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Microfluidic Device Enables CTC Single-Cell RNA Sequencing

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NEW YORK (GenomeWeb) – Researchers at the University of Michigan have developed a microfluidic device and technique called Hydro-Seq to collect single circulating tumor cells (CTCs) and prepare them for sequencing-based gene expression profiling.

The group is mulling multiple paths to commercialize the platform, including licensing the technology to large companies, partnering with sample prep firms, or establishing its own startup.

Max Wicha, a professor of oncology at the University of Michigan, explained that his lab has spent several years researching methods to identify and track circulating cancer stem cells (CSCs) in the bloodstream of breast cancer patients. Because CSCs are a very small part of tumor biopsies, he said, current tools are unable to track the cells in clinical trials.

"Once we put patients on actual therapeutic trials, we asked if we could monitor those patients and see if the trials were hitting the right cell populations," Wicha explained.  "We wanted something to efficiently extract cells and provide a more detailed analysis than just a simple indication of the cells' presence."

Wicha's team therefore developed the microfluidic Hydro-Seq CTC collection device, which he said greatly increases the efficiency of capturing circulating tumor cells based on their size and deformability, using hydrodynamic cell capture in parallel chambers.

In a validation study published in Nature earlier this month, Wicha and his team tested the device by extracting CTCs from metastatic breast cancer patients and performing single-cell RNA-seq on them. The team also tracked individual cells that express cancer stem cell biomarkers and biomarkers of epithelial/mesenchymal cell state transitions.

According to Wicha, the Hydro-Seq chip contains four major components: capture chambers where individual cells and beads are paired; microfluidic channels that cells, beads, and lysis buffer flow through; control valves that selectively close flow paths and segregate chambers during mRNA extraction; as well as inlet and outlet ports that researchers use to introduce samples and collect beads during mRNA extraction.

Each chamber contains a cell capture bead capture site, designed with an opening of 10 nm2. Wicha explained that the opening allows smaller cells — such as red blood cells — to pass through easily while capturing CTCs.

To allow cell lysis and the washing processes, the researchers added two extra washing channels to the entrance and exit of each branch channel. Wicha noted the washing step is crucial because it allows the system to achieve up to almost 95 percent pure cancer cells. 

Overall, the Hydro-Seq chip contains 16 branch channels, each with 50 cell-capture chambers.

To begin the workflow, a researcher performs an enrichment step on 10 ml of a patient's blood, resulting in about 5 percent CTCs and 95 percent contaminating blood cells, which are then fed into the microfluidic chip inlet. Within each capture chamber, the smaller red blood cells flow through the site, while a larger CTC cell blocks the channel and is captured. The resulting liquid flow diverts cells to other downstream chambers, guaranteeing one CTC per chamber.

After cell loading, a pneumatic valve is opened to wash the contaminants out of the chamber. The researchers then open the channel's valves and load the barcoded beads into the chip to pair with the captured cells. To lyse the cells, the researchers then close the chambers and pump lysis buffer into the chamber to break down the CTC. The released mRNA then hybridizes with the barcoded bead.

After waiting for 20 minutes, the researchers retrieve the beads by opening all valves with a back flow. The team then performs downstream sequencing procedures, including reverse transcription, amplification, library preparation, and paired-end sequencing, using a similar protocol as the Drop-seq method that was developed by researchers at Harvard Medical School.

"You can use informatics to see which transcript came from which cell and do RNA-seq together," Wicha said. "With a capture rate of over 90 percent, we get between 2,000 and 5,000 genes per cell."

In the study, the team demonstrated Hydro-Seq's potential clinical use by analyzing CTCs obtained from 21 patients with metastatic breast cancer, achieving single-cell transcriptome analysis of 666 CTCs. However, the researchers found that larger leukocytes were also captured at certain sites.

After sequencing the cells' mRNA, the team found that it was able to successfully screen out contaminating cell populations.

In order to determine intra-patient heterogeneity and discordant molecular profiles in CTCs, the team used Hydro-Seq to detect expression of clinical biomarkers including ER, PR, and HER2.

They found a small population of CSCs that simultaneously expressed epithelial (ALDH1A3) and mesenchymal (CD90 and THY1) biomarkers. In addition, they identified considerable intra-patient heterogeneity in the CTCs, noting that epithelial and mesenchymal CTCs had different activation of important CSC regulation pathways.

According to Wicha, Hydro-Seq only needs four to five hours to collect CTCs and capture the mRNA on beads, followed by a longer period for the downstream sequencing steps.

"Using the precisely controlled hydrodynamic capture operation, a small number of single cancer cells can be selectively captured and paired with barcoded beads for scRNA-seq with high purity, high efficiency, and high throughput," the study authors wrote. "These results demonstrate the potential of Hydro-Seq in downstream transcriptome analysis to provide insights into the biology of CTCs and cancer metastasis."

"In the primary tumor, [CSCs] represent 1 to 5 percent of the population," Wicha explained. "But we found that in the circulating tumor cells, they represent 30 to 50 percent of the population."

Wicha envisions the assay being used to follow cancer patients in clinical trials, especially to measure the effectiveness of drug agents that target CSCs in patients. In addition to breast cancer, his team has applied Hydro-Seq in liver, pancreatic, and lung cancer samples.

Wicha said his team plans to partner with other US academic groups to analyze cancer patient blood samples for larger-scale studies.

The researchers anticipate the assay's price to be comparable to or cheaper than that of existing microfluidic platforms in the market. It is also currently working with researchers at Genentech on a trial involving targeting cancer stem cells in HER2+ breast cancer.

"Researchers could take a tube of blood when a patient arrives in the clinic, perform the Hydro-Seq assay, and see if we're hitting the stem cells and their pathways," Wicha said. "We've started to apply this in patients in clinical trials and collecting blood samples from them."

He noted that his team has dealt with two major challenges, related to sample stability and cell clusters.

In order to receive blood samples from other labs for clinical studies, the researchers needed to develop a collection kit that ensured RNA stability. "Unlike DNA, RNA can easily be degraded," Wicha said. "We eventually found preservatives that might help, and we could also keep the samples at 4°C to maintain sample integrity."

They also found clusters of cells that it believed were crucial to metastasis, however they couldn’t extract the clusters in the microfluidic instrument. The team is now developing devices to capture clusters, inject them into Hydro-Seq, monitor the cells or take them apart within the cluster, and then perform single-cell analysis.

Wicha said the team has filed for IP for the Hydro-Seq assay and is examining, in collaboration with the University of Michigan's translational research partnership program, potential commercial routes for the technology, such as out-licensing it or creating a startup.

Ultimately, the group wants to develop a single turnkey device that involves a tube of blood that is "squirted in and comes out ready to be sequenced," Wicha said.

"First, we're going to work with a number of other leading cancer centers to share the technology in a non-commercial sphere to have them validate the assay," Wicha explained. "The next step is to demonstrate clinical utility for patients in clinical trials, which will let us know where it sits in commercialization."

As Wicha's group seeks to further develop and commercialize the Hydro-Seq assay, several companies in the cancer space currently offer their own methods for single CTC extraction and downstream single-cell analysis.

Fluidigm, for example, currently sells its high-throughput integrated fluidic circuit (IFC) for isolating medium-sized single cells, using its C1 system for mRNA sequencing. The preparation device captures up to 800 single cells and comes with live, on-IFC imaging for phenotypic analysis.

Also, researchers at Harvard Medical School, led by genetics professor Steven McCarroll, developed a droplet-based Drop-Seq in 2015 for profiling transcriptomes of thousands of cells. According to the lab's site, the system is open source and can be built in any research lab.

Harvard physics and applied physics professor David Weitz believes that Hydro-Seq's nuanced technology may eventually be able to provide unique data about a patient's cancer status. Weitz' own team is currently developing and commercializing a microfluidic approach called InDrop, which barcodes cells for single-cell RNA sequencing.

"The problem [Wicha's team is] addressing is really important and is a research topic of great current interest and effort," Weitz said. "It sounds like they have made good progress addressing one of the pressing needs of this topic."

While noting that companies and academics in general face high costs for developing tools to collect a very small number of CTCs, Weitz pointed out that Wicha's team may do a good job reducing Hydro-Seq's price as the technology develops further.

Wicha argued that while certain commercial and research devices claim to be useful to look at the heterogeneity of cancer cells, they are impractical for several reasons. First, he noted that all devices — while they enrich for CTCs — are highly contaminated by red blood cells. He said that some devices — such as Menarini-Silicon's CellSearch CTC system— only count the number of CTCs without providing additional characterization.

"Even when you're starting out with one tumor cell in a billion cells, the commercial devices can enrich those down to about 5 percent of total cells," Wicha said.

Wicha also highlighted that many blood-based assays use antibodies to pull CTCs out of a patient's bloodstream, as opposed to the sized-based separation of the Hydro-Seq chip.

"This is a biased technique since you have to know what is on the cells' surface, and it turns out that stem cells, which we believe are the seeds of metastasis, don't express the same molecules on their surface as do the other cells," he explained.

Wicha highlighted that by further examining how immune and cancer cells interact in the bloodstream, his team will discover more about how individual cells react to certain cancer therapies.

"For pharmaceutical companies, you can see this is really quite valuable, as the approach now is using targeted therapies or immunotherapies," Wicha explained. "Everyone, including the pharmaceutical groups, wants to know if their technology is really hitting the right targets and their effects on individual cells."

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