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Johns Hopkins Team Develops Microfluidic Assay to Predict Risk of Breast Cancer Metastasis


NEW YORK (GenomeWeb) – A group led by researchers at the Johns Hopkins University School of Medicine has developed a microfluidic assay to examine phenotypic behaviors of cancer cells isolated from biopsies at initial diagnosis.

The researchers hope to eventually develop a diagnostic based on the technology to predict the likelihood of metastasis in tumor cells in breast cancer patients, as well as support the development of new cancer treatments.

When clinicians identify abnormal tissue in a patient's body, they usually extract a biopsy to determine if the lesion is benign or malignant. While a pathologist can determine the tumor's status, current diagnostic technology cannot predict the metastatic risk of the primary tumor in the patient.

Johns Hopkins University professor Konstantinos Konstantopoulos explained that his lab had researched phenotypic behaviors of non-metastatic MCF7 cancer cells, including their ability to migrate through certain sized microfluidic feeder channels. Konstantopoulos realized that he could develop a prototype microfluidic device to potentially isolate and track the movement of metastasis-initiating cells.

"I was hoping to come up with a device that would ultimately reduce [radiotherapy] overtreatment and be able to predict the metastatic propensity or aggressiveness of the breast cancers, as well as identify which therapeutic agents may work effectively on a patient specific basis," Konstantopoulos explained.

Called the Microfluidic Assay for quantification of Cell Invasion (MAqCI), the team's device contains two parallel seeding and collection channels connected to Y-shaped microchannels that mimic aspects of a patient's in vivo microenvironment. Konstantopoulos noted that his team selected a relatively wide feeder channel (20 μm by 10 μm) because the researchers wanted to introduce as many cells from the tumor biopsy as possible. The feeder channel bifurcates into two narrow channels — 3 μm and 10 μm wide — in order to track metastatic cancer movement.  

"We came up with this design in light of our initial, unpublished observations years ago showing that non-metastatic breast cancer cells, although they can migrate inside the feeder channels, they cannot practically enter a narrow branch channel," Konstantopoulos said. Instead, he highlighted that the assay's narrow branches allow the movement of metastatic breast cancer cells.

Konstantopoulos noted that the assay essentially measures three tumor cell parameters: the ability to move into the microchannel, the ability to enter the narrow branch channels, and the ability to proliferate.

"We used two smaller branches of different widths because we envision that MAqCI will be used to distinguish aggressive from non-aggressive cancer cells from a wide range of solid tumors," Konstantopoulos said.

In a study published last week in Nature Biomedical Engineering, Konstantopoulos and his team aimed to use the assay to quantify the abundance and proliferative index of migratory cells in breast cancer specimens. The group wanted to assess the specimens' metastatic propensity and determine if that information could be used for rapid screening of potential anti-metastatic therapeutics.

Konstantopoulos and his team began by seeding the microfluidic device with 50,000 human breast cancer cells (mixtures of aggressive and non-aggressive cells at different ratios) and took time-lapse images of the microchannels in twenty-minute intervals over a period of 24 hours. Since breast cancer cell lines with a high metastatic potential contain a larger fraction of migratory cells than low-potential cell lines, the researchers classified the cells into two categories: non-migratory if they stayed in the feeder channel, or migratory if they reached the bifurcation region and entered one of the branch channels.

The group aimed to determine a threshold percentage of migratory cells that separates cell populations with low versus high metastatic potential. Using a range of threshold percentages of 7 percent to 9 percent and experiment duration of about 14 hours, the researchers found that MAqCI could produce prognostic results with a clinical sensitivity of 89 percent and specificity of 100 percent.        

In order to find the metastatic potential of migratory versus unsorted cells, the researchers injected the two human cell types into immunocompromised mice and performed bioluminescent imaging analysis after eight weeks. The group found an eightfold increase in the metastatic burden of the lung and liver of mice injected with migratory cells relative to those injected with the unsorted cell population.  

The study authors noted that the data indicates that "while migratory and unsorted cell populations both form tumors that grow at similar rates, migratory cells have a markedly enhanced ability to form spontaneous metastases."

In order to examine potential gene expression differences contributing to the migratory cells' increased motility in vitro and metastasis in vivo, the group isolated RNA from the cells and performed genome-wide transcription analysis using RNA sequencing. Identifying 1,433 differentially expressed genes between migratory and unsorted genes, the team found that migratory cells had gene expression changes in multiple pathways, including RAS/mitogen-activated phosphatidylinositol 3-kinase (PI3K)-AKT, tumor necrosis factor, FOXO, and several pathways related to metabolism.

The researchers then validated MAqCI's predictive ability by measuring the metastatic potential of independent breast cancer cell lines and patient-derived xenografts (PDX).

Injecting the independent cancer cell lines into the tails of mice, the team monitored the cell survival in the bloodstream and reattachment to the mice's lungs over a period of two days using bioluminescent imaging. They found that PTEN -/- KRAS (G12V) cells had a high metastatic potential, while MCF-10A, PTEN, and KRAS (G12V) cells failed to form metastasis in vivo, which the team noted the MAqCI accurately predicted.

Selecting two tumor specimens from patients with metastatic triple-negative breast cancer, the researchers injected the PDXs into mice. The group saw that PDX cells dissociated from the tumor in the mice migrated through the feeder channel and entered the branch channels. After each experiment, the team fixed the cells into the microfluidic device and immuno-stained them for human mitochondria to ensure they came from the cancer patient.

The researchers ultimately found that the human tumor specimens exceeded the threshold of migratory cells for each of the four combinations of time and percentage of migratory cells, demonstrating that MAqCI could be used to predict the metastatic potential of clinically relevant specimens.

Konstantopoulos and his group then aimed to see if they could use MAqCI to measure the ability of certain therapeutics currently in active clinical trials to inhibit the motility of breast cancer cells. The team selected trametinib (a MEK 1/2 inhibitor) and BKM120 (a PI3K inhibitor) and applied them to high metastatic potential SUM159, BT-549, and MDA-MB-231 triple negative breast cancer cell lines, inserting the cells into the MAqCI platform and monitoring their movement.

The group saw that trametinib reduced the percentage of migratory cells and decreased the migration velocity of all three cell lines, indicating the drug's potential to reduce the risk of metastasis in patients with the three cell lines.

While BKM120 reduced the percentage of migratory BT-549 and SUM159 cells, Konstantopoulos noted that the drug unexpectedly augmented the migratory potential of MDA-MB-231 cells in the MAqCI assay and therefore exacerbated metastasis risk.

Overall, the study authors noted that they demonstrated the "potential to use a phenotypic test to rapidly screen the efficacy of therapeutics for reducing metastatic potential without the need for genetic testing and analysis to attempt to predict the response."

Konstantopoulos explained that his team has been developing the microfluidic technology since 2012, publishing several studies on microfluidic channels as the group tried to understand how cancer cells migrate in different confined environments.  

According to Konstantopoulos, the researchers are planning to use MAqCI to investigate other solid tumors in future studies, with the intention to establish the tool's potential for diagnosis, prognosis, and precision care.

"We established the technique looking at 25 breast cancer cell lines, then we used another five breast cancer cell lines to document that our technique works well," Konstantopoulos said. "Eventually, we'd like to discuss with pharma or biotech and see how we could bring this tool to the clinic."

Konstantopoulos said that the researchers at JHU have received several patents from the US Patent and Trademark Office associated with the microfluidic technology for breast cancer applications.

Konstantopoulos believes that MAqCI distinguishes itself from other microfluidic assays to detect tumor cells because it can deliver results within 24 hours, hopefully predict the risk of metastasis before the cancer spreads, screens the effects of therapeutics on highly motile metastasis, and physically isolates the cells for further analysis. Acknowledging that circulating tumor cells can be used to monitor a patient's response to treatment after researchers administer therapeutics, Konstantopoulos argued that both MAqCI and CTC assays will be crucial for cancer patient care.

The study authors believe that MAqCI could be eventually integrated into the clinical setting to track the  metastasis status of patients' tumors. Konstantopoulos envisions pathologists applying the assays to quickly distinguish between aggressive and benign cancers in its earliest stages.

While Konstantopoulos' team does not have any definite plans to commercialize the MAqCI device, he said that the group is working with JHU clinicians and pharmaceutical groups to explore different situations to explore possible commercial avenues.

"A small piece of tumor could be outsourced to a company that does blood testing, which can use our assay to determine the metastatic risk, apply therapeutic agents, then come back and tell the pathologist the prognostic results," Konstantopoulos said. In addition, he believes clinicians could use the assay to select effective cancer treatment regimens without exposing the patient to multiple toxic drugs.

Konstantopoulos' team therefore wants to replace bulky and expensive microscopes with its compact and affordable MAqCI devices. He expects that MAqCI could complement pathology tests, anticipating the lab test would cost the end user about $500 to $600.

At the same time, Konstantopoulos said that his team will need to perform preclinical validation studies using larger groups of patients in order to confirm the probability threshold used to determine a patient's prognosis.