Tumor cell-based liquid biopsy using high-throughput microfluidic enrichment of entire leukapheresis product.

Circulating Tumor Cells (CTCs), interrogated by sampling blood from patients with cancer, contain multiple analytes, including intact RNA, high molecular weight DNA, proteins, and metabolic markers. However, the clinical utility of tumor cell-based liquid biopsy has been limited since CTCs are very rare, and current technologies cannot process the blood volumes required to isolate a sufficient number of tumor cells for in-depth assays. We previously described a high-throughput microfluidic prototype utilizing high-flow channels and amplification of cell sorting forces through magnetic lenses. Here, we apply this technology to analyze patient-derived leukapheresis products, interrogating a mean blood volume of 5.83 liters from patients with metastatic cancer, with a median of 2,799 CTCs purified per patient. Isolation of many CTCs from individual patients enables characterization of their morphological and molecular heterogeneity, including cell and nuclear size and RNA expression. It also allows robust detection of gene copy number variation, a definitive cancer marker with potential diagnostic applications. High-volume microfluidic enrichment of CTCs constitutes a new dimension in liquid biopsies.

A microfluidic transistor for automatic control of liquids.

Microfluidics have enabled notable advances in molecular biology1,2, synthetic chemistry3,4, diagnostics5,6 and tissue engineering7. However, there has long been a critical need in the field to manipulate fluids and suspended matter with the precision, modularity and scalability of electronic circuits8-10. Just as the electronic transistor enabled unprecedented advances in the automatic control of electricity on an electronic chip, a microfluidic analogue to the transistor could enable improvements in the automatic control of reagents, droplets and single cells on a microfluidic chip. Previous works on creating a microfluidic analogue to the electronic transistor11-13 did not replicate the transistor's saturation behaviour, and could not achieve proportional amplification14, which is fundamental to modern circuit design15. Here we exploit the fluidic phenomenon of flow limitation16 to develop a microfluidic element capable of proportional amplification with flow-pressure characteristics completely analogous to the current-voltage characteristics of the electronic transistor. We then use this microfluidic transistor to directly translate fundamental electronic circuits into the fluidic domain, including the amplifier, regulator, level shifter, logic gate and latch. We also combine these building blocks to create more complex fluidic controllers, such as timers and clocks. Finally, we demonstrate a particle dispenser circuit that senses single suspended particles, performs signal processing and accordingly controls the movement of each particle in a deterministic fashion without electronics. By leveraging the vast repertoire of electronic circuit design, microfluidic-transistor-based circuits enable fluidic automatic controllers to manipulate liquids and single suspended particles for lab-on-a-chip platforms.

A Microfluidic Transistor for Liquid Signal Processing.

Microfluidics have enabled significant advances in molecular biology 1-3 , synthetic chemistry 4,5 , diagnostics 6,7 , and tissue engineering 8 . However, there has long been a critical need in the field to manipulate fluids and suspended matter with the precision, modularity, and scalability of electronic circuits 9-11 . Just as the electronic transistor enabled unprecedented advances in the control of electricity on an electronic chip, a microfluidic analogue to the transistor could enable improvements in the complex, scalable control of reagents, droplets, and single cells on an autonomous microfluidic chip. Prior works on creating a microfluidic analogue to the electronic transistor 12-14 could not replicate the transistor's saturation behavior, which is crucial to perform analog amplification 15 and is fundamental to modern circuit design 16 . Here we exploit the fluidic phenomenon of flow-limitation 17 to develop a microfluidic element with flow-pressure characteristics completely analogous to the current-voltage characteristics of the electronic transistor. As this microfluidic transistor successfully replicates all of the key operating regimes of the electronic transistor (linear, cut-off and saturation), we are able to directly translate a variety of fundamental electronic circuit designs into the fluidic domain, including the amplifier, regulator, level shifter, logic gate, and latch. Finally, we demonstrate a "smart" particle dispenser that senses single suspended particles, performs liquid signal processing, and accordingly controls the movement of said particles in a purely fluidic system without electronics. By leveraging the vast repertoire of electronic circuit design, microfluidic transistor-based circuits are easy to integrate at scale, eliminate the need for external flow control, and enable uniquely complex liquid signal processing and single-particle manipulation for the next generation of chemical, biological, and clinical platforms.

Targeting breast and pancreatic cancer metastasis using a dual-cadherin antibody.

The successful application of antibody-based therapeutics in either primary or metastatic cancer depends upon the selection of rare cell surface epitopes that distinguish cancer cells from surrounding normal epithelial cells. By contrast, as circulating tumor cells (CTCs) transit through the bloodstream, they are surrounded by hematopoietic cells with dramatically distinct cell surface proteins, greatly expanding the number of targetable epitopes. Here, we show that an antibody (23C6) against cadherin proteins effectively suppresses blood-borne metastasis in mouse isogenic and xenograft models of triple negative breast and pancreatic cancers. The 23C6 antibody is remarkable in that it recognizes both the epithelial E-cadherin (CDH1) and mesenchymal OB-cadherin (CDH11), thus overcoming considerable heterogeneity across tumor cells. Despite its efficacy against single cells in circulation, the antibody does not suppress primary tumor formation, nor does it elicit detectable toxicity in normal epithelial organs, where cadherins may be engaged within intercellular junctions and hence inaccessible for antibody binding. Antibody-mediated suppression of metastasis is comparable in matched immunocompetent and immunodeficient mouse models. Together, these studies raise the possibility of antibody targeting CTCs within the vasculature, thereby suppressing blood-borne metastasis.

Isolation of circulating tumor cells.

Circulating tumor cells (CTCs) enter the vasculature from solid tumors and disseminate widely to initiate metastases. Mining the metastatic-enriched molecular signatures of CTCs before, during, and after treatment holds unique potential in personalized oncology. Their extreme rarity, however, requires isolation from large blood volumes at high yield and purity, yet they overlap leukocytes in size and other biophysical properties. Additionally, many CTCs lack EpCAM that underlies much of affinity-based capture, complicating their separation from blood. Here, we provide a comprehensive introduction of CTC isolation technology, by analyzing key separation modes and integrated isolation strategies. Attention is focused on recent progress in microfluidics, where an accelerating evolution is occurring in high-throughput sorting of cells along multiple dimensions.

Negative-Selection Enrichment of Circulating Tumor Cells from Peripheral Blood Using the Microfluidic CTC-iChip.

The ability to isolate and analyze rare circulating tumor cells (CTCs) holds the potential to increase our understanding of cancer evolution and allows monitoring of disease and therapeutic responses through a relatively non-invasive blood-based biopsy. While many methods have been described to isolate CTCs from the blood, the vast majority rely on size-based sorting or positive selection of CTCs based on surface markers, which introduces bias into the downstream product by making assumptions about these heterogenous cells. Here we describe a negative-selection protocol for enrichment of CTCs through removal of blood components including red blood cells, platelets, and white blood cells. This procedure results in a product that is amenable to downstream single-cell analytics including RNA-Seq, ATAC-Seq and DNA methylation, droplet digital PCR (ddPCR) for tumor specific transcripts, staining and extensive image analysis, and ex vivo culture of patient-derived CTCs.

Microfluidic capture of chromatin fibres measures neutrophil extracellular traps (NETs) released in a drop of human blood.

Neutrophils are the largest population of white blood cells in the circulation, and their primary function is to protect the body from microbes. They can release the chromatin in their nucleus, forming characteristic web structures and trap microbes, contributing to antimicrobial defenses. The chromatin webs are known as neutrophil extracellular traps (NETs). Importantly, neutrophils can also release NETs in pathological conditions related to rheumatic diseases, atherosclerosis, cancer, and sepsis. Thus, determining the concentration of NETs in the blood is increasingly important for monitoring patients, evaluating treatment efficacy, and understanding the pathology of various diseases. However, traditional methods for measuring NETs require separating cells and plasma from blood, are prone to sample preparation artifacts, and cannot distinguish between intact and degraded NETs. Here, we design a microfluidic analytical tool that captures NETs mechanically from a drop of blood and measures the amount of intact NETs unbiased by the presence of degraded NETs in the sample.

Ultrahigh-throughput magnetic sorting of large blood volumes for epitope-agnostic isolation of circulating tumor cells.

Circulating tumor cell (CTC)-based liquid biopsies provide unique opportunities for cancer diagnostics, treatment selection, and response monitoring, but even with advanced microfluidic technologies for rare cell detection the very low number of CTCs in standard 10-mL peripheral blood samples limits their clinical utility. Clinical leukapheresis can concentrate mononuclear cells from almost the entire blood volume, but such large numbers and concentrations of cells are incompatible with current rare cell enrichment technologies. Here, we describe an ultrahigh-throughput microfluidic chip, LPCTC-iChip, that rapidly sorts through an entire leukapheresis product of over 6 billion nucleated cells, increasing CTC isolation capacity by two orders of magnitude (86% recovery with 105 enrichment). Using soft iron-filled channels to act as magnetic microlenses, we intensify the field gradient within sorting channels. Increasing magnetic fields applied to inertially focused streams of cells effectively deplete massive numbers of magnetically labeled leukocytes within microfluidic channels. The negative depletion of antibody-tagged leukocytes enables isolation of potentially viable CTCs without bias for expression of specific tumor epitopes, making this platform applicable to all solid tumors. Thus, the initial enrichment by routine leukapheresis of mononuclear cells from very large blood volumes, followed by rapid flow, high-gradient magnetic sorting of untagged CTCs, provides a technology for noninvasive isolation of cancer cells in sufficient numbers for multiple clinical and experimental applications.

In-flow measurement of cell-cell adhesion using oscillatory inertial microfluidics.

Multicellular clusters in circulation can exhibit a substantially different function and biomarker significance compared to individual cells. Notably, clusters of circulating tumor cells (CTCs) are much more effective initiators of metastasis than single CTCs, and correlate with worse patient prognoses. Measuring the cell-cell adhesion strength of CTC clusters is a critical step towards understanding their subsistence in the circulation and mechanism of elevated tumorigenicity. However, measuring cell-cell adhesion forces in flow is elusive using existing methods. Here, we report an oscillatory inertial microfluidics system which exerts a repeating fluidic force profile on suspended cell doublets to determine their cell-cell adhesion strength (Fs), without any biophysical modifications to the cell surface and physiological morphology. Using our system, we analyzed a large number (N > 500) of doublets from a patient-derived breast cancer CTC line. We discovered that the cell-cell adhesion strength of CTC doublets varied almost 20-fold between the weakly adhered (Fs < 28 nN) and strongly bound subpopulations (Fs > 542 nN). Our system can be used with other cancer or noncancer cells without restrictions, and may be used for rapid screening of drugs aiming to disrupt the highly-metastatic CTC clusters in circulation.

Microfluidic concentration and separation of circulating tumor cell clusters from large blood volumes.

Circulating tumor cells (CTCs) are extremely rare in the blood, yet they account for metastasis. Notably, it was reported that CTC clusters (CTCCs) can be 50-100 times more metastatic than single CTCs, making them particularly salient as a liquid biopsy target. Yet they can split apart and are even rarer, complicating their recovery. Isolation by filtration risks loss when clusters squeeze through filter pores over time, and release of captured clusters can be difficult. Deterministic lateral displacement is continuous but requires channels not much larger than clusters, leading to clogging. Spiral inertial focusing requires large blood dilution factors (or lysis). Here, we report a microfluidic chip that continuously isolates untouched CTC clusters from large volumes of minimally (or undiluted) whole blood. An array of 100 µm-wide channels first concentrates clusters in the blood, and then a similar array transfers them into a small volume of buffer. The microscope-slide-sized PDMS device isolates individually-spiked CTC clusters from >30 mL per hour of whole blood with 80% efficiency into enumeration (fluorescence imaging), and on-chip yield approaches 100% (high speed video). Median blood cell removal (in base-10 logs) is 4.2 for leukocytes, 5.5 for red blood cells, and 4.9 for platelets, leaving less than 0.01% of leukocytes alongside CTC clusters in the product. We also demonstrate that cluster configurations are preserved. Gentle, high throughput concentration and separation of circulating tumor cell clusters from large blood volumes will enable cluster-specific diagnostics and speed the generation of patient-specific CTC cluster lines.

Oscillatory inertial focusing in infinite microchannels.

Inertial microfluidics (i.e., migration and focusing of particles in finite Reynolds number microchannel flows) is a passive, precise, and high-throughput method for microparticle manipulation and sorting. Therefore, it has been utilized in numerous biomedical applications including phenotypic cell screening, blood fractionation, and rare-cell isolation. Nonetheless, the applications of this technology have been limited to larger bioparticles such as blood cells, circulating tumor cells, and stem cells, because smaller particles require drastically longer channels for inertial focusing, which increases the pressure requirement and the footprint of the device to the extent that the system becomes unfeasible. Inertial manipulation of smaller bioparticles such as fungi, bacteria, viruses, and other pathogens or blood components such as platelets and exosomes is of significant interest. Here, we show that using oscillatory microfluidics, inertial focusing in practically "infinite channels" can be achieved, allowing for focusing of micron-scale (i.e. hundreds of nanometers) particles. This method enables manipulation of particles at extremely low particle Reynolds number (Rep < 0.005) flows that are otherwise unattainable by steady-flow inertial microfluidics (which has been limited to Rep > ∼10-1). Using this technique, we demonstrated that synthetic particles as small as 500 nm and a submicron bacterium, Staphylococcus aureus, can be inertially focused. Furthermore, we characterized the physics of inertial microfluidics in this newly enabled particle size and Rep range using a Peclet-like dimensionless number (α). We experimentally observed that α >> 1 is required to overcome diffusion and be able to inertially manipulate particles.

Anti-thrombotic strategies for microfluidic blood processing.

The redundant mechanisms involved in blood coagulation are crucial for rapid hemostasis. Yet they also create challenges in blood processing in medical devices and lab-on-a-chip systems. In this work, we investigate the effects of both shear stress and hypothermic blood storage on thrombus formation in microfluidic processing. For fresh blood, thrombosis occurs only at high shear, and the glycoprotein IIb/IIIa inhibitor tirofiban is highly effective in preventing thrombus formation. Blood storage generally activates platelets and primes them towards thrombosis via multiple mechanisms. Thrombus formation of stored blood at low shear can be adequately inhibited by glycoprotein IIb/IIIa inhibitors. At high shear, von Willebrand factor-mediated thrombosis contributes significantly and requires additional treatments with thiol-containing antioxidants-such as N acetylcysteine and reduced glutathione-that interfere with von Willebrand factor polymerization. We further demonstrate the effectiveness of these anti-thrombotic strategies in microfluidic devices made of cyclic olefin copolymer, a popular material used in the healthcare industry. This work identifies effective anti-thrombotic strategies that are applicable in a wide range of blood- and organ-on-a-chip applications.

Clusters of Circulating Tumor Cells: a Biophysical and Technological Perspective.

The vast majority of cancer associated deaths result from metastasis, yet the behaviors of its most potent cellular driver, circulating tumor cell clusters, are only beginning to be revealed. This review highlights recent advances to our understanding of tumor cell clusters with emphasis on enabling technologies. The importance of intercellular adhesions among cells in clusters have begun to be unraveled with the aid of promising microfluidic strategies for isolating clusters from patient blood. Due to their metastatic potency, the utility of circulating tumor cell clusters for cancer diagnosis, drug screening, precision oncology and as targets of antimetastatic therapeutics are being explored. The continued development of tools for exploring circulating tumor cell clusters will enhance our fundamental understanding of the metastatic process and may be instrumental in devising new strategies to suppress and eliminate metastasis.

A highly-occupied, single-cell trapping microarray for determination of cell membrane permeability.

Semi- and selective permeability is a fundamentally important characteristic of the cell membrane. Membrane permeability can be determined by monitoring the volumetric change of cells following exposure to a non-isotonic environment. For this purpose, several microfluidic perfusion chambers have been developed recently. However, these devices only allow the observation of one single cell or a group of cells that may interact with one another in an uncontrolled way. Some of these devices have integrated on-chip temperature control to investigate the temperature-dependence of membrane permeability, but they inevitably require sophisticated fabrication and assembly, and delicate temperature and pressure calibration. Therefore, it is highly desirable to design a simple single-cell trapping device that allows parallel monitoring of multiple separate, individual cells subjected to non-isotonic exposure at various temperatures. In this study, we developed a pumpless, single-layer microarray with high trap occupancy of single cells. The benchmark performance of the device was conducted by targeting spherical particles of 18.8 µm in diameter as a model, yielding trap occupancy of up to 86.8% with a row-to-row shift of 10-30 µm. It was also revealed that in each array the particles larger than a corresponding critical size would be excluded by the traps in a deterministic lateral displacement mode. Demonstrating the utility of this approach, we used the single-cell trapping device to determine the membrane permeability of rat hepatocytes and patient-derived circulating tumor cells (Brx-142) at 4, 22 and 37 °C. The membrane of rat hepatocytes was found to be highly permeable to water and small molecules such as DMSO and glycerol, via both lipid- and aquaporin-mediated pathways. Brx-142 cells, however, displayed lower membrane permeability than rat hepatocytes, which was associated with strong coupling of water and DMSO transport but less interaction between water and glycerol. The membrane permeability data reported here provide new insights into the biophysics of membrane transport such as aquaporin expression and coupling transport of water and solutes, as well as providing essential data for the ultimate goal of biobanking rare cells and precious tissues.

Non-equilibrium Inertial Separation Array for High-throughput, Large-volume Blood Fractionation.

Microfluidic blood processing is used in a range of applications from cancer therapeutics to infectious disease diagnostics. As these applications are being translated to clinical use, processing larger volumes of blood in shorter timescales with high-reliability and robustness is becoming a pressing need. In this work, we report a scaled, label-free cell separation mechanism called non-equilibrium inertial separation array (NISA). The NISA mechanism consists of an array of islands that exert a passive inertial lift force on proximate cells, thus enabling gentler manipulation of the cells without the need of physical contact. As the cells follow their size-based, deterministic path to their equilibrium positions, a preset fraction of the flow is siphoned to separate the smaller cells from the main flow. The NISA device was used to fractionate 400 mL of whole blood in less than 3 hours, and produce an ultrapure buffy coat (96.6% white blood cell yield, 0.0059% red blood cell carryover) by processing whole blood at 3 mL/min, or ∼300 million cells/second. This device presents a feasible alternative for fractionating blood for transfusion, cellular therapy and blood-based diagnostics, and could significantly improve the sensitivity of rare cell isolation devices by increasing the processed whole blood volume.

Monolithic Chip for High-throughput Blood Cell Depletion to Sort Rare Circulating Tumor Cells.

Circulating tumor cells (CTCs) are a treasure trove of information regarding the location, type and stage of cancer and are being pursued as both a diagnostic target and a means of guiding personalized treatment. Most isolation technologies utilize properties of the CTCs themselves such as surface antigens (e.g., epithelial cell adhesion molecule or EpCAM) or size to separate them from blood cell populations. We present an automated monolithic chip with 128 multiplexed deterministic lateral displacement devices containing ~1.5 million microfabricated features (12 µm-50 µm) used to first deplete red blood cells and platelets. The outputs from these devices are serially integrated with an inertial focusing system to line up all nucleated cells for multi-stage magnetophoresis to remove magnetically-labeled white blood cells. The monolithic CTC-iChip enables debulking of blood samples at 15-20 million cells per second while yielding an output of highly purified CTCs. We quantified the size and EpCAM expression of over 2,500 CTCs from 38 patient samples obtained from breast, prostate, lung cancers, and melanoma. The results show significant heterogeneity between and within single patients. Unbiased, rapid, and automated isolation of CTCs using monolithic CTC-iChip will enable the detailed measurement of their physicochemical and biological properties and their role in metastasis.

Microfluidic Isolation of Circulating Tumor Cell Clusters by Size and Asymmetry.

Circulating tumor cell clusters (CTC clusters) are potent initiators of metastasis and potentially useful clinical markers for patients with cancer. Although there are numerous devices developed to isolate individual circulating tumor cells from blood, these devices are ineffective at capturing CTC clusters, incapable of separating clusters from single cells and/or cause cluster damage or dissociation during processing. The only device currently able to specifically isolate CTC clusters from single CTCs and blood cells relies on the batch immobilization of clusters onto micropillars which necessitates long residence times and causes damage to clusters during release. Here, we present a two-stage continuous microfluidic chip that isolates and recovers viable CTC clusters from blood. This approach uses deterministic lateral displacement to sort clusters by capitalizing on two geometric properties: size and asymmetry. Cultured breast cancer CTC clusters containing between 2-100 + cells were recovered from whole blood using this integrated two-stage device with minimal cluster dissociation, 99% recovery of large clusters, cell viabilities over 87% and greater than five-log depletion of red blood cells. This continuous-flow cluster chip will enable further studies examining CTC clusters in research and clinical applications.

Bacterial Ice Nucleation in Monodisperse D2O and H2O-in-Oil Emulsions.

Ice nucleation is of fundamental significance in many areas, including atmospheric science, food technology, and cryobiology. In this study, we investigated the ice-nucleation characteristics of picoliter-sized drops consisting of different D2O and H2O mixtures with and without the ice-nucleating bacteria Pseudomonas syringae. We also studied the effects of commonly used cryoprotectants such as ethylene glycol, propylene glycol, and trehalose on the nucleation characteristics of D2O and H2O mixtures. The results show that the median freezing temperature of the suspension containing 1 mg/mL of a lyophilized preparation of P. syringae is as high as -4.6 °C for 100% D2O, compared to -8.9 °C for 100% H2O. As the D2O concentration increases every 25% (v/v), the profile of the ice-nucleation kinetics of D2O + H2O mixtures containing 1 mg/mL Snomax shifts by about 1 °C, suggesting an ideal mixing behavior of D2O and H2O. Furthermore, all of the cryoprotectants investigated in this study are found to depress the freezing phenomenon. Both the homogeneous and heterogeneous freezing temperatures of these aqueous solutions depend on the water activity and are independent of the nature of the solute. These findings enrich our fundamental knowledge of D2O-related ice nucleation and suggest that the combination of D2O and ice-nucleating agents could be a potential self-ice-nucleating formulation. The implications of self-nucleation include a higher, precisely controlled ice seeding temperature for slow freezing that would significantly improve the viability of many ice-assisted cryopreservation protocols.

Retina-on-a-chip: a microfluidic platform for point access signaling studies.

We report on a microfluidic platform for culture of whole organs or tissue slices with the capability of point access reagent delivery to probe the transport of signaling events. Whole mice retina were maintained for multiple days with negative pressure applied to tightly but gently bind the bottom of the retina to a thin poly-(dimethylsiloxane) membrane, through which twelve 100 µm diameter through-holes served as fluidic access points. Staining with toluidine blue, transport of locally applied cholera toxin beta, and transient response to lipopolysaccharide in the retina demonstrated the capability of the microfluidic platform. The point access fluidic delivery capability could enable new assays in the study of various kinds of excised tissues, including retina.

Inertio-elastic focusing of bioparticles in microchannels at high throughput.

Controlled manipulation of particles from very large volumes of fluid at high throughput is critical for many biomedical, environmental and industrial applications. One promising approach is to use microfluidic technologies that rely on fluid inertia or elasticity to drive lateral migration of particles to stable equilibrium positions in a microchannel. Here, we report on a hydrodynamic approach that enables deterministic focusing of beads, mammalian cells and anisotropic hydrogel particles in a microchannel at extremely high flow rates. We show that on addition of micromolar concentrations of hyaluronic acid, the resulting fluid viscoelasticity can be used to control the focal position of particles at Reynolds numbers up to Re≈10,000 with corresponding flow rates and particle velocities up to 50 ml min(-1) and 130 m s(-1). This study explores a previously unattained regime of inertio-elastic fluid flow and demonstrates bioparticle focusing at flow rates that are the highest yet achieved.