A Capillary-Force-Driven, Single-Cell Transfer Method for Studying Rare Cells.

The characterization of individual cells within heterogeneous populations (e.g., rare tumor cells in healthy blood cells) has a great impact on biomedical research. To investigate the properties of these specific cells, such as genetic biomarkers and/or phenotypic characteristics, methods are often developed for isolating rare cells among a large number of background cells before studying their genetic makeup and others. Prior to using real-world samples, these methods are often evaluated and validated by spiking cells of interest (e.g., tumor cells) into a sample matrix (e.g., healthy blood) as model samples. However, spiking tumor cells at extremely low concentrations is challenging in a standard laboratory setting. People often circumvent the problem by diluting a solution of high-concentration cells, but the concentration becomes inaccurate after series dilution due to the fact that a cell suspension solution can be inhomogeneous, especially when the cell concentration is very low. We report on an alternative method for low-cost, accurate, and reproducible low-concentration cell spiking without the use of external pumping systems. By inducing a capillary force from sudden pressure drops, a small portion of the cellular membrane was aspirated into the reservoir tip, allowing for non-destructive single-cell transfer. We investigated the surface membrane tensions induced by cellular aspiration and studied a range of tip/tumor cell diameter combinations, ensuring that our method does not affect cell viability. In addition, we performed single-cell capture and transfer control experiments using human acute lymphoblastic leukemia cells (CCRF-CEM) to develop calibrated data for the general production of low-concentration samples. Finally, we performed affinity-based tumor cell isolation using this method to generate accurate concentrations ranging from 1 to 15 cells/mL.

Investigating surface proteins and antibody combinations for detecting circulating tumor cells of various sarcomas.

Circulating tumor cells (CTCs) have gathered attention as a biomarker for carcinomas. However, CTCs in sarcomas have received little attention. In this work, we investigated cell surface proteins and antibody combinations for immunofluorescence detection of sarcoma CTCs. A microfluidic device that combines filtration and immunoaffinity using gangliosides 2 and cell surface vimentin (CSV) antibodies was employed to capture CTCs. For CTC detection, antibodies against cytokeratins 7 and 8 (CK), pan-cytokeratin (panCK), or a combination of panCK and CSV were used. Thirty-nine blood samples were collected from 21 patients of various sarcoma subtypes. In the independent samples study, samples were subjected to one of three antibody combination choices. Significant difference in CTC enumeration was found between CK and panCK + CSV, and between panCK and panCK + CSV. Upon stratification of CK+ samples, those of metastatic disease had a higher CTC number than those of localized disease. In the paired samples study involving cytokeratin-positive sarcoma subtypes, using panCK antibody detected more CTCs than CK. Similarly, for osteosarcoma, using panCK + CSV combination resulted in a higher CTC count than panCK. This study emphasized deliberate selection of cell surface proteins for sarcoma CTC detection and subtype stratification for studying cancers as heterogeneous as sarcomas.

Microfluidics-Enabled Isolation and Single-Cell Analysis of Circulating Tumor Cells.

Microfluidic platforms enable the enrichment and analysis of circulating tumor cells (CTCs), a potential biomarker for cancer diagnosis, prognosis, and theragnosis. Combined with immunocytochemistry/immunofluorescence (ICC/IF) assays for CTCs, microfluidics-enabled detection presents a unique opportunity to study tumor heterogeneity and predict treatment response, both of which can help cancer drug development. In this chapter, we detail the protocols and methods employed to fabricate and use a microfluidic device for the enrichment, detection, and analysis of single CTCs from the blood samples of sarcoma patients.

Gold Nanoparticle-Based Microfluidic Chips for Capture and Detection of Circulating Tumor Cells.

Liquid biopsy has progressed to its current use to diagnose and monitor cancer. Despite the recent advances in investigating cancer detection and diagnosis strategies, there is still a room for improvements in capturing CTCs. We developed an efficient CTC detection system by integrating gold nanoparticles with a microfluidic platform, which can achieve CTC capture within 120 min. Here, we report our development of a simple and effective way to isolate CTCs using antibodies attached on gold nanoparticles to the surface of a lateral filter array (LFA) microdevice. Our method was optimized using three pancreatic tumor cell lines, enabling the capture with high efficiency (90% ± 3.2%). The platform was further demonstrated for isolating CTCs from patients with metastatic pancreatic cancer. Our method and platform enables the production of functionalized, patterned surfaces that interact with tumor cells, enhancing the selective capture of CTCs for biological assays.

Lateral Filter Array Microfluidic Devices for Detecting Circulating Tumor Cells.

Circulating tumor cells (CTCs) are an important liquid biopsy biomarker for next-generation cancer diagnosis and prognosis. However, their clinical usage is hindered by the rarity of CTCs in patient's peripheral blood. Microfluidics has shown unique advantages in CTC isolation and detection. We have developed lateral filter array microfluidic (LFAM) devices for highly efficient CTC isolation. In this chapter, we describe in detail the design and fabrication of the LFAM devices and their applications for CTC enumeration from clinical blood samples.

Comparison of sample preparation methods for rare cell isolation in microfluidic devices.

The analysis of circulating tumor cells (CTCs) is important for cancer diagnosis and prognosis. Microfluidics has been employed for CTC analysis due to its scaling advantages and high performance. However, pre-analytical methods for CTC sample preparation are often combined with microfluidic platforms because a large sample volume is required to detect extremely rare CTCs. Among pre-analytical methods, Ficoll-Paque™, OncoQuick™, and RosetteSep™ are commonly used to separate cells of interest. To compare their performance, we spiked L3.6pl pancreatic cancer cells into healthy blood samples and then employed each technique to prepare blood samples, followed by using a microfluidic platform to capture and detect L3.6pl cells. We found these three methods have similar performance, though the slight edge of RosetteSep™ over Ficoll-Paque™ is statistically significant. We also studied the effects of the tumor cell concentrations on the performance of the frequently used Ficoll-Paque™ method. Furthermore, we examined the repeatability and variability of each pre-analytical technique and the microfluidics-enabled detection. This study will provide researchers and clinicians with comparative data that can influence the choice of sample preparation method, help estimate CTC loss in each pre-analytical method, and correlate the results of clinical studies that employ different techniques.

L'analyse des cellules tumorales circulantes (CTC) est une pierre angulaire du diagnostic et du pronostic du cancer. On recourt à la microfluidique pour l'analyse des CTC en raison des avantages qu'elle offre pour la mise à l'échelle et de sa grande performance. Par ailleurs, les méthodes préanalytiques pour la préparation d'échantillons de CTC font souvent appel à des plateformes microfluidiques, car il faut un grand volume d'échantillon pour détecter des CTC extrêmement rares. Parmi les méthodes préanalytiques couramment utilisées pour séparer les cellules sanguines d'intérêt, notons Ficoll-PaqueMC, OncoQuickMC et RosetteSepMC. Afin de comparer les performances de ces méthodes, nous avons additionné de cellules de cancer du pancreas L3,6pl des échantillons de sang sains, puis nous avons utilisé les trois méthodes pour préparer les échantillons sanguins, que nous avons ensuite soumis à une plateforme microfluidique pour isoler et détecter les cellules L3,6pl. Nos résultats montrent que les performances de ces trois méthodes sont similaires, bien que le léger avantage de RosetteSepMC par rapport à Ficoll-PaqueMC soit statistiquement significatif. Nous avons également étudié les effets des concentrations de cellules tumorales sur la performance de la méthode Ficoll-PaqueMC, qui est la plus fréquemment utilisée. En outre, nous avons examiné la répétabilité et la variabilité de chaque méthode préanalytique et les caractéristiques de détection que permet d'obtenir la microfluidique. Cette étude fournit aux chercheurs et aux cliniciens des données comparatives qui peuvent influencer leur choix de la méthode de préparation des échantillons, et permet d'estimer la perte de CTC propre à chaque méthode préanalytique et de corréler les résultats des études cliniques qui utilisent différentes techniques. [Traduit par la Rédaction].

Longitudinal Study of Circulating Biomarkers in Patients with Resectable Pancreatic Ductal Adenocarcinoma.

While patients with resectable pancreatic ductal adenocarcinoma (PDAC) show improved survival compared to their non-resectable counterparts, survival remains low owing to occult metastatic disease and treatment resistance. Liquid biopsy based on circulating tumor cells (CTCs) and cell-free DNA (cfDNA) has been shown to predict recurrence and treatment resistance in various types of cancers, but their utility has not been fully demonstrated in resectable PDAC. We have simultaneously tracked three circulating biomarkers, including CTCs, cfDNA, and circulating tumor DNA (ctDNA), over a period of cancer treatment using a microfluidic device and droplet digital PCR (ddPCR). The microfluidic device is based on the combination of filtration and immunoaffinity mechanisms. We have measured CTCs, cfDNA, and ctDNA in a cohort of seven resectable PDAC patients undergoing neoadjuvant therapy followed by surgery, and each patient was followed up to 10 time points over a period of 4 months. CTCs were detectable in all patients (100%) at some point during treatment but were detectable in only three out of six patients (50%) prior to the start of treatment. Median cfDNA concentrations remained comparable to negative controls throughout treatment. ddPCR was able to find KRAS mutations in six of seven patients (86%); however, these mutations were present in only two of seven patients (29%) prior to treatment. Overall, the majority of circulating biomarkers (81% for CTCs and 91% for cfDNA/ctDNA) were detected after the start of neoadjuvant therapy but before surgery. This study suggests that a longitudinal study of circulating biomarkers throughout treatment provides more useful information than those single time-point tests for resectable PDAC patients.

SARS-CoV-2 in residential rooms of two self-isolating persons with COVID-19.

Individuals with COVID-19 are advised to self-isolate at their residences unless they require hospitalization. Persons sharing a dwelling with someone who has COVID-19 have a substantial risk of being exposed to the virus. However, environmental monitoring for the detection of virus in such settings is limited. We present a pilot study on environmental sampling for SARS-CoV-2 virions in the residential rooms of two volunteers with COVID-19 who self-quarantined. Apart from standard surface swab sampling, based on availability, four air samplers positioned 0.3-2.2 m from the volunteers were used: a VIable Virus Aerosol Sampler (VIVAS), an inline air sampler that traps particles on polytetrafluoroethylene (PTFE) filters, a NIOSH 2-stage cyclone sampler (BC-251), and a Sioutas personal cascade impactor sampler (PCIS). The latter two selectively collect particles of specific size ranges. SARS-CoV-2 RNA was detected by real-time Reverse-Transcription quantitative Polymerase Chain Reaction (rRT-qPCR) analyses of particles in one air sample from the room of volunteer A and in various air and surface samples from that of volunteer B. The one positive sample collected by the NIOSH sampler from volunteer A's room had a quantitation cycle (Cq) of 38.21 for the N-gene, indicating a low amount of airborne virus [5.69E-02 SARS-CoV-2 genome equivalents (GE)/cm3 of air]. In contrast, air samples and surface samples collected off the mobile phone in volunteer B's room yielded Cq values ranging from 14.58 to 24.73 and 21.01 to 24.74, respectively, on the first day of sampling, indicating that this volunteer was actively shedding relatively high amounts of SARS-CoV-2 at that time. The SARS-CoV-2 GE/cm3 of air for the air samples collected by the PCIS was in the range 6.84E+04 to 3.04E+05 using the LED-N primer system, the highest being from the stage 4 filter, and similarly, ranged from 2.54E+03 to 1.68E+05 GE/cm3 in air collected by the NIOSH sampler. Attempts to isolate the virus in cell culture from the samples from volunteer B's room with the aforementioned Cq values were unsuccessful due to out-competition by a co-infecting Human adenovirus B3 (HAdVB3) that killed the Vero E6 cell cultures within 4 days of their inoculation, although Cq values of 34.56-37.32 were measured upon rRT-qPCR analyses of vRNA purified from the cell culture medium. The size distribution of SARS-CoV-2-laden aerosol particles collected from the air of volunteer B's room was >0.25 µm and >0.1 µm as recorded by the PCIS and the NIOSH sampler, respectively, suggesting a risk of aerosol transmission since these particles can remain suspended in air for an extended time and travel over long distances. The detection of virus in surface samples also underscores the potential for fomite transmission of SARS-CoV-2 in indoor settings.

Phase II Study of 5-Fluorouracil, Oxaliplatin plus Dasatinib (FOLFOX-D) in First-Line Metastatic Pancreatic Adenocarcinoma.

LESSONS LEARNED: Preclinical studies have demonstrated that Src inhibition through dasatinib synergistically enhances the antitumor effects of oxaliplatin. In this phase II, single-arm study, FOLFOX with dasatinib in previously untreated patients with mPC only showed only modest clinical activity, with a progressive-free survival of 4 months and overall survival of 10.6 months. Continued investigation is ongoing to better understand the role of Src inhibition with concurrent 5-fluorouracil and oxaliplatin in a subset of exceptional responders. BACKGROUND: Src tyrosine kinase activity is overexpressed in many human cancers, including metastatic pancreatic cancer (mPC). Dasatinib is a potent inhibitor of Src family of tyrosine kinases. This study was designed to investigate whether dasatinib can synergistically enhance antitumor effects of FOLFOX regimen (FOLFOX-D). METHODS: In this single-arm, phase II study, previously untreated patients received dasatinib 150 mg oral daily on days 1-14, oxaliplatin 85 mg/m2 intravenous (IV) on day 1 every 14 days, leucovorin (LV) 400 mg/m2 IV on day 1 every 14 days, 5-fluorouracil (5-FU) bolus 400 mg/m2 on day 1 every 14 days, and 5-FU continuous infusion 2,400 mg/m2 on day 1 every 14 days. Primary endpoint was progression-free survival (PFS) with preplanned comparison to historical controls. RESULTS: Forty-four patients enrolled with an estimated median PFS of 4.0 (95% confidence interval [CI], 2.3-8.5) months and overall survival (OS) of 10.6 (95% CI, 6.9-12.7) months. Overall response rate (ORR) was 22.7% (n = 10): one patient (2.3%) with complete response (CR) and nine patients (20.5%) with partial response (PR). Fifteen patients (34.1%) had stable disease (SD). Nausea was the most common adverse event (AE) seen in 35 patients (79.5%). CONCLUSION: The addition of dasatinib did not appear to add incremental clinical benefit to FOLFOX in untreated patients with mPC.

Using a combination of gangliosides and cell surface vimentin as surface biomarkers for isolating osteosarcoma cells in microfluidic devices.

BACKGROUND: Osteosarcoma (OS) is the most common primary bone tumor and the third leading cause of pediatric cancer deaths. Liquid biopsies are an alternative to current diagnostic imaging modalities that can be used to monitor treatment efficacy and the development of metastases. This study addresses the use of novel biomarkers to detect circulating osteosarcoma cells. PROCEDURES: Flow cytometry was used to evaluate the relative expression of epithelial cell adhesion molecule (EpCAM), ganglioside 2 and 3 (GD2/3), and cell surface vimentin (CSV) on a panel of OS cell lines. A microfluidic device was used to affirm the efficacy of GD2/3 and CSV to capture CTCs. Once captured, CTCs on the device are enumerated and the capture efficiency for each marker is measured. Patient samples were captured using the LFAM chip. RESULTS: We report the evaluation of GD2, GD3, and CSV as markers for OS cell capture in cell lines and in patient samples. The results of our capture studies correlate with our flow cytometry data and have shown a low capture efficiency of OS cells using EpCAM antibodies, while showing a moderate capture efficiency of OS cells using the GD2, GD3, and CSV antibodies independently. The combination of biomarkers demonstrate a high capture efficiency of approximately 80%. This is further supported by the detection of 1-1.5 CTCs per mL of blood using GD2 + CSV in OS patient samples. CONCLUSIONS: The combination of GD2 + CSV significantly increased the capture efficacy of OS cells. The detection of CTCs through routine blood sampling may be used clinically for earlier detection of metastases and monitoring the therapeutic effect of treatments in metastatic osteosarcomas.

Exosome isolation using nanostructures and microfluidic devices.

Exosomes contain cargoes of proteins, lipids, micro-ribonucleic acids, and functional messenger RNAs, and they play a key role in cell-to-cell communication and hold valuable information about biological processes such as disease pathology. To harvest their potentials in disease diagnostics, prognostics, and therapeutics, exosome isolation is a crucial first step in providing pure and intact samples for both research and clinical purposes. Unfortunately, conventional methods for exosome separation suffer from low purity, low capture efficiency, long processing time, large sample volume requirement, the need for dedicated equipment and trained personnel, and high cost. In the last decade, microfluidic devices, especially those that incorporate nanostructures, have emerged as superior alternatives for exosome isolation and detection. In this review, we examine microfluidic platforms, dividing them into six categories based on their capture mechanisms: passive-structure-based affinity, immunomagnetic-based affinity, filtration, acoustofluidics, electrokinetics, and optofluidics. Here, we start out exploring the research and clinical needs that translate into important performance parameters for new exosome isolation designs. Then, we briefly introduce the conventional methods and discuss how their failure to meet those performance standards sparks an intense interest in microfluidic device innovations. The essence of this review is to lead an in-depth discussion on not only the technicality of those microfluidic platforms, but also their strengths and weaknesses with regards to the performance parameters set forth. To close the conversation, we call for the inclusion of exosome confirmation and contamination evaluation as part of future device development and performance assessment process, so that collectively, efforts towards microfluidics and nanotechnology for exosome isolation and analysis may soon see the light of real-world applications.

Incorporation of lateral microfiltration with immunoaffinity for enhancing the capture efficiency of rare cells.

The methods for isolating rare cells such as circulating tumor cells (CTCs) can be generally classified into two categories: those based on physical properties (e.g., size) and methods based on biological properties (e.g., immunoaffinity). CellSearch, the only FDA-approved method for the CTC-based cancer prognosis, relies on immunoaffinity interactions between CTCs and antibodies immobilized on magnetic particles. Immunoaffinity-based CTC isolation has also been employed in microfluidic devices, which show higher capture efficiency than CellSearch. We report here our investigation of combining size-based microfiltration into a microfluidic device with immunoaffinity for enhanced capture efficiency of CTCs. The device consists of four serpentine main channels, and each channel contains an array of lateral filters that create a two-dimensional flow. The main flow is through the serpentine channel, allowing the majority of the sample to pass by while the secondary flow goes through the lateral filters. The device design is optimized to make all fluid particles interact with filters. The filter sizes range from 24 to 12 µm, being slightly larger than or having similar dimension of CTCs. These filters are immobilized with antibodies specific to CTCs and thus they function as gates, allowing normal blood cells to pass by while forcing the interactions between CTCs and antibodies on the filter surfaces. The hydrodynamic force experienced by a CTC was also studied for optimal experimental conditions to ensure immunoaffinity-enabled cell capture. The device was evaluated by capturing two types of tumor cells spiked in healthy blood or a buffer, and we found that their capture efficiency was between 87.2 and 93.5%. The platform was further validated by isolating CTCs from blood samples of patients with metastatic pancreatic cancer.

Engineering magnetic nanoparticles and their integration with microfluidics for cell isolation.

Isolation of cancer cells, bacteria, and viruses from peripheral blood has important applications in cancer diagnosis, therapy monitoring, and drug development. Magnetic particles functionalized with antibodies that target receptors of cancer cells have been shown to isolate such entities using magnetic field gradients. Here, we report enhancement in capture efficiency and specificity by engineering magnetic nanoparticles and integrating them with microfluidics for the enumeration of tumor cells. Nanoparticles were made from iron oxide, coated with poly(ethylene glycol), and conjugated through avidin-biotin chemistry with antibody specifically against epithelial cell adhesion molecule (EpCAM). On exposure of targeted nanoparticles to tumor cells, specific uptake by EpCAM-expressing tumor cells (e.g., BxPC3, a pancreatic cancer cell) was observed, whereas there was negligible uptake by cells with low EpCAM expression (e.g., CCRF-CEM, a leukemia cell). Using an arrangement of magnets called a Halbach array, capture efficiency and specificity towards BxPC3 cells tagged with magnetic nanoparticles were enhanced, compared to conditions without the magnetic field gradient and/or without magnetic nanoparticles, either in buffer or in whole blood. These results illustrate that engineered magnetic nanoparticles and their integration with microfluidics have great potential for tumor cell enumeration and cancer prognosis.

Integration of Lateral Filter Arrays with Immunoaffinity for Circulating-Tumor-Cell Isolation.

Circulating tumor cells (CTCs) are an important biomarker for cancer prognosis and treatment monitoring. However, the heterogeneity of the physical and biological properties of CTCs limits the efficiency of various approaches used to isolate small numbers of CTCs from billions of normal blood cells. To address this challenge, we developed a lateral filter array microfluidic (LFAM) device to integrate size-based separation with immunoaffinity-based CTC isolation. The LFAM device consists of a serpentine main channel, through which most of a sample passes, and an array of lateral filters for CTC isolation. The unique device design produces a two-dimensional flow, which reduces nonspecific, geometric capture of normal cells as typically observed in vertical filters. The LFAM device was further functionalized by immobilizing antibodies that are specific to the target cells. The resulting devices captured pancreatic cancer cells spiked in blood samples with (98.7±1.2) % efficiency and were used to isolate CTCs from patients with metastatic colorectal cancer.

Microfluidic Isolation of Circulating Tumor Cells and Cancer Stem-Like Cells from Patients with Pancreatic Ductal Adenocarcinoma.

Background: Pancreatic ductal adenocarcinoma (PDAC) requires multimodal therapeutic approaches and disease monitoring for effective treatment. Liquid biopsy biomarkers, including circulating tumor cells (CTCs) and cancer stem-like cells (CSCs), hold promise for evaluating treatment response promptly and guiding therapeutic modifications. Methods: From 24 patients with metastatic PDAC (stage IV, M1) undergoing active systemic treatment, we collected 78 blood samples at different time points for CTC and CSC isolation using a microfluidic platform functionalized with antibodies against a CTC biomarker, epithelial cell adhesion molecule (EpCAM), or a CSC biomarker, CD133. These isolated cells were further verified, via fluorescent staining and imaging, using cytokeratin (CK), CD45, and nucleic acid stain 4',6-diamidino-2-phenylindole (DAPI). Results: The majority (84.4%) of patient blood samples were positive for CTCs (EpCAM+CK+CD45-DAPI+) and 70.8% of patient blood samples were positive for CSCs (CD133+CK+CD45-DAPI+), using the highest baseline value of healthy samples as threshold. The CTC subtypes (EpCAM+CK+CD45-DAPI+CD133+ and EpCAM+CK+CD45-DAPI+CD133-) and CSC subtypes (CD133+CK+CD45-DAPI+EpCAM+ and CD133+CK+CD45-DAPI+EpCAM-) were also analyzed using immunochemical methods. In several cases, CSCs exhibited cytokeratin expression that did not express EpCAM, indicating that they will not be detected using EpCAM-based isolation. Conclusion: The microfluidic platform enabled the reliable isolation of CTCs and CSCs from PDAC patient samples, as well as their subtypes. Complementary assessment of both CTCs and CSCs appears advantageous to assess the profile of tumor progressing in some cases. This research has important implications for the application and interpretation of approved methods to detect CTCs.

A universal tumor cell isolation method enabled by fibrin-coated microchannels.

We report a simple but effective strategy to capture tumor cells using fibrin-immobilized microchannels. It is a universal method since it shows an ability to capture both epithelial and mesenchymal tumor cells. The cell capture efficiency is up to 90%.

An ensemble of aptamers and antibodies for multivalent capture of cancer cells.

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).

Cotinine concentration in serum correlates with tobacco smoke-induced emphysema in mice.

Secondhand smoke (SHS) has been associated with a variety of adverse health outcomes in nonsmokers, including emphysema (a chronic obstructive pulmonary disease). One way to detect SHS exposure is to measure the concentration of cotinine, the primary metabolite of nicotine, in bodily fluids. We have developed a method for cotinine analysis by combining micellar electrokinetic chromatography with enrichment techniques. We employed the method to measure cotinine concentrations in serum samples of mice exposed to tobacco smoke for 12 or 24 weeks and found that it was 3.1-fold or 4.8-fold higher than those exposed to room air for the same period. Further, we investigated the morphological changes in lungs of mice and observed tobacco smoke induced emphysema. Our results indicate that the method can be used to measure cotinine and there is an association between the serum cotinine concentration and tobacco smoke-induced emphysema in mice.

Capture, release and culture of circulating tumor cells from pancreatic cancer patients using an enhanced mixing chip.

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.