For biologists, a single cell is a world of its own: It can form a harmonious part of a tissue, or go rogue and take on a diseased state, like cancer. But biologists have long struggled to identify and track the many different types of cells hiding within tissues.

Researchers at the 91̽»¨ and the have developed a new method to classify and track the multitude of cells in a tissue sample. In a published March 15 in the journal , the team reports that this new approach — known as SPLiT-seq — reliably tracks gene activity in a tissue down to the level of single cells.

“Cells differ from each other based on the activity of their genes — which genes are switched off or switched on,â€� said senior author , a 91̽»¨associate professor in both the Department of Electrical Engineering and the Paul G. Allen School of Computer Science & Engineering. “Using SPLiT-seq, it becomes possible to measure gene activity in individual cells, even if there are hundreds of thousands of different cells in a tissue sample.â€�

SPLiT-seq — which stands for Split Pool Ligation-based Transcriptome sequencing — combines a traditional approach to measuring gene expression with a new twist. For more than a decade, scientists have measured gene expression in tissues by sequencing the genetic “letters� of RNA, the DNA-like molecule that is the first step in gene expression. This standard approach — known as RNA-sequencing — profiles RNA across the whole tissue. But this approach does not tell researchers how cells within the tissue differ from one another. Single-cell RNA-sequencing addresses this by sequencing RNA from isolated cells, but existing methods are costly and do not scale well.

SPLiT-seq makes it possible to perform single-cell RNA-sequencing without ever isolating individual cells. The researchers put the cells through four rounds of “shuffling� — splitting them into separate pools and mixing them back together. At each shuffling step, they labeled the RNA in each pool with its own unique DNA “barcode.� At the end of four rounds of shuffling and labeling, RNA from each cell essentially contained its own unique combination of barcodes — and that barcode combination is included in the bulk sequencing of all the RNA in the tissue.

“With these ‘split-pool barcoding steps,’ we solve a big problem in measuring gene expression: reliably identifying which RNA molecules came from which cell in the original tissue sample,â€� said , who is also a researcher in the 91̽»¨Molecular Engineering & Sciences Institute.

“With that problem addressed, we can begin to ask biological questions about the different types of cells we define in the tissue,� said co-author , Associate Director of Molecular Genetics at the Allen Institute for Brain Science.

The team performed SPLiT-seq on brain and spinal cord tissue samples from laboratory mice. Using SPLiT-seq, they could measure the gene activity of over 156,000 cells. Based on patterns of gene activity, they estimated that more than 100 different types of cells were present in those tissue samples – including neurons and glial cells at various stages of development and differentiation.

SPLiT-seq can deliver this rich array of biological data at a cost of “just a penny per cell,� said Seelig in a  by the Allen Institute for Brain Science. This is a significantly lower cost than other single-cell RNA sequencing approaches, according to the researchers.

The researchers say that SPLiT-seq could answer important questions about how tissues develop, and identify minute changes in gene expression that precede the onset of complex diseases like Parkinson’s disease or cancer.

Co-lead authors on the paper are 91̽»¨electrical engineering postdoctoral researcher and , a 91̽»¨doctoral student in the Department of Bioengineering. Additional 91̽»¨co-authors are Richard Muscat, Anna Kuchina, Paul Sample and Sumit Mukherjee in the Department of Electrical Engineering; David Peeler in the Department of Bioengineering; Wei Chen in the Molecular Engineering & Sciences Institute; , a professor of bioengineering; and Drew Sellers, a research assistant professor of bioengineering and scientist with the 91̽»¨Institute for Stem Cell and Regenerative Medicine. Additional co-authors from Allen Institute for Brain Science are Zizhen Yao and Lucas Gray. The research was funded by the National Institutes of Health, the National Science Foundation and the Allen Institute for Brain Science.

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For more information, contact Rosenberg at alex.b.rosenberg@gmail.com or 773-294-4109 and Seelig at gseelig@uw.edu or 206-294-8180.

Most medical advancements of modern life started in the laboratory. Some, like the polio vaccine, sprouted from years of research directed toward combating a specific disease. Other breakthroughs, like penicillin, were discoveries in “basic” research later shown to have a clinical benefit. But translating a discovery into a clinical treatment can take years — though penicillin was first isolated in 1928, it took about 15 years to fully realize its clinical potential and develop a pipeline for mass production.

Researchers at the 91̽»¨ are among the winners of a startup challenge to shorten the transition time from lab bench to patient. The team, including members of professor ‘s in the 91̽»¨Department of Bioengineering, was selected based on its proposal and business plan to develop a targeted drug delivery system for breast cancer. As one of 10 teams winning the National Cancer Institute’s , the group will receive assistance and support from NCI and other partners in the often daunting transition into the business world of drug development.

Pun credits , the UW’s hub for innovation and spinoffs, for facilitating their entry into the challenge.

CoMotion connected Pun and 91̽»¨bioengineering graduate student Chayanon Ngambenjawong with Elizabeth Cho-Fertikh, a scientist and founder of Washington, D.C.-based , and together they drafted a business proposal for a drug delivery system targeting a specific population of tumor-promoting cells present in most solid cancers.

Their team proposal relied on years of research into these tumor-promoting cells, known as tumor-associated macrophages or TAMs. When this proposal in the startup challenge, they then developed a business plan and pitched it to a panel of experts and judges. As a winner of this second and final round in the competition, Pun and partners will be ushered through a multistep process of incorporating a company, meeting investors and launching their startup.

Pun and her team believe targeting TAMs may help improve the effectiveness of chemotherapy and other anticancer drugs.

“TAMs are a specific type of cell found within tumors, and past research indicates that they can interfere with the body’s own antitumor activity and clinical treatments,” said Pun. “So if you can find a way to specifically target and eliminate TAMs, this could help boost the efficacy of cancer treatments.”

Pun’s group a short peptide sequence displayed on the surface of TAMs in mouse tumors. Human TAMs should contain a similar sequence, and the to exploit this sequence as the basis for a novel drug delivery system against TAMs in breast cancer. But that could just be the beginning.

“TAMs play an important role in almost any type of major cancer,” said Pun. “So, our hope is that the lessons we learn in developing this treatment against breast cancer TAMs can be applied to other types of tumors.”

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For more information, contact Pun at spun@uw.edu.

In some cases, there’s not much medics can do — a tourniquet won’t stop bleeding from a chest wound, and clotting treatments that require refrigerated or frozen blood products aren’t always available in the field.

That’s why 91̽»¨ researchers have developed a new injectable polymer that strengthens blood clots, called PolySTAT. Administered in a simple shot, the polymer finds any unseen or internal injuries and starts working immediately.

The new polymer, described featured on the cover of the March 4 issue of could become a first line of defense in everything from battlefield injuries to rural car accidents to search and rescue missions deep in the mountains. It has been tested in rats, and researchers say it could reach human trials in five years.

In the initial study with rats, 100 percent of animals injected with PolySTAT survived a typically-lethal injury to the femoral artery. Only 20 percent of rats treated with a natural protein that helps blood clot survived.

“Most of the patients who die from bleeding die quickly,” said co-author , an assistant professor of who teamed with 91̽»¨ and to develop the macromolecule.

“This is something you could potentially put in a syringe inside a backpack and give right away to reduce blood loss and keep people alive long enough to make it to medical care,” he said.

The 91̽»¨team was inspired by , a natural protein found in the body that helps strengthen blood clots.

Normally after an injury, platelets in the blood begin to congregate at the wound and form an initial barrier. Then a network of specialized fibers — — start weaving themselves throughout the clot to reinforce it.

If that scaffolding can’t withstand the pressure of blood pushing against it, the clot breaks apart and the patient keeps bleeding.

Both PolySTAT and factor XIII strengthen clots by binding fibrin strands together and adding “cross-links” that reinforce the latticework of that natural bandage.

“It’s like the difference between twisting two ropes together and weaving a net,” said co-author , the UW’s Robert J. Rushmer Professor of Bioengineering. “The cross-linked net is much stronger.”

But the synthetic PolySTAT offers greater protection against natural enzymes that dissolve blood clots. Those help during the healing process, but they work against doctors trying to keep patients from bleeding to death.

The enzymes, which cut fibrin strands, don’t target the synthetic PolySTAT bonds that are now integrated into the clot. That helps keep the blood clots intact in the critical hours after an injury.

“We were really testing how robust the clots were that formed,” said lead author , a 91̽»¨doctoral student in bioengineering. “The animals injected with PolySTAT bled much less, and 100 percent of them lived.”

The synthetic polymer offers other advantages over conventional hemorrhaging treatments, said White, who also treats trauma patients at Harborview Medical Center.

Blood products are expensive, need careful storage, and they can grow bacteria or carry infectious diseases, he said. Plus, the hundreds of proteins introduced into a patient’s body during a transfusion can have unintended consequences.

After a traumatic injury, the body also begins to lose a protein that’s critical to forming fibrin. Once those levels drop below a certain threshold, existing treatments stop working and patients are more likely to die.

In the study, researchers found PolySTAT worked to strengthen clots even in cases where those fibrin building blocks were critically low.

The 91̽»¨team also used a highly specific peptide that only binds to fibrin at the wound site. It does not bind to a precursor of fibrin that circulates throughout the body. That means PolySTAT shouldn’t form dangerous clots that can lead to a stroke or embolism.

Though the polymer’s initial safety profile looks promising, researchers said, next steps include testing on larger animals and additional screening to find out if it binds to any other unintended substances. They also plan to investigate its potential for treating hemophilia and for integration into bandages.

Other co-authors are Xu Wang in 91̽»¨emergency medicine, Hua Wei in 91̽»¨bioengineering, and in 91̽»¨chemical engineering.

Funding came from the National Institutes of Health and its National Center for Advancing Translational Science, the 91̽»¨, the Washington Research Foundation, an NIH-supported 91̽»¨Bioengineering Cardiovascular Training Grant and discretionary funds from private donations.

For more information, contact Pun at spun@uw.edu or White at whiten4@uw.edu.

Now, scientists at the 91̽»¨ have developed a strategy to slow tumor growth and prolong survival in mice with cancer by targeting and destroying a type of cell that dampens the body’s immune response to cancer. The researchers published the week of Sept. 16 in the .

“We’re really enthusiastic about these results because they suggest an alternative drug target that could be synergistic with current treatments,” said co-author , a 91̽»¨associate professor of bioengineering.

Our immune system normally patrols for and eliminates abnormal cells. are a type of helpful immune cell that can be converted to the “dark side” by signals they receive from a tumor. When inside a tumor, macrophages can switch from helping the immune system to suppressing the body’s immune response to cancer. Several studies show a correlation between the number of macrophages in tumor biopsies and poor prognosis for patients, Pun said.

The 91̽»¨team developed a method to target and eliminate the cancer-supporting macrophages in mouse tumors. Researchers predict this strategy could be used along with current treatments such as chemotherapy for cancer patients.

“We think this would amplify cancer treatments and hopefully make them better,” Pun said.

Scientists have a strong understanding of the behavior of macrophages in tumors, but most current methods to remove them do away with all macrophages in the body indiscriminately instead of targeting only the harmful ones that live in tumors.

In this study, 91̽»¨bioengineering doctoral student Maryelise Cieslewicz designed a method to find a specific amino-acid sequence – or a peptide – that binds only the harmful macrophages in tumors and ignores helpful ones in the bodies of mice. When this sequence was injected into mice with cancer, the research team found that the peptide collected in the macrophage cells within tumors, leaving alone other healthy organs.

Once they discovered they could deliver the peptide sequence to specific cells, the researchers attached another peptide to successfully kill the harmful macrophages without affecting other cells. The mice had slower tumor growth and better survival when treated with this material.

The research team plans to test this method with existing cancer drugs to hopefully boost the success of other treatments.

The peptide sequence that successfully bound to harmful macrophages in mice doesn’t bind to their counterparts in humans, Pun said, but the researchers expect soon to find a similar peptide that targets human cells. They plan to use this method to investigate treatments for other types of cancer, including breast and pancreatic cancers.

The Pun research team collaborated with the 91̽»¨labs of Elaine Raines in pathology and André Lieber in medical genetics on this study.

The research was funded by the National Institutes of Health and a National Science Foundation fellowship.

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For more information, contact Pun at spun@uw.edu or 206-685-3488.