Circulating tumor cells (CTCs) from peripheral blood hold important information for cancer diagnosis and disease monitoring. Analysis of this “liquid biopsy” holds the promise to usher in a new era of personalized therapeutic treatments and real-time monitoring for cancer patients. But the extreme rarity of CTCs in blood makes their isolation and characterization technologically challenging. This paper reports the development of a geometrically enhanced mixing (GEM) chip for high-efficiency and high-purity tumor cell capture. We also successfully demonstrated the release and culture of the captured tumor cells, as well as the isolation of CTCs from cancer patients. The high-performance microchip is based on geometrically optimized micromixer structures, which enhance the transverse flow and flow folding, maximizing the interaction between CTCs and antibody-coated surfaces. With the optimized channel geometry and flow rate, the capture efficiency reached >90% with a purity of >84% when capturing spiked tumor cells in buffer. The system was further validated by isolating a wide range of spiked tumor cells (50–50,000) in 1 mL of lysed blood and whole blood. With the combination of trypsinization and high flow rate washing, captured tumor cells were efficiently released. The released cells were viable and able to proliferate, and showed no difference compared with intact cells that were not subjected to the capture and release process. Furthermore, we applied the device for detecting CTCs from metastatic pancreatic cancer patients’ blood; and CTCs were found from 17 out of 18 samples (>94%). We also tested the potential utility of the device in monitoring the response to anti-cancer drug treatment in pancreatic cancer patients, and the CTC numbers correlated with the clinical computed tomograms (CT scans) of tumors. The presented technology shows great promise for accurate CTC enumeration, biological studies of CTCs and cancer metastasis, as well as for cancer diagnosis and treatment monitoring.
Isolation of Circulating tumor cells (CTCs) from peripheral blood or cancer cells from bone marrow has significant applications in cancer diagnosis, therapy monitoring and drug development. CTCs are cancer cells shed from primary tumors; they circulate in the bloodstream, leading to metastasis. The extraordinary rarity of CTCs in the bloodstream makes their isolation a significant technological challenge. Herein, we report the development of a platform combining multivalent DNA aptamer nanospheres with microfluidic devices for efficient isolation of cancer cells from blood. Gold nanoparticles (AuNPs) were used as an efficient platform for assembling a number of aptamers for high-efficiency cell capture. Up to 95 aptamers were attached onto each AuNP, resulting in enhanced molecular recognition capability. An increase of 39-fold in binding affinity was confirmed by flow cytometry for AuNP-aptamer conjugates (AuNP-aptamer) when compared with aptamer alone. With a laminar flow flat channel microfluidic device, the capture efficiency of human acute leukemia cells from a cell mixture in buffer increased from 49% using aptamer alone to 92% using AuNP-aptamer. We also employed AuNP-aptamer in a microfluidic device with herringbone mixing microstructures for isolation of leukemia cells in whole blood. The cell capture efficiency was also significantly increased with the AuNP-aptamer over aptamer alone, especially at high flow rates. Our results show that the platform combining DNA nanostructures with microfluidics has a great potential for sensitive isolation of CTCs, and is promising for cancer diagnosis and prognosis.
Circulating tumor cells (CTC) in the peripheral blood could provide important information for diagnosis of cancer metastasis and monitoring treatment progress. However, CTC are extremely rare in the bloodstream, making their detection and characterization technically challenging. We report here the development of an aptamer-mediated, micropillar-based microfluidic device that is able to efficiently isolate tumor cells from unprocessed whole blood. High-affinity aptamers were used as an alternative to antibodies for cancer cell isolation. The microscope-slide-sized device consists of >59,000 micropillars, which enhanced the probability of the interactions between aptamers and target cancer cells. The device geometry and the flow rate were investigated and optimized by studying their effects on the isolation of target leukemia cells from a cell mixture. The device yielded a capture efficiency of >95% with purity of ~81% at the optimum flow rate of 600 nL/s. Further, we exploited the device for isolating colorectal tumor cells from non-processed whole blood; as few as 10 tumor cells were captured from 1 mL of whole blood. We also addressed the question of low throughput of a typical microfluidic device by processing 1 mL of blood within 28 minutes. In addition, we found that ~93% of the captured cells were viable, making them suitable for subsequent molecular and cellular studies.
We developed an optimized ensemble of aptamers and antibodies that functions as a multivalent adhesive domain for the capture and isolation of cancer cells. When incorporated into a microfluidic device, the ensemble showed not only high capture efficiency, but also superior capture selectivity at a high shear stress (or high flow rate).
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