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'Click Chip' CTC Purification Method Enables Identification of Cancer-Linked Genetic Arrangements

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NEW YORK – A group led by University of California, Los Angeles researchers has developed a method combining antibody-based circulating tumor cell (CTC) capture and disulfide cleavage-driven CTC release to efficiently and rapidly purify the cells for downstream molecular analysis. 

The researchers envision using the covalent chemistry-based nanostructured silicon substrate, called "Click Chip," in microRNA assays to identify genetic rearrangements for potentially evaluating treatment responses and disease progression in lung cancer. 

"In the past, we have coated these capture antibodies onto nanofibers in our microfluidic chips, which increases the contact between the antibodies and antigens on the CTCs," UCLA molecular and medical pharmacology professor Hsian-Rong Tseng explained. "While the chip selectively captures CTCs in the bloodstream, some white blood cells (WBCs) are stuck non-specifically as well, [requiring] staining to visually distinguish the CTC and WBCs."

"We wanted to find a better way to reduce the consumption of the antibody, as well as improv[e] the tool's sensitivity while minimizing WBC background noise," Tseng added.

In a proof-of-concept study published last month in Science Advances, Tseng and his team demonstrated Click Chip's ability to quantify genetic rearrangements in CTCs derived from non-small-cell lung cancer (NSCLC) samples. 

The UCLA team designed a custom microfluidic chip that integrates tetrazine antibody (Tz)-grafted silicon nanowire substrates with a network of microchannels modified to induce chaotic mixing. 

In order to perform biorthogonal ligation-mediated CTC capture, the researchers graft trans-cyclooctene (TCO) modified capture antibodies to the CTCs in a blood sample. When a blood sample runs through the chip, the Tz and TCO react and instantly snag the CTCs. Tseng likened TCO and Tz to the male and female parts of a seatbelt, respectively, that "click" together.

Tseng explained that the researchers then expose the molecular "seatbelt" to a disulfide cleaving agent that snips the antibody-based tether holding the targeted CTC, while ignoring non-specifically caught WBCs. Released from the molecular bonds, the CTCs flow out of the chip with minimal impurities.

According to Tseng, researchers can use between 100 μl and 3 ml of a liquid sample on the Click Chip during each run. 

In the study, Tseng and his team optimized Click Chip by spiking 200 EpCAM-positive NSCLC cells with ROS1 mutations into a culture medium with human WBCs. After washing away excess antibodies, the team ran the CTCs through the Click Chips and later counted the cells using a fluorescence microscope. 

The group then compared Click Chip's biorthogonal ligation-mediated CTC capture ability with that of a conventional anti-EpCAM-mediated CTC capture called NanvoVelcro, which Tseng's team previously developed, using flow rates of 1 ml/hour. 

The team found that Click Chip had CTC capture efficiencies of about 94 percent using 0.1 ng of TCO-anti-EpCAM. In contrast, the researchers saw that NanoVelcro Chips had a lower capture efficiency of about 46 percent and higher WBC contamination when using the same amount of antibodies. 

Tseng's team then examined the effect of different flow rates on Click Chip capture efficiency. Finding an optimum flow rate of 1 ml/hour, the group then compared Click Chip's performance to a magnetic bead-based cell sorting approach. The team found that the magnetic bead-based tool only exhibited about a 24 percent capture efficiency and much higher WBC contamination. 

The researchers then measured Click Chip's ability to release CTCs. Injecting 200 μl of the disulfide cleaving agent into the device, the team saw that a flow rate of 1 ml/hour provided the highest and most robust number of cells. 

The group then tested the ability to detect and quantify ALK and ROS1 oncogenic gene rearrangements in CTCs isolated from patients with NSCLC using Click Chip. The researchers collected blood samples from 12 NSCLC patients before and after crizotinib cancer drug therapy, as well as samples from six healthy controls. Seven of the NSCLC patients had ALK rearrangements and five had ROS1 rearrangements. 

Tseng's team used two tubes of 2-ml blood samples from each patient to perform CTC capture, immunostaining, CTC enumeration, and CTC purification in the Click Chip, followed by reverse transcriptase(RT) Droplet Digital PCR analysis to detect and quantify the copy number of rearranged ALK or ROS1 transcripts. 

The UCLA researchers found that each NSCLC patient had anywhere from 0 to 36 CTCs in their blood samples. They also detected positive ALK or ROS1 rearrangements in all 12 patients, which was consistent with tissue biopsies collected at initial diagnosis. 

Tseng's group also monitored the treatment of two NSCLC patients with ALK or ROS1 rearrangement during crizotinib treatment. In both patients, the team found that patients' CTC counts measured over time were consistent with radiographic observations of lung tumor burden. The researchers therefore believe that Click Chips could serve as a tool to evaluate treatment responses and disease progression in critically ill cancer patients. 

According to senior author and UCLA assistant project scientist Yazhen Zhu, the Click Chip platform can purify CTCs from samples within 30 minutes.  

Meanwhile, Tseng acknowledged that his team encountered some limitations in the study, including its attempts to gather enough useful data from the small patient cohort. He noted that his team will require a bigger patient cohort to validate the tool's feasibility, sensitivity, and specificity in future work. 

However, Tseng said that his team has been banking blood samples for six different kinds of solid tumors that he believes will help future retrospective studies. The firm is now working with multiple academic groups, including the Cedars-Sinai Medical Center and the UCLA School of Medicine, to collect blood samples for future studies. 

Zhu said that the researchers will initially use Click Chip to capture and purify CTCs from patients with solid tumor samples derived from lung, liver, and prostate cancers, eventually expanding to other cancers such as breast, ovarian, and pancreatic. 

While the group is initially focusing on applying Click Chip to cancer research, Tseng believes that the method could potentially be used in the noninvasive prenatal testing [NIPT] space. Tseng's team has previously used NanoVelcro to isolate circulating fetal nucleated cells from maternal cells. 

"With the improved rare-cell purification performance observed for Click Chips, it is conceivable that the devices can be adopted for purification of rare circulating fetal nucleated cells, [such as] circulating trophoblasts for downstream single-cell whole genome profiling, [paving] the way for implementation of NIPT," Tseng said. 

Tseng noted that UCLA initially filed provisional patents for the IP associated with Click Chip in 2018, and that he expects the university to receive patent approval by 2023. 

Tseng said that Click Chip can potentially be more cost effective than other CTC capture methods due to its efficiency. He also highlighted that the platform consumes very little capture agent and quickly and gently recovers pooled CTCs with well-preserved mRNA, facilitating downstream molecular analysis. 

While the researchers do not have concrete plans to commercialize Chick Chip, Tseng said that UCLA is open to commercialization partners. In addition, he speculated that the team might further develop the technology through CytoLumina, a startup he previously launched to develop NanoVelcro technology for isolating CTCs.

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