New tech could find tiny RNA cancer beacons in blood

By Nicole Casal Moore
Michigan Engineering

Cancerous tumors cast off tiny telltale genetic molecules known as microRNAs and a team of University of Michigan researchers has come up with an efficient way to detect them in blood.

The researchers say their approach could open the door to a single, inexpensive blood test to simultaneously screen for multiple types of cancer – eventually perhaps more than 100 different kinds.

“This could lead to technology that enables earlier detection in individuals at risk for cancer, earlier detection of recurrences in cancer survivors, and also better and earlier assessment of how well cancer therapies are working in patients,” said Muneesh Tewari, the Ray and Ruth Anderson-Laurence M. Sprague Memorial research professor of internal medicine at the U-M Medical School and an associate professor of biomedical engineering in the College of Engineering.

It would be years, if not a decade, before this could be available for routine clinical use. But the researchers have high hopes for their ultrasensitive technique that can pick out a single one of these nanoscale snippets in a speck of fluid.

“What we have done is develop a new paradigm, a new principle for detecting any sort of RNA in blood,” said Nils Walter, a professor of chemistry and biophysics in the College of Literature, Science and the Arts. Walter and Tewari are the senior authors of a paper on the work published in Nature Biotechnology.

RNA stands for ribonucleic acid, a class of molecule whose members play important roles in building living things from their DNA blueprints. For decades, scientists thought RNA was mainly a messenger: It ferried genetic information from DNA to the sites where cells make proteins – the workhorse molecules that essentially carry out the directions encoded in our genes.

But when scientists finished sequencing the human genome around 2003, they learned that 90 percent of it contains directions for making RNA. And most of that RNA is not the messenger kind that helps make proteins.

“The field of biochemistry is about 100 years old,” Walter said. “And for the longest time, we were focusing on proteins. It is almost as if we were studying the wrong thing. RNA is profoundly important for understanding and manipulating mammalian and human life, yet it is arguably the least studied genetic material in the mammalian cell. We’re just at the beginning of big discoveries of its functions.”

MicroRNA molecules, for example, are short strands that can bind to the messenger RNA, intercepting the dispatch and preventing bits of genetic code from being put into action. More than 1,000 varieties exist in our bodies. They directly or indirectly control virtually all major life processes, the researchers say. Having too little or too much of a particular microRNA can fuel tumor growth.

Cancerous cells are descendent from haywire healthy ones, so they have microRNA in them too. The tiny strands of genetic material have been detected in blood before (though not very efficiently) and scientists have several hypotheses about how they get there. They may be released when a cancerous cell dies and breaks down. And cells, including cancerous ones, may communicate with one another through microRNAs they send into the bloodstream to act as hormones. Blood-borne microRNAs from both mechanisms would be the cancer beacons the new technique could efficiently detect in patients, the researchers say.

In their experiments, they coated a glass slide with molecules called “capture probes” that would grab onto microRNAs in their vicinity. Then, in different trials, they dropped onto the slide samples of solutions containing five different microRNAs. In one case, the solution that carried the microRNAs was human blood serum – the fluid component with the blood cells removed.

To tell them RNA had been captured by one of the probes, they relied on a third type of molecule – fluorescent DNA strands that bind to the microRNA and emit light when they do. Only specific DNA sequences will bind to particular RNAs, so by varying the arrangement of the building blocks that make the DNA, they engineered strands that would attach to the different microRNAs.

What makes their method unique is that the DNA and RNA connect so weakly they don’t stay stuck. DNA strings latch onto and detach from RNA in particular rhythms. When the researchers observe this through a super-sensitive fluorescence microscope, it looks like a firefly blinking. They can confirm the capture of different microRNAs based on the blink rate – its “kinetic fingerprint.”

Although microRNAs have been detected in blood serum before, this approach is more direct and suffers virtually no false positives.

The paper is titled “Kinetic fingerprinting to identify and count single nucleic acids.” Other contributors include first authors Alexander Johnson-Buck of U-M and the Dana Farber Cancer Institute and Xin Su at Peking University, as well as Maria Giraldez at U-M Medical School and Meiping Zhao at Peking University. The work was funded in part by the U.S. Department of Defense. U-M is pursuing patent protection for the intellectual property, and is seeking commercialization partners to help bring the technology to market.


New technology could lead to tailor-made cancer treatments

By Gabe Cherry
Michigan Engineering

Tailor-made cancer treatments? New cell culture technique paves the way.

Electron microscopy shows cultured CTC cells (circled in red) growing on the chip device used in the study. Photo Credit: Jennifer Zhuo Zhang

In a development that could lead to a deeper understanding of cancer and better early-stage treatment of the disease, University of Michigan researchers have devised a reliable way to grow a certain type of cancer cells from patients outside the body for study. The new technique is more than three times as effective as previous methods.

Researchers say it’s a major step forward in the study of circulating tumor cells, which are shed from tumors and circulate through the blood of cancer patients. They’re believed to cause metastasis, the spread of cancer through the body that’s responsible for nearly 90 percent of cancer-related deaths.

The cells also hold valuable genetic information that could lead doctors to more informed treatment decisions and even tailor-made therapies for individual patients. And because the cells circulate in the blood, they can be gathered with a blood draw rather than a more invasive tissue biopsy. But progress has been slow, largely because the cells are rare in early-stage cancer patients.

U-M researcher Jennifer Zhuo Zhang runs an experiment using the microfluidic chip device. Photo Credit: Jennifer Zhuo Zhang

The new capture and culture method changes this by providing a reliable way to get usable numbers of circulating tumor cells from even early-stage patients. It grew new cells from 73 percent of the patients in a recent study, more than three times the success rate of previous methods and a first for early-stage cancers. It’s a major game changer, according to Sunitha Nagrath, an assistant professor of chemical engineering who is working to develop the new technology.

“This culture method gives clinicians a way to study each patient’s cancer much earlier and much more frequently,” Nagrath said. “We can look for resistance to therapy and test potential therapeutics. It also moves us closer to being able to predict metastasis.”

The technique may also bring doctors closer to their goal of capturing cancer cells for diagnosis with a quick, non-invasive “blood biopsy” instead of the tissue biopsies that are currently used. This could enable them to keep closer tabs on each patient’s status and make more informed treatment decisions.

“We envision a point-of-care solution in four to five years,” said Nithya Ramnath, an associate professor of medical oncology at U-M. “You’d give blood and a short time later, doctors would have a whole repertoire of what’s going on with your tumor.”

The capture and culture process starts with a microfluidic chip device that captures cancer cells as a blood sample is pumped across it. The research team used a chip made of polydimethylsiloxane (PDMS) on a 1-inch by 3-inch glass slide. They covered the chip with microscopic posts that slow and trap cells, then coated it with antibodies that bind to the cancer cells.

After the cancer cells were captured on the chip, the team pumped in a mixture of collagen and Matrigel growth medium. They also added cancer-associated fibroblast cells that were grown in the lab of Diane Simeone, surgical director at the Multidisciplinary Pancreatic Cancer Clinic of the U-M Cancer Center. This created a three-dimensional environment that closely mimics the conditions inside the body of a patient.

The captured cancer cells prospered in the mixture, reproducing additional cells in 73 percent of tested samples. It was a dramatic improvement over earlier methods, which studied later-stage cancer patients and saw success rates of only around 20 percent.

“Primary cancer cells don’t grow well on a flat surface, and like people, they need neighbors to really prosper,” Nagrath said. “The collagen and Matrigel provide a three-dimensional environment for the cells to grow, while the cancer-associated fibroblasts give them the neighboring cells they need.”

The technology can be applied to most cancers, including breast, lung, pancreatic and others. It could enable doctors to follow the progression of each patient’s disease much more closely, says Max Wicha, M.D., Distinguished Professor of Oncology and director of the U-M Comprehensive Cancer Center, who is working to develop the technology.

“Cancer cells change constantly and they can quickly develop resistance to a given treatment,” Wicha said. “A device like this will enable us to follow the cancer’s progression in real time. If a cancer develops resistance to one therapy, we’ll be able to quickly change to a different treatment.”

The next step for the researchers is to fine-tune the design of the chip device, optimizing its ability to both capture cells and grow new ones. They also envision larger studies that test more treatments on cultured cells and examine the correlation between the ability to grow cancer cells from individual patients and those patients’ long-term outcomes.

A paper on the findings titled “Expansion of CTCs from early stage lung cancer patients using a micro-fluidic co-culture model” is published online in Oncotarget. It will appear in a forthcoming print edition. The research is conducted under the Translational Oncology Program, which brings together scientists from across U-M to translate research findings into potential new treatments for cancer. Funding is provided by the National Institutes of Health (NIH) Director’s New Innovator Award (1DP2OD006672-01).


Liquid biopsy could improve cancer diagnosis and treatment

 
By Kate McAlpine
Michigan Engineering

A microfluidic chip developed at the University of Michigan is among the best at capturing elusive circulating tumor cells from blood—and it can support the cells' growth for further analysis.

The device, believed to be the first to pair these functions, uses the advanced electronics material graphene oxide. In clinics, such a device could one day help doctors diagnose cancers, give more accurate prognoses and test treatment options on cultured cells without subjecting patients to traditional biopsies.

"If we can get these technologies to work, it will advance new cancer drugs and revolutionize the treatment of cancer patients," said Max Wicha, M.D., Distinguished Professor of Oncology and director of the U-M Comprehensive Cancer Center and co-author of a paper on the new device, published online this week in Nature Nanotechnology.

"Circulating tumor cells will play a significant role in the early diagnosis of cancer and to help us understand if treatments are working in our cancer patients by serving as a 'liquid' biopsy to assess treatment responses in real time," said co-author Diane Simeone, M.D., the Lazar J. Greenfield Professor of Surgery at the U-M Medical School and director of the Translational Oncology Program.

"Studies of circulating tumor cells will also help us understand the basic biologic mechanisms by which cancer cells metastasize or spread to distant organs—the major cause of death in cancer patients."

Yet these cells aren't living up to their promise in medicine because they are so difficult to separate from a blood sample, the researchers say. In the blood of early-stage cancer patients, they account for less than one in every billion cells, so catching them is tougher than finding the proverbial needle in a haystack.

"I can burn the haystack or use a huge magnet," said Sunitha Nagrath, an assistant professor of chemical engineering, who led the research. "When it comes to circulating tumor cells, they almost look like—feel like—any other blood cell."

On their microfluidic chip, Nagrath's team grew dense forests of molecular chains, each equipped with an antibody to grab onto cancer cells.

Even after the cells are caught, it's still hard to run a robust analysis on just a handful of them, the researchers say. That's why this demonstration of highly sensitive tumor cell capture, combined with the ability to grow the cells in the same device, is so promising.

"If we can get these technologies to work, it will advance new cancer drugs and revolutionize the treatment of cancer patients." Max Wicha

Hyeun Joong Yoon, a postdoctoral researcher in the Nagrath lab with a background in electrical engineering, was instrumental in making the microfluidic chip. He started with a silicon base and added a grid of nearly 60,000 flat gold shapes, like four-petaled flowers, each no wider than a strand of hair.

The gold flowers naturally attracted a relatively new material called graphene oxide. These sheets of carbon and oxygen, just a few atoms thick, layered themselves over the gold. This layered formation allowed the team to grow the tumor-cell-catching molecular chains so densely.

"It's almost like each graphene has many nano-arms to capture cells," Nagrath said.

To test the device, the team ran one-milliliter samples of blood through the chip's thin chamber. Even when they had added just three-to-five cancer cells to the 5-10 billion blood cells, the chip was able to capture all of the cells in the sample half the time, with an average of 73 percent over 10 trials.

"That's the highest anybody has shown in the literature for spiking such a low number of cells," Nagrath said.

The team counted the captured cancer cells by tagging them with fluorescent molecules and viewing them through a microscope. These tags made the cancer cells easy to distinguish from accidentally caught blood cells. They also grew breast cancer cells over six days, using an electron microscope to see how they spread across the gold flowers.

"When you have individual cells, the amount of material in each cell is often so small that it's hard to develop molecular assays," Wicha said. "This device allows the cells to be grown into larger quantities so you can do a genetic analysis more easily."

The chip could capture pancreatic, breast and lung cancer cells from patient samples. Nagrath was surprised that the device was able to catch about four tumor cells per milliliter of blood from the lung cancer patients, even though they had the early-stage form of the disease.

Working in a team that comprises both engineers and medical professionals at U-M, Nagrath is optimistic that the new technique could reach clinics in three years.

The paper is titled "Sensitive capture of circulating tumor cells by functionalized graphene oxide nanosheets." The university is pursuing patent protection for the intellectual property and is seeking commercialization partners to help bring the technology to market.

This research is supported by the National Institutes of Health Director's New Innovator Award No. 1DP2OD006672-01.

Graduate student Tae Hyun Kim runs a blood sample through the chip while Hyeun Joong Yoon, the post-doc who made the chips, looks on. Also pictured, from left: chemical engineering graduate students Yang Wang and Molly Kozminsky, mechanical engineering undergraduate student Courtney Riley.

Graduate student Tae Hyun Kim runs a blood sample through the chip while Hyeun Joong Yoon, the post-doc who made the chips, looks on. Also pictured, from left: chemical engineering graduate students Yang Wang and Molly Kozminsky, mechanical engineering undergraduate student Courtney Riley.