Skip to main content
Premium Trial:

Request an Annual Quote

WUSTL Develops Method That Could Result in Brain Cancer Detection With Blood Test


NEW YORK (360Dx) – Researchers at the Washington University in St.Louis have developed a proof-of-concept technique to identify brain cancer biomarkers from a patient's bloodstream, which eventually could allow clinicians to detect tumor biomarkers with a simple blood test.

The technology, described in a study published last month in Nature, allows specific biomarkers to pass through the blood-brain barrier into a patient's bloodstream using a noninvasive focused ultrasound (FUS) and microbubbles.

Typically, surgeons diagnose brain cancer by examining tissue biopsies and searching for specific biomarkers. In order to collect living brain tissue, however, surgeons must surgically extract a sample by making a small incision in the skull, inserting a needle, and slicing off a piece of the tumor from the brain.

In the study, the researchers sought to determine whether FUS in combination with microbubbles could enhance the release of biomarkers from the brain tumor to blood circulation. Hong Chen, assistant professor in the biomedical engineering department and radiation oncology department at Wash U, and her team treated nine mice with two glioblastoma tumor models, U87 and GL261, by performing FUS in the presence of injected microbubbles.

The team's technology uses "focused" ultrasonic energy to target tissue deep in the body without incisions or radiation, concentrating the energy to a tiny spot in the brain. While researchers have used microbubbles in tandem with FUS to guide drug delivery into the brain through the blood brain barrier (BBB), Chen noted that the major challenge for her work was dislodging cancer biomarkers out of the brain into the bloodstream.

When microbubbles reach the targeted tumor, they oscillate back and forth and eventually pop, causing tiny ruptures of the blood-brain barrier (BBB). The fissure allows vesicles in the brain tumor to pass through the barrier and into the bloodstream. Chen explained that the microbubbles, made of a perfluorocarbon gas core with a lipid outer shell, are small enough to harmlessly move through the bloodstream.

In the study, the team used qPCR to determine the levels of circulating eGFP mRNA, a fluorescent biomarker expressed only in glioblastoma cells in the mice's plasma samples. Requiring 0.5 to 0.8 milliliters of plasma, qPCR detected eGFP mRNA in the FUS-treated U87 mice instead of the untreated U87 control samples.

The researchers then replicated the results in GL261 mice across three acoustic pressures,1.52MPa, 2.74 MPa, and 3.53 MPa. At higher acoustic pressures, they noticed that vascular damage linked to FUS treatment hinders efficient passage of tumor biomarkers into the bloodstream.

"We tried to be aggressive in selecting ultrasound pressures to ensure we saw something in our results," Chen explained.

Chen noted that like similar preparation needed for standard ultrasound techniques, the team's sonographers needed to clean off the mice's skin and apply ultrasound gel. The researchers then used magnetic reasoning to identify the tumor's location, and then performed the FUS process in less than two minutes. From sample prep to biomarker detection via qPCR, the overall process takes about three days in total.

The end result of their method is that the tumor-specific biomarkers would be released into the bloodstream, from which a blood-based biopsy could be used to detect the brain cancer.

Chen declined to disclose the technique's clinical sensitivity or specificity, but said that her team will publish quantified results in two to three months. However, at the lowest acoustic pressure, the team noticed the expression levels significantly increased compared to the expression levels of the other two groups for eGFP A and eGFP B, suggesting that the relatively lower pressure is more efficient at releasing eGFP mRNA from the tumor than relatively higher pressures.

In addition, the team saw that the circulating levels of eGFP mRNA were 1,500-times to 4,800-times higher in the FUS-treated GL261 than that of untreated mice for all three acoustic pressures.

"As a proof-of-concept study, we are trying relatively high pressures, selected below what we usually do to induce BBB opening without damage," Chen explained. "Now, we are working back to decrease the ultrasound energy to find out the optimal pressure to balance biomarker efficiency and safety."

Chen said her team chose to work with glioblastoma as a tumor model because they saw the brain as an organ that requires much more precise, noninvasive, and localized techniques than other parts of the body. With FUS, Chen believes the technique will lead to minimal hemorrhaging and tissue damage than standard brain surgeries. In addition, she noted that glioblastoma is the most common and deadliest brain cancer in the adult population.

Chen also noted that her team is working with Wash U's technical transfer office in order to apply for a patent for the technology her team developed as part of the Nature study.

In the study, the team ran into several limitations using FUS to search for tumor cells. First, the researchers noted that they will need to improve the technique by examining the efficiency of biomarker release under different FUS and microbubble parameters, as FUS could potentially release biomarkers under lower pressures. The researchers will also need to examine short- and long-term safety issues associated with FUS brain liquid biopsies, including the risk of hemorrhage.

"When we showed the histology results [to] new surgeons, [however], we found that they weren't worried [about hemorrhaging] at all," Chen explained. "They consider it would [cause] much less hemorrhaging than what occurs in tissue biopsies, where they have to physically slice the tissue, which can lead to complications."

Chen also noted that the terminal cardiac puncture performed in the study has only a small total volume, lacking the ability to perform repeated blood sample collection. In the future, the researchers will need larger animal models to collect blood samples at multiple time points after FUS treatment as a precursor for detection in human samples.

"We were hoping that contrasting MRI would show us that we'd be able to indicate the probability of the BBB, and hopefully that can give us a lot to predict how much of the biomarker is released," Chen said. "However, we didn't find [this] correlation in the proof-of-concept study."

Because the magnetic reasoning scanner used in the study was not dedicated to small animals, the researchers speculated that the technique may not have produced accurate biomarker results. Chen reasoned that future studies will need to determine whether contrast-enhanced MRI is a useful tool in predicting the number of biomarkers in FUS release.

Tatiana Khokhlova, research assistant professor at the University of Washington, noted that Chen's team sought to "hit the sample as hard as it [could], and then optimize by scal[ing] back on the acoustic pressure and bubble activity." Highlighting that the FUS technique dealt with "hemorrhaging in all frequencies," Khokhlova wondered whether "it would be possible to achieve any measurable increase [in biomarker release] if there isn't any disruption of blood vessels."

Chen pointed out that her team does not understand the exact mechanism behind FUS-enabled release of mRNA biomarkers. While Chen proposed that a "two-way door" situation exists with the BBB, she noted that that the group will have to further explore the potential mechanisms of FUS-enabled biomarker release.

John Lewis, associate professor of experimental oncology at the University of Alberta, argued that the approach is the first of its kind to tackle the BBB and extract potential cancer biomarkers. Like Khokhlova, he agreed that the technique needs to minimize hemorrhaging.

"So there's some more work that needs to be done in order to optimize the ultrasound parameters so that you don't damage the brain at the same time," Lewis explained. "With FUS, you can change the number of treatments depending on how strong they are and space them out over time to minimize damage."

While Chen and her team are using FUS to search for brain cancer biomarkers, other researchers have been applying similar methods to detect tumor samples in patients. Khokhlova and her colleagues, for example, have used ultrasound technology to detect biomarkers for prostate and ovarian cancer in patients. In a study published in RSNA Radiology in April 2017, Khokhlova' team identified candidate mRNA biomarkers in a rat's cell line by comparing the abilities of three FUS to release tumor-derived microRNA into the circulating in vivo and observe release dynamics. Khokhlova, however, noted that using FUS in general is risky because it can can cause tumor cells to metastasize.

"If you create holes in the tumor tissue, people worry that tumor cells may enter the bloodstream and undergo metastasis," Khokhlova explained. "While the process is safer, less invasive, and less perturbing than needle biopsy, we see the [risk] as the biggest hurdle to clinical application."

Lewis and his team has applied a similar technology to potentially detect circulating tumor cells in patients with prostate cancer. In a study published in January 2017 in Cancer Research, the researchers applied FUS and nanodroplets, which expand to microbbules when exposed to ultrasound, in a chick embryo to liberate blood vesicles from tumor tissue.

In addition to detecting brain tumors, Chen envisions the technology being used to monitor glioblastoma patients for treatment over time. She believes it could eventually help detect other brain diseases including Alzheimer's and amyotrophic lateral sclerosis.

"The next step is to look at natural biomarkers and [demonstrate] that the technique still works," Chen explained. She aims to integrate the tool with a navigation technique to replace standard technology used by neurosurgeons for liquid biopsy applications.