer Insight

Pancreatic Cancer Insight

Study reveals gene expression patterns in circulating tumor cells

By SUE McGREEVEY
Wed, 2014-09-24

An earlier example of circulating tumor cells captured by a microfluidic isolation device. Image: Mass General

An analysis of circulating tumor cells (CTCs) in a mouse model of pancreatic cancer has identified distinct patterns of gene expression in several groups of these cells, including significant differences from the primary tumor that may contribute to their ability to spread. In a paper published in Cell Reports, Harvard Medical School investigators at the Massachusetts General Hospital Cancer Center pinpointed several different classes of pancreatic CTCs and found unexpected factors that may prove to be targets for improved treatment of the deadly tumor.

“Our ability to combine a novel microfluidic CTC isolation device, developed here at Mass General, with single-cell RNA sequencing has given us new biological insights into these cells and revealed novel avenues to try to block the spread of cancer,” said lead author David T. Ting, HMS assistant professor of medicine at Mass General. 

Pancreatic cancer is among the most deadly of tumors because it spreads rapidly via CTCs carried in the bloodstream. The earliest technologies for isolating CTCs from blood samples relied on interactions with known tumor-specific marker proteins, potentially missing cells that did not express those particular markers. 

The device used in the current study, called the CTC-iChip, enables the isolation of all CTCs in a blood sample, regardless of the proteins they express on their surface, by removing all other components. Since the CTCs collected are in solution, unlike with previous CTC capture devices, they are suitable for advanced RNA-sequencing techniques to reveal the gene expression patterns of each individual cell.

Using a well-known mouse model of pancreatic cancer, the researchers first isolated 168 single CTCs from the blood of five individual mice. Analysis of the RNA transcripts of each CTC revealed several different subsets of CTCs based on gene expression patterns that were different from each other and from the primary tumor. 

The largest subset, which the authors call “classic CTCs,” was found to have elevated expression of a stem cell gene called Aldh1 a2. This subset of CTCs also had genes characteristic of two basic cell types—epithelial and mesenchymal. The transition between epithelial and mesenchymal cell types has been associated with tumor metastasis. 

Another gene expressed by almost all classic CTCs, Igfbp5, is expressed only in primary tumors at locations where epithelial cancer cells interface with the supporting stromal cells that provide a nurturing microenvironment. This observation suggests that those regions may be the source of CTCs. 

The research team was most surprised to observe that extracellular matrix (ECM) genes in general—usually expressed primarily in stromal, or connective tissue, cells—were highly expressed in all classic CTCs. Previous studies have suggested that the establishment of metastases depends on the appropriate cellular microenvironment—the  “soil” in which CTCs can plant themselves as “seeds”—and that the expression of ECM genes is an important aspect of that environment. Expression of ECM genes by CTCs themselves suggests that the blood-borne cells may provide or help prepare their own “soil.”

Analysis of CTCs from blood samples of human patients with pancreatic, breast or prostate cancer also found elevated expression of several ECM genes. One particular gene, SPARC, was highly expressed in all pancreatic CTCs as well as in 31 percent of breast CTCs. Further experiments revealed that suppressing SPARC expression in human pancreatic cancer cells reduced their ability to migrate and invade tissue, and significantly fewer metastases were generated when SPARC-suppressed pancreatic tumors were implanted into a mouse model, supporting the protein’s role in a tumor’s metastatic potential.

“Given our limited therapeutic options for pancreatic cancer, understanding the role of the ECM in this tumor seems to be of great importance,” said Ting. “Much effort has been focused on targeting the microenvironment to improve the efficacy of chemotherapy, and data indicating that environmental stromal cells can enhance a tumor’s metastatic ability indicate that ECM proteins are important whether they are produced in stroma or within the tumor cells themselves. Now we need to investigate whether therapeutically targeting ECM can destroy both the tumor microenvironment and CTCs before they have a chance to metastasize.”

In 2011 Mass General entered a collaborative agreement with Janssen Diagnostics to establish a center of excellence in CTC research. Development of RNA in situ hybridization biomarkers was done with sponsored research support from Affymetrix Inc. Additional support for the current study includes a “Dream Team” grant from Stand Up to Cancer and grants from the Howard Hughes Medical Institute, Burroughs Wellcome Fund, National Institute of Biomedical Imaging and Bioengineering grant 5R01EB008047, and National Cancer Institute grant 2R01CA129933.

Adapted from a Mass General news release.

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Targeting Bone Cancer

Targeting Bone Cancer

HMS researchers have utilized nanomedicine technologies to develop a drug-delivery system that can attack cancer cells in the bone.

By MARJORIE MONTEMAYOR-QUELLENBERG
Tue, 2014-07-01

Image: iStock/xrender

A collaboration between Harvard Medical School researchers at Brigham and Women’s Hospital and Dana-Farber Cancer Institute has utilized nanomedicine technologies to develop a drug-delivery system that can precisely target and attack cancer cells in the bone, as well as increase bone strength and volume to prevent bone cancer progression.

The study was published the week of June 30, 2014 in Proceedings of the National Academy of Sciences.

“Bone is a favorable microenvironment for the growth of cancer cells that migrate from tumors in distant organs of the body, such as breast, prostate and blood, during disease progression,” said Archana Swami, an HMS research fellow in anesthesia at the BWH Laboratory of Nanomedicine and Biomaterials and co-lead study author.

 “We engineered and tested a bone-targeted nanoparticle system to selectively target the bone microenvironment and release a therapeutic drug in a spatiotemporally controlled manner, leading to bone microenvironment remodeling and prevention of disease progression,” Swami said. 

“There are limited treatment options for bone cancers,” added Michaela Reagan, an HMS research fellow in Medicine at DFCI Center for Hematologic Oncology and co-lead study author.

“Our engineered targeted therapies manipulate the tumor cells in the bone and the surrounding microenvironment to effectively prevent cancer from spreading in bone with minimal off-target effects,” Reagan said. 

Stealth Nanoparticles

The scientists developed stealth nanoparticles made of a combination of clinically validated biodegradable polymers and alendronate, a clinically validated therapeutic agent, which belongs to the bisphosphonate class of drugs.

Bisphosphonates bind to calcium. The largest store of calcium in the human body is in bones, so bisphosphonates accumulate in high concentration in bones.

By decorating the surface of the nanoparticles with alendronate, the nanoparticles could home to bone tissue to deliver drugs that are encapsulated within the nanoparticles and kill tumor cells, as well as stimulate healthy bone tissue growth.

Furthermore, bisphosphonates are commonly utilized during the treatment course of cancers with bone metastasis, and thus alendronate plays a dual role in the context of these targeted nanoparticles.

The scientists tested their drug-toting nanoparticles in mice with multiple myeloma, a type of bone cancer. The mice were first pre-treated with nanoparticles loaded with the anti-cancer drug, bortezomib, before being injected with myeloma cells.

Slower myeloma growth

The treatment resulted in slower myeloma growth and prolonged survival. Moreover, the researchers also observed that bortezomib, as a pre-treatment regimen, changed the make-up of bone, enhancing its strength and volume.

“These findings suggest that bone-targeted nanoparticle anti-cancer therapies offers a novel way to deliver a concentrated amount of drug in a controlled and target-specific manner to prevent tumor progression in multiple myeloma,” said Omid Farokhzad, HMS associate professor of anesthesia and director of the BWH Laboratory of Nanomedicine and Biomaterials, and co-senior study author.

“This approach may prove useful in treatment of incidence of bone metastasis, common in 60 to 80 percent of cancer patients and for treatment of early stages of multiple myeloma,” Farokhzad said.

 “This study provides the proof of concept that targeting the bone marrow niche can prevent or delay bone metastasis," added Irene Ghobrial, HMS associate professor of medicine at DFCI Center for Hematologic Oncology and co-senior study author.

"This work will pave the way for the development of innovative clinical trials in patients with myeloma to prevent progression from early precursor stages or in patients with breast, prostate or lung cancer who are at high-risk to develop bone metastasis,” she said.

This research was supported by the United States Department of Defense (W81XWH-13-1-0390), National Institutes of Health (CA151884, CA133799), Movember-PCF Challenge Award, David Koch-Prostate Cancer Foundation Award in Nanotherapeutics, and a Friends of the Farber Grant.

 

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The Mystery of LAM

The Mystery of LAM

Rare disease in women pulls together community of researchers and patients

By ELIZABETH COONEY
Fri, 2014-03-21

Finding a treatment for LAM has galvanized a vibrant community of scientists, physicians and patients. Watch the story.

It’s not cancer, but it grows and spreads to distant organs.

It’s not malignant, but women die when it destroys their lungs.

It has no cure, but scientists, physicians and patients are converging to change that.

Lymphangioleiomyomatosis — LAM for short — is a rare disease in which abnormal, smooth muscle-like cells grow out of control, usually in the kidney, lymph nodes and lungs. It develops almost exclusively in women during their childbearing years.

At first, they might feel shortness of breath, which is sometimes confused with asthma and sometimes caused by a collapsed lung. Some women find out they have the disease when CT scans their doctors order for other reasons reveal LAM’s telltale cysts, invisible on ordinary chest X rays but sprinkled throughout their lungs.

LAM is in many ways a mystery defined by what it is not. But the story of LAM has evolved rapidly over the past 15 years. That momentum accelerated when a team led by a physician-scientist now at Harvard Medical School identified the genetic mutation at its core and then developed a model that could be used to study it. Their work allowed the molecular machinery to be nailed down by scientists across the world.

Madeline Nolan, the New England liaison for the LAM Foundation. Image: Rick Groleau

A vibrant community of researchers has grown at HMS and the LAM Center at Brigham and Women’s Hospital. They in turn are linked to other research labs and hospital clinics around the country united by the LAM Foundation, a driving force in patient support and research funding.

“Our lab was in the right place at the right time to really start to make new contributions to understanding the pathology of LAM and the evolution of the disease,” said John Blenis, HMS professor of cell biology. “It turns out that two molecular pathways we’ve been studying for 20-plus years converge to contribute to LAM.”

At a meeting 15 years ago convened by the father of a patient, Blenis learned about the challenge from, among others, Elizabeth Henske, HMS professor of medicine at Brigham and Women’s and director of the LAM Center. An oncologist, Henske worked on tuberous sclerosis during her postdoctoral training. Tuberous sclerosis can cause LAM and severe cognitive problems, as well as unusual kidney tumors filled with blood vessels, smooth muscle tissue and fat cells. She had been puzzled because similar tumors occur in women with LAM.

Henske has devoted her career to solving the mystery of LAM. While a rare disease, new ways to treat it may have implications for more common disorders.

“These are tumor-like cells, but they’re not malignant-appearing. So it seems like it should be easier to fix than a cancer. And it affects almost only women, so there may be a hormonal element. It just seemed like something we ought to be able to figure out,” Henske said. “That’s what hooked me in and that’s still what I think about all the time.”

Shocking diagnosis

Madeline Nolan learned about LAM at the end of a months-long series of referrals to figure out why she was so fatigued and why her bloodwork looked so odd. A hematologist had sent her to a pulmonologist after ruling out a blood disorder. She was 47 years old, she taught phys. ed., and she was shocked.

“I don’t smoke, I’m not around toxic chemicals, how do I have a lung disease?” Nolan  recalls wondering.

Nolan’s pulmonologist couldn’t tell her in 1999 why she had a disease that damages her lungs. With tears in his eyes, he told her she might have three or four years before needing a double-lung transplant.

She hasn’t gone down that road, although she now uses oxygen and takes a drug that has stalled her disease’s progression. Her story mirrors the mystery that LAM still is today.

Now teaching health to high schoolers in Waterbury, Conn., Nolan jokes that the pimples— a side effect from her medication—make her fit in with her students. She talks warmly about other LAM patients, who get together around their appointments at the LAM clinic in Boston as well at other events throughout New England. Her mission is to promote awareness, support patients and push research as the New England liaison for the LAM Foundation. 

“I want to do everything I can to make a cure happen faster,” she said. “I want to do this for the next 20-year-old who gets diagnosed.”

Scientific serendipity

Tuberous sclerosis and LAM share more than idiosyncratic kidney tumors. Tuberous sclerosis is an inherited disease caused by mutations in genes called TSC1 and TSC2, which patients carry in every cell in their bodies. About 40 percent of girls with tuberous sclerosis also go on to develop LAM in their childbearing years. Women without tuberous sclerosis have a sporadic form of LAM: They carry mutations only in the TSC2 gene and only in their tumor cells.

Henske participated in the cloning of the TSC1 gene in 1999, and she was the one who discovered in 2000 (while working at Fox Chase Cancer Center) that LAM is caused by mutations in TSC2.

At that point, no one knew what TSC2 did. But in what Henske calls a moment almost too good to be true, scientists studying flies to find genes that enlarge cells in the eye discovered that TSC genes control cell size. Other scientists determined that these genes regulate an enzyme called mTOR, a key player in a series of molecular events involved in cancer and one of two major signaling systems studied in the Blenis lab since the early 1990s.

The link between TSC and mTOR led quickly to a clinical trial showing that rapamycin, a natural product that blocks the mTOR pathway (and gives the enzyme its name: mammalian target of rapamycin complex), could shrink kidney tumors in women with LAM. A later clinical trial found that rapamycin could also stabilize lung function in women with LAM.  It’s not a cure—the disease continues to destroy the lungs if a woman stops taking the drug—but it stalls further lung damage.

“This is a really beautiful example of pure science,” Henske said. “Looking for regulators of cell size in the fly eye led in five years to the completion of a trial in a disease that is just devastating. You can’t imagine what it’s like for a young woman who’s already on oxygen.”

Meeting LAM face to face

Henske thinks about those women all the time. To get to her desk, she walks by a quilt hung outside her office honoring women with LAM. Squares depict lives stitched together at LAM Foundation events where scientists, women with LAM and their families all rub shoulders and form bonds. Physicians and researchers give presentations at annual meetings, but patients speak too, telling their stories and touching the hearts of basic scientists who don’t always see the people whose lives they hope to help.

Henske’s colleague in a nearby office, Jane Yu, proudly wears a new navy blue fleece top embroidered with the LAM Center’s name, a gift from Henske on the center’s fifth anniversary. HMS assistant professor of medicine at Brigham and Women’s, Yu led pioneering research that created the first animal model that closely recapitulates how LAM metastasizes under the influence of estrogen. She later showed that a drug can block LAM cells from spreading to the lung by inhibiting estrogen, confirming the hormone’s role in the disease.

Yu finds the LAM meetings unusual—and inspiring.

“Once you are there, you are family: Your sisters are there, your aunts are there. I wouldn’t say grandmothers—yet,” she said. “You feel that you are one of them and you want to help. They never give up, and every year I think we should try harder.”

Blenis said by going to LAM meetings, he became more interested in researching the disease. “I get very emotional when I go to these meetings, and I think all my postdocs who have ever gone have felt the same way.”

Xiaoxiao Gu, a postdoc in the Blenis lab, is also a LAM Foundation fellow. She had two questions she wanted to answer when she started her three-year fellowship in 2011.  “Because the hypothesis is that these kidney tumor cells are migrating to the lung in the presence of estrogen, how can I test estrogen responsiveness in those cells? And why is an inhibitor like rapamycin not enough to block disease progression?” 

Convergence

In a PNAS paper published in September 2013, Gu and Blenis offered an explanation: The two pathways that the Blenis lab has played a major role in defining actually converge in LAM in a way that requires their precise spatial and temporal regulation to generate the disease.

It takes more than the mTOR pathway—the set of molecular activities that rapamycin can inhibit, thereby blocking runaway cell metabolism and growth. It also takes a signaling pathway involved in the development of cancer: a chain of communicating proteins called the estrogen receptor-MAP (ERK-MAP) kinase pathway that transmits signals from the cell surface to DNA in the nucleus of the cell.

The team built on work by Yu and Henske showing that estrogen increases the migration of cells with TSC2 mutations via the MAP kinase pathway. Gu and Blenis found that estrogen signaling in the ERK-MAP kinase pathway is active in LAM cells, establishing that estrogen collaborates with inappropriate regulation of mTOR, converging to generate disease.

The ERK-MAP kinase pathway, activated by estrogen, starts a process that makes cells more likely to migrate, invade and survive. The mTOR pathway provides the energy and building blocks to support LAM cell growth and to enhance the estrogen-stimulated events.

“It’s this amazing integration of different signals at different times that contributes to disease progression,” Blenis said. “By discovering how these two pathways converge, it also suggests possible therapeutic options.”

The MAP kinase pathway has already been targeted by pharmaceutical companies for other reasons, Blenis said.

“We’re defining mechanistically how these pathways converge and verifying the choice of using an estrogen inhibitor in combination with mTOR inhibitors such as rapamycin,” Blenis said. “One drug may have some effects, but the combination of the two is going to be significantly greater at treating the disease.”

LAM’s lessons

Blenis, Henske, Yu and Gu all profess optimism with the field’s rapid progress, confident that what they learn about LAM may be applied to other diseases, including cancer.

“The tuberous sclerosis genes are in the center of the universe in terms of how a cell regulates almost everything it does, from metabolism to growth to its decision to divide. Everything depends on mTOR,” Henske said. “Many of us who study rare diseases have the very strong belief that the way you figure out a common disease is almost always by starting with a rare disease.”

Knowing the one faulty gene where a rare disease starts is much better than looking at 10 or 100 or 1,000 genes involved in a common disease, Henske said. “That’s kind of like looking at a crashed car and trying to figure out if it was the steering wheel or the brakes.”

In the Blenis lab, LAM is directly linked to his work on breast cancer metastasis while remaining significant in its own right. LAM patients’ pull on the scientists is strong.

“Clinician-scientists have more of a direct impact on patients,” Blenis said. “For a basic scientist, this is the whole reason to get into the field: We hope our work will have an impact down the road.”

Madeline Nolan and her LAM sisters are waiting.

“We’ve come such a long way in a short time.”

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Bone Repair by Silk

Bone Repair by Silk

Silkworm-derived surgical devices may be superior to metal and synthetics-based plates and screws in bone.

By BONNIE PRESCOTT
Tue, 2014-03-11

When a person suffers a broken bone, treatment may call for the surgeon to insert screws and plates to help bond the broken sections and enable the fracture to heal. These “fixation devices” are usually made of metal alloys.

These metal devices have disadvantages: Because they are stiff and unyielding, they can cause stress to underlying bone. They also pose an increased risk of infection and poor wound healing. In some cases, the metal implants must be removed following fracture healing, necessitating a second surgery. Resorbable fixation devices, made of synthetic polymers, avoid some of these problems but may pose a risk of inflammatory reactions and are difficult to implant.

Surgical screws made from silk protein. Image: Samuel Lin Lab/BIDMC

Now, using pure silk protein derived from silkworm cocoons, a team of investigators from Harvard Medical School, Beth Israel Deaconess Medical Center and Tufts University School of Engineering has developed surgical plates and screws that may offer improved bone remodeling following injury. Equally important, the devices can also be absorbed by the body over time, eliminating the need for surgical removal.

The findings, demonstrated in vitro and in a rodent model, are described in the March 4 issue of Nature Communications.

“Unlike metal, the composition of silk protein may be similar to bone composition,” said co-senior author Samuel Lin, HMS associate professor of surgery in the Division of Plastic and Reconstructive Surgery at Beth Israel Deaconess. “Silk materials are extremely robust. They maintain structural stability under very high temperatures and withstand other extreme conditions, and they can be readily sterilized.”

Lin worked with co-senior author and Tufts chair of biomedical engineering David Kaplan. “One of the other big advantages of silk is that it can stabilize and deliver bioactive components, so that plates and screws made of silk could actually deliver antibiotics to prevent infection, pharmaceuticals to enhance bone regrowth and other therapeutics to support healing,” said Kaplan.

First author Gabriel Perrone, of the Department of Biomedical Engineering at Tufts, used silk protein obtained from Bombyx mori silkworm cocoons to form the surgical plates and screws. Produced from the glands of the silkworm, the silk protein is folded in complex ways that give it unique properties of both exceptional strength and versatility.

To test the new devices, the investigators implanted a total of 28 silk-based screws in six laboratory rats. Insertion of screws was straightforward and assessments were then conducted at four weeks and eight weeks, post-implantation.

“No screws failed during implantation,” said Perrone, explaining that because silk is slow to swell, the new devices maintained their mechanical integrity even when coming into contact with fluids and surrounding tissue during surgery. The outcomes suggest that the use of silk plates and screws can spare patients the complications that can develop when metal or synthetic polymer devices come into contact with fluids.

“Having a resorbable, long-lasting plate and screw system has potentially huge applications,” said Lin. While the initial aim is to use silk-based screws to treat facial injuries, which occur at a rate of several hundred thousand each year, the devices have potential for the treatment of a variety of different types of bone fractures.

“Because the silk screws are inherently radiolucent (not seen on X-ray) it may be easier for the surgeon to see how the fracture is progressing during the post-op period, without the impediment of metal devices,” added Lin. “And having an effective system in which screws and plates ‘melt away’ once the fracture is healed may be of enormous benefit. We’re extremely excited to continue this work in larger animal models and ultimately in human clinical trials.”

This research was supported by the National Institutes of Health (EB002520).

Adapted from a Beth Israel Deaconess news release.

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Translating Success

Translating Success

Three centers launched to battle infectious pathogens

By ELIZABETH COONEY
Tue, 2014-03-11

In order to better combat infectious disease, Harvard Medical School is creating three Centers of Excellence for Translational Research: one in tuberculosis, one in bacteriology and one in virology.

Three five-year grants totaling up to $15 million per year from the National Institute of Allergy and Infectious Diseases, part of the National Institutes of Health, will allow HMS researchers to move discoveries about TB and emerging infections closer to applications in diagnosis, treatment and prevention.

Image: iStock

Improving diagnosis, fighting drug resistance

The TB center will focus on improving diagnostics, especially in children, and on combatting drug resistance. Megan Murray, HMS professor of global health and social medicine, will lead the center.

Megan Murray“The TB epidemic is still fueled by the fact that people are diagnosed relatively late in the course of their disease and a lot of transmission happens before diagnosis,” said Murray, who is also HMS associate professor of medicine at Brigham and Women’s Hospital and director of research at Partners in Health. “There’s no single therapy for TB, so there’s a big need to know which drugs people are resistant to.”

The TB center will build on previous work that used whole-genome sequencing to identify genetic mutations associated with drug resistance. Using bioinformatics and evolutionary techniques, they will study some 1,500 TB strains collected from an ongoing clinical research study in Peru to characterize resistance mechanisms. The scientists will correlate what they find with a measure called quantitative drug resistance, or the specific amount of a drug to which a strain becomes resistant.

Another project in the center, to be led by Eric Rubin, professor of immunology and infectious disease at the Harvard School of Public Health, will apply functional genomics to the mutations found by sequencing to see if the mutations confer drug resistance.

The TB center will work with an industry partner, Akonni Biosystems, to develop a diagnostic tool to be used in the field. Led by Chief Scientific Officer Darrell Chandler and Director of Engineering Christopher Cooney, the molecular diagnostics company will optimize a microarray for TB to test mutations associated with drug resistance.  

The TB center’s fourth of four projects may be its most ambitious, Murray said. Inspired in part by techniques used to interpret archeological DNA in Neanderthal samples, the scientists hope to capture fragments of TB’s genetic material from children. The standard TB test looks at sputum, but children can rarely cough up a useful sample. What if clinicians could examine bits of DNA in blood or urine and do other diagnostic tests as well?

“In the Neanderthal project, the challenge has been to take very degraded mammalian DNA mixed with bacterial DNA,” she said. “In our case, we’ve got the other problem: We try to pull microbial DNA mixed with human DNA in urine. Can we sequence that?”

Searching for new defenses

The two new centers in bacteriology and virology will build on the success of the New England Regional Center for Excellence, a regional research hub established at HMS after anthrax attacks in 2001 heightened national concerns about biological threats. Dennis Kasper, the HMS William Ellery Channing Professor of Medicine at Brigham and Women’s Hospital and professor of microbiology and immunobiology at HMS, is the principal investigator on the NERCE grant.

Dennis KasperHMS researchers will continue to focus on pathogens and diseases for which no vaccines or therapies exist—and microbes that resist current drugs. These include the bacterium Francisella tularensis, considered a potential agent of bioterrorism, as well as dengue fever, a significant disease in much of the world, which is now creeping into Florida.

“In NERCE, there was a lot of basic research on biodefense pathogens and later on emerging infectious disease pathogens,” added Kasper. “These new centers are now here to take those discoveries to the next step: translation.” Kasper will head the bacteriology CETR, a program that will focus on tackling the cell envelope that surrounds bacteria in such microbes as burkholderia, brucella, Vibrio cholerae and methicillin-resistant Staphylococcus aureus.

The virology CETR will be led by Sean Whelan, professor of microbiology and immunobiology and associate head of the Harvard Program in Virology. “This really is an opportunity to make a significant impact in understanding the entry mechanisms of emerging viral pathogens,” Whelan said. “We’re going to learn new biology and understand how some small molecules block viral entry.”

Sean WhelanViral entry is a target of our natural antibody response to viral infection, a step in the viral replication cycle that has not been well exploited by synthetic inhibitors.

One virology project involves small molecules that inhibit dengue virus replication and another concerns small molecules that block the Ebola virus from getting into cells. A third effort will search for cellular molecules that many viruses use to reach and enter cells. A fourth project will explore how viruses move from one cellular compartment to another before infecting cells with their genes.

New science for emerging threats

“We are looking at a very broad spectrum of emerging viruses that are threats to human health and potential biodefense agents, asking what molecules and cellular factors are needed to get into cells,” Whelan said. “We’re hoping that we will identify both pathogen-specific host factors as well as those that are shared among different viruses.”

In a similar vein, scientists in the bacteriology CETR hope to find common weaknesses to exploit.

“A lot of antibiotic research addresses enzymes or proteins that are involved in the synthesis of the cell wall of bacteria. There are key proteins that are very similar between different bacteria that could potentially serve as targets,” Kasper said. “Vaccines tend to be organism-specific, but we’re developing platforms that can be applied to any organism for which you want to develop a vaccine.”

“For many years it was sufficient for people to talk about their basic research as perhaps leading to a drug or to a vaccine someday,” said Gerald Beltz, administrative director for the two centers in the Department of Microbiology and Immunobiology. “Now it has to be much more direct, and I think these centers are part of that.”

HMS investigators within the bacteriology CETR are John Mekalanos, the Adele Lehman Professor of Microbiology and Molecular Genetics and head of the department of microbiology and immunobiology; Suzanne Walker and Stephen Lory, both professors of microbiology and immunobiology; Thomas  Bernhardt and  David Rudner, associate professors of microbiology and immunobiology; and Daniel Kahne, professor of biological chemistry and molecular pharmacology.

Virology CETR investigators at HMS include James Cunningham, associate professor of medicine (microbiology and immunobiology) at Brigham and Women’s Hospital; Stephen Harrison, the Giovanni Armenise-Harvard Professor of Basic Biomedical Science; Tomas Kirchhausen, professor of cell biology; Priscilla Yang, associate professor of microbiology and immunobiology; and Nathanael Gray, professor of biological chemistry and molecular pharmacology.

Other researchers involved in the TB center include Max Salfinger, lab director of mycobacteriology and pharmacokinetics at National Jewish Health in Denver, who will lead quantitative genomics project. In the later years of the TB grant, scientists will work with Ann Goldfeld, HMS professor of medicine at Boston Children’s Hospital, who heads a clinical research site in Cambodia. Louise Ivers, HMS associate professor of Global Health and Social Medicine and associate professor of medicine at Brigham and Women’s Hospital, will lead similar clinical research in children in Haiti.

James Sacchettini, professor of biochemistry and biophysics and of chemistry at Texas A&M University, and Thomas Ioerger, associate professor of computer science at Texas A&M, will lead gene sequencing.

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Stimulating Mechanisms

Stimulating Mechanisms

 

HMS researchers are unlocking the secrets of a hormone known to spur weight loss and improve glucose metabolism.

 

By Stephanie M. McPherson
Thu, 2014-01-09

Brown fat tissue. Image: Wikipedia

Harvard Medical School researchers at Joslin Diabetes Center have discovered that a hormone long associated with weight loss and improved glucose metabolism is linked to activation of calorie-burning brown fat. This finding could have implications for production of new medications for type 2 diabetes and obesity.

The results are published in the January issue of the Journal of Clinical Investigation in a paper titled “Interplay between FGF21 and Insulin Action in the Liver for the Regulation of Metabolism.”

For the past decade, the hormone FGF21 (fibroblast growth factor 21) has been known to play a role in metabolic regulation. Its mechanism of action, however, remained unidentified.

“So what we were interested in learning is how does FGF21 both stimulate weight loss and improve glucose metabolism,” said corresponding author on the paper C. Ronald Kahn, Mary K. Iacocca Professor of Medicine at HMS and chief academic officer at Joslin.

“And this study shows that one big factor in this is the ability of FGF21 to stimulate what’s called browning of white fat. That is where the white fat becomes more energetically active and begins to burn energy rather than store energy.”

In 2009, Joslin researchers showed that brown fat exists in humans and burns calories to produce heat. White fat can act in a similar manner when stimulated, a process known as “browning.”

Determining stimulation mechanisms can provide researchers with a first step toward using brown fat as a treatment for obesity and type 2 diabetes.

FGF21 is secreted from the liver, prompting Kahn and colleagues to question if its metabolic-related activity depended on molecular interactions within the liver tissues. They tested their hypothesis using insulin-resistant animal models created through two different methods: feeding the mice a high-fat diet or knocking out the insulin signaling in the mice’s liver tissues.

The investigators then introduced FGF21 to both model groups continuously for two weeks via an inserted pump. During that time, they monitored weight, blood glucose levels and lipid levels. After the two weeks ended, they analyzed the liver tissue.

“What we found was that even without insulin signaling in the liver, FGF21 could still improve glucose metabolism,” said Kahn.

To determine that the improvements were due to the browning of white fat, rather than the activation of brown fat, they removed the animals’ brown fat pads.

“So, in those animals, where most of the brown fat is removed, FGF21 still works on the remaining white fat because of browning,” Kahn said.

FGF21 also regulates lipid metabolism, and the researchers determined that that process is dependent on functioning insulin signaling in the liver.

Proving that FGF21 activates the browning of white fat is a large step forward in understanding the process of how variations of brown fat assist in metabolic regulation.

Identifying this hormone as a major player in this activation has implications for the eventual creation of a brown fat-stimulating drug.

“As with any new drug or hormone, of course, we need to learn not only its good effects, but also any potential side effects,” said Kahn. “And I think that’s where a lot of the effort is now … by pharmaceutical companies.”

In addition to its drug potential, Kahn thinks this discovery is interesting from a basic biology point of view.

“FGF21 wasn’t even known to exist until 10 years ago, and now we know it is a new circulating hormone that is regulated in feeding and fasting,” he said.

“And I think that this is another piece of evidence that we don’t understand all there is to know yet about metabolic regulation even though people have been studying it for literally hundreds of years.”

Adapted from a Joslin Diabetes Center news release

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Lurie Foundation Offering Fellowships

Lurie Foundation Offering Fellowships

Application deadline is March 22

Wed, 2013-02-06

The Nancy Lurie Marks Family Foundation and Harvard Medical School invite candidates to submit applications for two fellowships related to autism research. 

Postdoctoral Fellowship

The Nancy Lurie Marks Postdoctoral Fellowship is intended to provide salary support to a postdoctoral fellow and limited funds for research supplies. To be eligible, candidates must hold an MD and/or PhD; must be affiliated with Harvard Medical School or a Harvard-affiliated hospital; must have at least two years’ prior postdoctoral research experience and must be actively engaged in research related to autism.

To apply, applicants should send a single PDF to NLM_fellowship@hms.harvard.edu containing:

  • A cover letter with contact information
  • A CV
  • A five-page proposal including one page of specific aims, one page of background and significance, and three pages describing the research plan and experimental approach (references can be on a separate page).

Applicants should also arrange to have three letters of recommendation sent directly to NLM_fellowship@hms.harvard.edu. The letters should be from individuals familiar with the applicant’s research and future potential as an investigator, including a letter of support from the applicant’s postdoctoral adviser for the proposed project.

The deadline for applications and letters of recommendation is Friday, March 22, 2013.

Junior Faculty Fellowship

The foundation is also offering a fellowship intended to support junior faculty as they develop an independent research program related to autism. Funding provides $75,000 per year for two years, plus $10,000 per year for supplies.

To be eligible, applicants must hold an MD or MD/PhD; must be a junior faculty member (within first five years) at Harvard Medical School or a Harvard-affiliated institution; must have at least two years’ prior postdoctoral research experience and must be actively engaged in research related to autism.

Candidates should submit applications in a single PDF to NLM_fellowship@hms.harvard.edu containing:

  •  A cover letter with contact information;
  • A 250-word abstract;
  •  An NIH Biosketch;
  • A five-page proposal (no smaller than 11 point font) including one page of specific aims, one page of background and significance, and three pages describing the research plan and experimental approach (references can be on a separate page).

Applicants should also arrange to have three letters of recommendation from individuals familiar with their research and their future potential as an independent investigator sent directly to NLM_fellowships@hms.harvard.edu. Applications and letters of recommendation are due by Friday, March 22, 2013.

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Blood Cleanser for Sepsis

Blood Cleanser for Sepsis

Dialysis-like ‘biospleen’ quickly filters bacteria, fungi and toxins

By KRISTEN KUSEK
Mon, 2014-09-15

Wyss Institute Founding Director Don Ingber, Senior Staff Scientist Michael Super and Technology Development Fellow Joo Kang explain how they engineered the mannose-binding lectin (MBL) protein to bind to a wide range of sepsis-causing pathogens and then safely remove the pathogens from the bloodstream using a novel microfluidic spleen-like device.

Things can go downhill fast when a patient has sepsis, a life-threatening condition in which bacteria or fungi multiply in the blood—often too fast for antibiotics to help.

A new device inspired by the human spleen and developed by a team at Harvard’s Wyss Institute for Biologically Inspired Engineering may radically transform the way doctors treat sepsis.

“Even with the best current treatments, sepsis patients are dying in intensive care units at least 30 percent of the time,” said Mike Super, senior staff scientist at the Wyss Institute. “We need a new approach.”

Sepsis kills at least 8 million people worldwide each year and is the leading cause of hospital deaths.

The device, called a “biospleen,” exceeded the team’s expectations with its ability to cleanse human blood tested in the laboratory and to increase survival in animals with infected blood, as reported in Nature Medicine  on Sept. 14. In a matter of hours, it was able to filter live and dead pathogens from the blood, as well as dangerous toxins released from the pathogens. 

Sepsis occurs when a patient’s immune system overreacts to a bloodstream infection, triggering a chain reaction that can cause inflammation, blood clotting, organ damage and death. It can arise from a variety of infections, including appendicitis, urinary tract infections and skin or lung infections, as well as from contaminated IV lines, surgical sites and catheters.

Identifying the specific pathogen responsible for a patient’s sepsis can take several days, and in most cases the causative agent is never identified. If doctors are unable to pinpoint which types of bacteria or fungi are causing the infection, they treat sepsis patients empirically with broad-spectrum antibiotics—but these often fail, and they can have devastating side effects.

The sepsis treatment challenge continues to grow more complex as the prevalence of drug-resistant bacteria increases while the development of new antibiotics lags. 

“This is setting the stage for a perfect storm,” said Super, who was part of a team led by Wyss Institute Founding Director Don Ingber, the Judah Folkman Professor of Vascular Biology at Harvard Medical School and at the Vascular Biology Program at Boston Children’s Hospital. The team also included Wyss Institute Technology Development Fellow Joo Kang and colleagues from HMS, Boston Children’s and Massachusetts General Hospital. 

Kang, who is also a research associate at Harvard’s School of Engineering and Applied Sciences (SEAS) and a research fellow in the Vascular Biology Program at Boston Children’s, set out with the team to build a fluidic device that works outside the body like a dialysis machine, removing living and dead microbes of all varieties—as well as toxins. They modeled it after the microarchitecture of the human spleen, an organ that removes pathogens and dead cells from the blood through a series of tiny, interwoven blood channels.

The spleen-like blood-cleansing device makes use of genetically modified blood proteins coated on magnetic nanoparticles and magnetic forces to pull a variety of pathogens from the blood. Image: Harvard’s Wyss Institute

The biospleen is a microfluidic device that consists of two adjacent hollow channels that are connected to each other by a series of slits. One channel contains flowing blood, and the other has a saline solution that collects and removes the pathogens that travel through the slits.

Key to the success of the device are tiny, nanometer-sized magnetic beads that are coated with a genetically engineered version of a natural immune system protein called mannose-binding lectin (MBL).   

In its innate state, MBL has a branch-like head and a stick-like tail. In the body, the head binds to specific sugars on the surfaces of all sorts of bacteria, fungi, viruses, protozoa and toxins, and the tail cues the immune system to destroy them.

However, sometimes other immune system proteins bind to the MBL tail and activate clotting and organ damage. Super used genetic engineering tools to lop off the tail and graft on a similar one from an antibody protein that does not cause these problems.

The team then attached the hybrid proteins to magnetic beads 128 nanometers in diameter¾approximately one-five hundredths the width of a human hair¾to create novel beads that could be added to blood of an infected patient to bind to the pathogens and toxins without having to first identify the type of infectious agent.

The team then added a magnet to the sepsis device that pulls the pathogen-coated magnetic beads through the channels to cleanse the blood flowing through the device. The cleansed blood is then returned.

The team first tested their blood-cleansing system in the laboratory using human blood that was spiked with pathogens. They were able to filter blood much faster than ever before, and the magnets efficiently pulled the pathogen-coated beads out of the blood. In fact, more than 90 percent of key sepsis pathogens were bound and removed when the blood flowed through a single device at a rate of about a half- to one liter per hour.

Many devices can be linked together to obtain levels required for human blood cleansing at dialysis-like rates. 

This image demonstrates the effectiveness of the genetically engineered protein-coated magnetic beads binding to pathogens.  Here, the magnetic beads (128 nm) are bound to two pathogens (E. coli on the left and S. aureus on the right). Image: Harvard’s Wyss Institute

Next, the researchers tested the device in rats that were infected with E. coli, S. aureus, and toxins—mimicking many of the bloodstream infections that human sepsis patients experience. Quite similar to the tests on human blood, after just five hours of filtering, about 90 percent of the bacteria and toxins were removed from the rats’ blood.  

“We didn’t have to kill the pathogens. We just captured and removed them,” Super said.

What’s more, 90 percent of the treated animals survived, compared to 14 percent of the controls. And thanks to the team’s modified MBL, the animals’ immune systems had not overreacted.

“Sepsis is a major medical threat, which is increasing because of antibiotic resistance,” said Ingber, who is also a professor of bioengineering at SEAS. “We’re excited by the biospleen because it potentially provides a way to treat patients quickly without having to wait days to identify the source of infection, and it works equally well with antibiotic-resistant organisms. We hope to move this towards human testing to advancing to large animal studies as quickly as possible.”

The work was funded by the Defense Advanced Research Projects Agency (DARPA) Dialysis-Like Therapeutics program, the Department of Defense/Center for Integration of Medicine and Innovative Technology (CIMIT) and the Wyss Institute for Biologically Inspired Engineering at Harvard University.

Adapted from a Wyss news release.

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Fast Forwarding Research

Fast Forwarding Research

 

Mass Eye and Ear fast-tracks a multisite study on Marathon bombing casualties

By LORI SHRIDHARE
Wed, 2014-06-18

Alicia Quesnel, HMS instructor in otology and laryngology at Mass Eye and Ear

As doctors at Massachusetts Eye and Ear treated patients in the aftermath of the 2013 Boston Marathon bombings, they realized that the tragedy presented a rare opportunity for learning.

Working in collaboration with several other Boston hospitals, principal investigator Alicia Quesnel, HMS instructor in otology and laryngology at Mass Eye and Ear, co-investigator Daniel Lee, HMS associate professor of otology and laryngology, and Aaron Remenschneider, then a fifth-year resident in the HMS otolaryngology residency program, launched a study in April 2013 that is examining post-blast trauma to the ear. The team is following approximately 100 patients who are receiving ongoing care and long-term monitoring at eight Boston-area hospitals. The first data are expected later this year.

Launching a multisite study is typically a time-consuming, cumbersome task, but as Remenschneider began preparing the IRB review process, he learned that Harvard Catalyst offered a method that allows all institutions participating in a study to cede the IRB review to one institution—in this case, Mass Eye and Ear.

“Through working with colleagues at Harvard Catalyst, we were able to submit a unified application and then get site approval at the respective institutions,” said Remenschneider. “The result was that we had a very solid and thorough application and study review that involved individuals at all the different sites.”

This form, available as a free resource to Harvard-affiliated researchers, continues to facilitate additional multisite studies as part of the New England Reliance Agreement set up by Harvard Catalyst’s Regulatory Foundations, Ethics, and Law Program.

Originally an agreement among only Harvard-affiliated institutions, the New England Reliance Agreement now counts Tufts University, Dartmouth College and MIT among its participating institutions, bringing the number of signatory institutions to 26.

Close to 12 more institutions from across New England are actively considering joining the agreement—further expanding the opportunity for streamlined review of collaborative research projects.

For more information, click on the video link above.

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Biomarkers Under the Scope

Biomarkers Under the Scope

Harvard Catalyst course explores burgeoning field of study

By SHRADDHA CHAKRADHAR
Wed, 2014-05-14

Harvard Catalyst course "Understanding Biomarker Science: From Molecules to Images."  Image: Courtesy Harvard Catalyst

They provide a window into disease, a way to peer into an organism and mine its secrets.  They can illuminate the inner workings of a heart muscle or indicate the presence of infection, and interest in how they work has been growing.

The study of biomarkers may be where the future of biomedical research is headed. Since a 1973 paper first mentioned the term “biomarker,” there has been a two-fold increase in the number of NIH-funded grants that deal with biomarkers.

Between 2000 and 2011, the NIH gave out nearly $10.7 billion worth of grants to roughly 30,000 studies that all had some form of biomarker science, a tenth of which pertained to biomarker discovery, with another 10 percent dedicated to the validation of pre-existing biomarkers.

Nearly 100 members from around the Harvard Medical School community gathered at the Harvard School of Engineering and Applied Sciences this spring to learn more about biomarkers during four days of coursework offered by Harvard Catalyst, Harvard’s clinical and translational science center.

The course, formally titled, “Understanding Biomarker Science: From Molecules to Images,” was filled with information ranging from discovering biomarkers to patenting them and everything in between.

“A biomarker is nothing but a way to classify your body’s abnormal response,” said Vishal S. Vaidya, HMS assistant professor of medicine at Brigham and Women’s Hospital and the director of the course.

“Fever, blood pressure, cholesterol levels, X-Ray images, or mutations in genes could be examples of biomarkers that allow us to understand and classify diseases,” he said.

Given this diversity of what constitutes a biomarker, it may seem like our current body of medical knowledge is chock full of biomarkers. Vaidya pointed out, however, that for many diseases, good biomarkers simply fail to exist. Several of the biomarkers currently used to diagnose and treat disease are based on science from decades ago and often can’t detect disease at an early stage.

This lack of viable biomarkers, and the fact that there was no formal biomarker community at Harvard, or even in Boston, led Vaidya to create the course.

“My vision was to start a course on understanding biomarker science for the Harvard community that offers a one-stop-shop to learn about the field,” said Vaidya.

He hopes that the creation of a biomarker community at HMS is the first step toward bringing researchers from different disciplines together to catalyze innovative solutions using biomarkers.

“A problem so complex requires team science. That is, basic scientists and clinicians. To not only talk to one another, but to also involve engineers and material scientists. To devise the best ways to measure biomarkers at the bedside,” said Vaidya.

One of the ways in which Vaidya and his team illustrated this interdisciplinary process was by designing sessions that addressed the use of biomarkers within specific diseases.

James Januzzi, HMS professor medicine at Massachusetts General Hospital and the director of Mass General’s Cardiac Intensive Unit, led a session on heart disease and offered his unique perspective on finding biomarkers for what continues to be the biggest killer of both men and women in the U.S.

“We’ve gotten very good at treating heart attacks,” said Januzzi, “but there is still no gold standard for evaluating heart failure.”

As an example, he cited one of the biggest tests for heart failure currently, the electrocardiogram or ECG. While effective, the test also has several shortcomings: it is not easy to interpret nor is it conclusive in its findings. 

There isn’t, however, a lack of proposed biomarkers to address the problem: nearly 150,000 papers are published in medical journals yearly, each documenting thousands of biomarkers for heart disease.

The problem, however, is what Januzzi called clinical inertia: many doctors are resistant to the idea of integrating better biomarker science into their current regimen and, as a result, only about 100 of these suggested biomarkers are used in clinical settings. 

There are other challenges with diseases such as cancer. Given that cancer affects many different processes in the body, it is harder to identify sensitive and specific biomarkers that are concrete and easy to measure.  The same is true for Alzheimer’s. Since the disease is only just beginning to be understood, the identification of a reliable biomarker is still a distant dream.

During her session on Alzheimer’s, Reisa Sperling, HMS professor of neurology at Brigham and Women’s Hospital, emphasized the need for good biomarkers that would aid in prevention, rather than in treatment, of Alzheimer’s. She was recently awarded an $8 million grant by the Alzheimer’s Association to continue her work on identifying the disease in people who are at risk for it.

“Intervention prior to dementia will likely have a better chance of changing how we treat Alzheimer’s,” said Sperling. “If we can delay dementia by even five years, we can cut the cost of Medicare to treat Alzheimer’s by nearly 50 percent.”

Whether used in prevention or for treatment, biomarkers have to be recognized as telling features before they can be evaluated. Imaging of biomarkers, therefore, was another major focus of the course.

Speakers included Jeffrey Yap, co-director of this course and associate director of the Center for Quantitative Cancer Imaging at the University of Utah, as well as Tina Kapur, executive director of the Image Guided Therapy Program in the Department of Radiology at Brigham and Women's Hospital.

Kapur’s recent work has focused on the development and use of 3D Slicer, an open-source imaging software that allows users to visualize and examine digital data from specimen three-dimensionally.

The software is being used in a variety of diseases, including cancer, COPD and Huntington’s disease, to better detect the presence and progress of disease.  Since this software is open-source, Kapur encouraged the course attendees to consider using it to help identify biomarkers for their own work.

“Not only is this software available for everyone to use, there is also an extensive community around it,” said Kapur. “It serves as another tool at your disposal to help identify biomarkers.”

Ultimately, learning about the existence of such tools, and acquiring the know-how to identify viable biomarkers, brought attendees to the course. As such, the course ended with a career panel of successful scientists fielding questions about navigating the complicated web of translational biomarker research and offering their own views on the process.

Kapur, for instance, advised the group to keep the passion—whether for research or application—alive.

“If research is something you like to do, you’ll find a way to come back to it,” Kapur said.

Shashi Ramaiah, the Translational Biomarker Head for Pfizer Pharmaceuticals and a faculty member at Texas A&M University, offered insight on the industrial side of biomarker discovery.

“When a pharmaceutical company wants to develop a drug, they approach a biomarker scientist to help them figure out not only what the target biomarker is, but also what the safety biomarker is,” he said.

A safety biomarker, he explained, is something that would need to be kept in check while the drug is in body. “So it might be useful for those of you trying to isolate biomarkers to look beyond just a target biomarker and also identify safety biomarkers before approaching the industry.”

How do you know when a biomarker is integral to a disease’s natural history?

“A good biomarker tears up all the standard paradigms. It should free your mind a bit and give you information to show how wrong you were, so you can have new directions to explore,” asserted Robert Thomas, HMS assistant professor of medicine at Beth Israel Deaconess Medical Center, and one of the attendees of the course.

“Understanding Biomarker Science: From Molecules to Images,” is one of over a dozen courses led by Harvard Catalyst’s Education and Training Program, which continues to make new offerings available each year. 

“Our collective understanding of biomarkers is critical for every facet of research, which is why we launched this course,” said Elliott Antman, director of Harvard Catalyst’s Education and Training Program.

“Our hope is that this course will continue to stimulate discussion in this area, and ultimately, help to strengthen the biomarker community at HMS,” Antman said. 

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