Evaluating the biological effectiveness of boron neutron capture therapy by using microfluidics-based pancreatic tumor spheroids.

Background: The recent success of boron neutron capture therapy (BNCT) for cancer treatment has attracted considerable attention. Because irradiated neutrons penetrate deep into solid tumor tissue, BNCT efficacy is strongly influenced by cell pathophysiology in tumors. The tumor microenvironment critically influences tumor pathophysiology, but its effects on BNCT remain unexplored. Methods: We used a pancreatic tumor as a model to develop a high-throughput 3D tumor spheroid platform for evaluating BNCT efficacy under different microenvironment conditions. We expanded our system to serve as a transwell-like device in order to investigate the influence of stromal fibroblasts in the tumor microenvironment. Results: With the use of the proposed microfluidic chip and a laboratory pipette, more than 40 spheroids with controllable diameters (standard deviation <10%) could be cultured on a chip for more than 10 days. The response to BNCT from each spheroid can be monitored in real time. By using pancreatic tumor spheroids of two different diameters, we found that large spheroids, characterized by more hypoxic microenvironments, exhibited lower BNCT susceptibility. The cells in the hypoxic region expressed the HIF1-α signal, which is crucial in many therapeutic resistance signal pathways. In addition, the heterogeneous presence of stemness markers (Oct-4, Sox-2, and CD 44) implied that the underlying BNCT resistance mechanism was sophisticated. In the presence of fibroblasts, we found an association between ß-catenin nuclear translocation and BNCT resistance; membrane contacts from fibroblasts were found to be indispensable for translocation activation. Conclusions: In summary, by means of easily accessible microfluidic engineering, we developed tumor spheroids to recapture the pathophysiological characteristics of pancreatic tumors. Our data suggest that hypoxia and fibrosis can reduce BNCT efficacy in pancreatic cancer treatment. Considering the growing requirement for drug screening in personalized medicine, our findings and the developed method are expected to improve the fundamental understanding of BNCT and facilitate broad applications of BNCT in clinical settings.

A Portable Controllable Compressive Stress Device to Monitor Human Breast Cancer Cell Protrusions at Single-Cell Resolution.

In vitro devices offer more numerous methods than in vivo models to investigate how cells respond to pressure stress and quantify those responses. Several in vitro devices have been developed to study the cell response to compression force. However, they are unable to observe morphological changes of cells in real-time. There is also a concern about cell damage during the process of harvesting cells from 3D gels. Here we report a device employing transparent, thin gel layers to clamp cells between the interfaces and applied a controllable compression force by stacking multiple layers on the top. In this approach, cells can be monitored for alteration of cellular protrusions, whose diversity has been proven to promote cancer cell dissemination, with single-cell resolution under compression force. Furthermore, p-Rac-1 and rhodamine staining on the device directly to confirm the actin filaments of lamellipodia. The method was able to fulfill real-time live-cell observation at single-cell resolution and can be readily used for versatile cell analysis. MDA-MB-231 and MCF7 breast cancer cells were utilized to demonstrate the utility of the device, and the results showed that the stimuli of compression force induce MDA-MB-231 and MCF7 to form lamellipodia and bleb protrusions, respectively. We envision the device may be used as a tool to explore mechanisms of membrane protrusion transitions and to screen drug candidates for inhibiting cancer cell protrusion plasticity for cancer therapy.

A microfluidic hanging drop-based spheroid co-culture platform for probing tumor angiogenesis.

Co-culturing of embryoid bodies (EBs) and tumor spheroids (TSs) allows mimicking tumor angiogenesis in vitro. Here, we report a microfluidic hanging drop-based spheroid co-culture device (µ-CCD) that permits the generation and co-culturing of EBs and TSs using a simple manual operation procedure and setup. In brief, uniform-sized EBs and TSs can be generated on the device in eight pairs of hanging droplets from adjacent microfluidic channels, followed by the confrontation of EB and TS pairs by merging the droplet pairs to culture the EB-TS spheroids to investigate tumor-induced angiogenic sprouting. The physical parameters of the device were optimized to maintain the long-term stability of hanging droplets for up to ten days. The mouse embryonic stem cell line ES-D3 and breast cancer cell lines MDA-MB-231 and MCF-7 were used to generate EBs, invasive TSs, and non-invasive TSs respectively. Confocal imaging results showed that the vessel percentage area and total vessel length which are linked to tumor angiogenesis increased after 6 days of co-culturing. An anti-angiogenesis drug testing on the co-cultured EB-TS spheroids was also demonstrated in the device. The µ-CCD provides a simple yet high-efficiency method to generate and co-culture cell spheroids and may also be useful for other applications involving spheroid co-culturing.

The preparation of cell-containing microbubble scaffolds to mimic alveoli structure as a 3D drug-screening system for lung cancer.

Cancer is the leading cause of mortality worldwide, and lung cancer is the most malignant. However, the high failure rate in oncology drug development from in vitro studies to in vivo preclinical models indicates that the modern methods of evaluating drug efficacies in vitro are not reliable. Traditional 2D cell culture has proved inadequate to mimic real physiological conditions. Current 3D cell culture methods do not represent the delicate structure of lung alveoli. To mimic lung alveoli structure, a cell-containing enzyme-crosslinked gelatin microbubble scaffold was produced by mixing surfactant-containing gelatin solution with microbial transglutaminase (mTGase)-mixed A549 cell suspension in a four-channel flow-focusing microfluidic device. With uniform pore size of about 100 µm in diameter, this gelatin microbubble scaffold resembled the lung alveoli in structure and in mechanical properties with good biocompatibility. Effective gemcitabine concentration required to induce cell death in microbubble scaffolds was significantly higher than in 2D culture together with a longer treatment time. Cell death mechanisms were confirmed to be gemcitabine-induced cell apoptosis through Western blotting and real-time polymerase chain reaction. H&E staining and TUNEL assay showed rounded cells with DNA damage in drug-treated scaffolds. Taken together, the cell-containing microbubble scaffolds successfully mimicked lung alveoli in structure and cellular responses after gemcitabine treatment were similar to clinical regimen of treating lung carcinoma. The microbubble scaffold is promising to facilitate anticancer drug discovery by providing more accurate preclinical predictions.

Rabies virus glycoprotein-amplified hierarchical targeted hybrids capable of magneto-electric penetration delivery to orthotopic brain tumor.

Compact nanohybrids can potentially unite various therapeutic features and reduce side effects for precise cancer therapy. However, the poor accumulation and limited tumor penetration of drugs at the tumor impede the manifestation of nanomedicine. We developed a rabies virus glycoprotein (RVG)-amplified hierarchical targeted hybrid that acts as a stealthy and magnetolytic carrier that transports dual tumor-penetrating agents incorporating two drugs (boron-doped graphene quantum dots (B-GQDs)/doxorubicin and pH-responsive dendrimers (pH-Den)/palbociclib). The developed RVG-decorated hybrids (RVG-hybrids) enhance the accumulation of drugs at tumor by partially bypassing the BBB via spinal cord transportation and pH-induced aggregation of hierarchical targeting. The penetrated delivery of dual pH-Den and B-GQD drugs to deep tumors is actuated by magnetoelectric effect, which are able to generate electrons to achieve electrostatic repulsion and disassemble the hybrids into components of a few nanometers in size. The synergy of magnetoelectric drug penetration and chemotherapy was achieved by delivery of the B-GQDs and pH-Den to orthotopic tumors, which prolonged the host survival time. This RVG-amplified dual hierarchical delivery integrated with controlled and penetrated release from this hybrid improve the distribution of the therapeutic agents at the brain tumor for synergistic therapy, exhibiting potential for clinic use.

Establishing Single-Cell Based Co-Cultures in a Deterministic Manner with a Microfluidic Chip.

Cell co-culture assays have been widely used for studying cell-cell interactions between different cell types to better understand the biology of diseases including cancer. However, it is challenging to clarify the complex mechanism of intercellular interactions in highly heterogeneous cell populations using conventional co-culture systems because the heterogeneity of the cell subpopulation is obscured by the average values; the conventional co-culture systems can only be used to describe the population signal, but are incapable of tracking individual cells behavior. Furthermore, conventional single-cell experimental methods have low efficiency in cell manipulation because of the Poisson distribution. Microfabricated devices are an emerging technology for single-cell studies because they can accurately manipulate single cells at high-throughput and can reduce sample and reagent consumption. Here, we describe the concept and application of a microfluidic chip for multiple single-cell co-cultures. The chip can efficiently capture multiple types of single cells in a culture chamber (~46%) and has a sufficient culture space useful to study the cells' behavior (e.g., migration, proliferation, etc.) under cell-cell interaction at the single-cell level. Lymphatic endothelial cells and oral squamous cell carcinoma were used to perform a single-cell co-culture experiment on the microfluidic platform for live multiple single-cell interaction studies.

Multilayer architecture microfluidic network array for combinatorial drug testing on 3D-cultured cells.

In vitro testing of drug compounds on cell models during the drug development process represents an indispensable step in the initial screening process. Although drug testing on three-dimensional (3D) cultured cells may provide a more accurate prediction of drug efficacy, it is relatively costly and time-consuming to perform compared with conventional 2D cultures due to the thick z-axis of the 3D models. In this study, we have presented a microfluidic platform with integrated pneumatic valves for producing a thin-gel 3D cell culture-based combinatorial drug screening array (3D-µCDS array). The multilayer architecture and microfluidic layout has a smaller device footprint than a single-layer microfluidic channel arrangement, making it well suited to scaling up for high-throughput combinatorial drug screening on 3D cell model. We performed 8 × 8 combination drug screening experiments with the device using two anti-cancer drugs (doxorubicin and paclitaxel) on MDA-MB-231 and MCF-7 breast cancer cell lines for demonstration. Our results indicate that our 3D-µCDS array device allows the successful screening of multiple drug combinations while reducing the operation time and the number of sample/reagents required, making it an ideal tool for general combinatorial drug screening, as well as for applications using valuable tissues and clinical samples.

Hydrodynamic shuttling for deterministic high-efficiency multiple single-cell capture in a microfluidic chip.

Studies on cellular heterogeneity have emerged as a powerful approach for developing new strategies to treat diseases including cancer. However, it is difficult to set up an in vitro co-culture experiment to study the interaction of individual live cells. In this paper, we report a hydrodynamic shuttling chip (HSC) which can deterministically capture single cells into microfluidic chambers to set up multiple single-cell co-culture experiments in which individual live cells can be microscopically observed. Using this chip device, we demonstrated a triple single-cell culture of oral squamous cell carcinoma and lymphatic endothelial cells to observe the effect of cell-cell interaction on the cell motility. Triple, single-cell pairing efficiency by our HSC device was eightfold higher than that of the probabilistic method. Using this HSC device, we were able to perform triple-culture experiments to show the cell type-dependent motility of oral squamous cell carcinoma and lymphatic endothelial cells, which was not observed in co-culture experiments.

The effects of aromatherapy massage on improvement of anxiety among patients receiving palliative care: A systematic review of randomized controlled trials.

BACKGROUND: Anxiety in patients receiving palliative care is a noteworthy concern because it may affect their quality of life. Aromatherapy has been widely utilized to improve anxiety among patients receiving palliative care. OBJECTIVE: To investigate the effectiveness of anxiety improvement in patients receiving palliative care by comparing the intervention group (aromatherapy massage) with the control group (common massage alone). METHODS: A literature search was performed using PubMed, Cochrane Library, Embase, MEDLINE, and CINAHL for all related studies from inception through November 30, 2018 without restriction on language. A quantitative synthesis of randomized controlled trials (RCTs) was conducted to compare the difference in effectiveness scores between the aromatherapy massage and only common massage groups by employing a random-effect model. RESULTS: We included three RCTs with a total of 160 participants (81 in the intervention group and 79 in the control group) in our systematic review and conducted a quantitative synthesis. The secondary data from the reviewed trials were then pooled using a random-effect model. Anxiety (mean difference = -2.60 [95% confidence interval: -7.82, 2.63], P = .33) was assessed using anxiety scores from the State-Trait Anxiety Inventory. CONCLUSION: Compared with common massage alone, aromatherapy massage does not provide significant effectiveness of anxiety improvement among patients receiving palliative care.

Protective effects of aucubin on osteoarthritic chondrocyte model induced by hydrogen peroxide and mechanical stimulus.

BACKGROUND: During the onset of osteoarthritis (OA), certain biochemical events have been shown to accelerate cartilage degradation, including the dysregulation of cartilage ECM anabolism, abnormal generation of reactive oxygen species (ROS) and overproduction of proteolytic enzymes and inflammatory cytokines. The potency of aucubin in protecting cellular components against oxidative stress, inflammation and apoptosis effects are well documented, which makes it a potential candidate for OA treatment. In this study, we aimed to evaluate the protective benefits of aucubin against OA using H2O2 and compression induced OA-like chondrocyte models. METHODS: The effects of aucubin were studied in porcine chondrocytes after 1 mM H2O2 stimulation for 30 min or sustained compression for 24 h. Effects of aucubin on cell proliferation and cytotoxicity of chondrocytes were measured with WST-1 and LDH assays. ROS production was evaluated by the Total ROS/Superoxide Detection Kit. Caspase-3 activity was evaluated by the CaspACE assay system. The levels of apoptosis were evaluated by the Annexin V-FITC apoptosis detection kit. OA-related gene expression was measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Total DNA quantification was evaluated by the DNeasy Blood and Tissue kit. Sulfated-glycosaminoglycans (sGAGs) production and content were evaluated by DMMB assay and Alcian blue staining. RESULTS: The results showed that the ROS scavenge effects of aucubin appeared after 1 h of pretreatment. Aucubin could reduce the caspase-3 activity induced by H2O2, and reduced the apoptosis cell population in flowcytometry. In RT-qPCR results, aucubin could maintain ACAN and COL2A1 gene expressions, and prevent IL6 and MMP13 gene up-regulation induced by H2O2 and compression stimulations. In the DMMB assay and Alcian blue staining, aucubin could maintain the sGAG content and protect chondrocytes against compressive stress, but not oxidative stress from H2O2. CONCLUSIONS: These results indicated that aucubin has protective effects in an osteoarthritic chondrocyte model induced by H2O2 and mechanical stimulus.

C-phycocyanin alleviates osteoarthritic injury in chondrocytes stimulated with H2O2 and compressive stress.

During the progression of osteoarthritis (OA), dysregulation of extracellular matrix anabolism, abnormal generation of reactive oxygen species (ROS) and inflammatory cytokines have been shown to accelerate the degradation process of cartilage. The potency of c-phycocyanin (C-PC) to protect cellular components against oxidative stress, along with its anti-inflammation and anti-apoptosis effects, are well documented; however, effects of C-PC on OA are still unclear. In this study, we aimed to investigate the effects of C-PC on OA using H2O2 or compression-stimulated OA-like porcine chondrocyte models. The results showed that C-PC had the ability to inhibit ROS production, reverse caspase-3 activity, and reduce apoptosis cell population. C-PC also reversed aggrecan and type II collagen gene expressions after stimulation with 1mM H2O2 or 60psi of compression. Inhibition of IL-6 and MMP-13 genes was observed in compression-stimulated chondrocytes but not in H2O2-treated cells. In dimethylmethylene blue assay and alcian blue staining, C-PC maintained the sulfated-glycosaminoglycan (sGAG) content after stimulation with compression. We concluded that C-PC can prevent early signs of OA caused by compressive stress and attenuate H2O2-induced oxidative stress. Therefore, we suggest that C-PC can be used as a potential drug candidate for chronic OA treatment.

Continuous microfluidic assortment of interactive ligands (CMAIL).

Finding an interactive ligand-receptor pair is crucial to many applications, including the development of monoclonal antibodies. Biopanning, a commonly used technique for affinity screening, involves a series of washing steps and is lengthy and tedious. Here we present an approach termed continuous microfluidic assortment of interactive ligands, or CMAIL, for the screening and sorting of antigen-binding single-chain variable antibody fragments (scFv) displayed on bacteriophages (phages). Phages carrying native negative charges on their coat proteins were electrophoresed through a hydrogel matrix functionalized with target antigens under two alternating orthogonal electric fields. During the weak horizontal electric field phase, phages were differentially swept laterally depending on their affinity for the antigen, and all phages were electrophoresed down to be collected during the strong vertical electric field phase. Phages of different affinity were spatially separated, allowing the continuous operation. More than 10(5) CFU (colony forming unit) antigen-interacting phages were isolated with ~100% specificity from a phage library containing 3 × 10(9) individual members within 40 minutes of sorting using CMAIL. CMAIL is rapid, sensitive, specific, and does not employ washing, elution or magnetic beads. In conclusion, we have developed an efficient and cost-effective method for isolating and sorting affinity reagents involving phage display.

Laminin modification subretinal bio-scaffold remodels retinal pigment epithelium-driven microenvironment in vitro and in vivo.

Advanced age-related macular degeneration (AMD) may lead to geographic atrophy or fibrovascular scar at macular, dysfunctional retinal microenvironment, and cause profound visual loss. Recent clinical trials have implied the potential application of pluripotent cell-differentiated retinal pigment epithelial cells (dRPEs) and membranous scaffolds implantation in repairing the degenerated retina in AMD. However, the efficacy of implanted membrane in immobilization and supporting the viability and functions of dRPEs, as well as maintaining the retinal microenvironment is still unclear. Herein we generated a biomimetic scaffold mimicking subretinal Bruch's basement from plasma modified polydimethylsiloxane (PDMS) sheet with laminin coating (PDMS-PmL), and investigated its potential functions to provide a subretinal environment for dRPE-monolayer grown on it. Firstly, compared to non-modified PDMS, PDMS-PmL enhanced the attachment, proliferation, polarization, and maturation of dRPEs. Second, PDMS-PmL increased the polarized tight junction, PEDF secretion, melanosome pigment deposit, and phagocytotic-ability of dRPEs. Third, PDMS-PmL was able to carry a dRPEs/photoreceptor-precursors multilayer retina tissue. Finally, the in vivo subretinal implantation of PDMS-PmL in porcine eyes showed well-biocompatibility up to 2-year follow-up. Notably, multifocal ERGs at 2-year follow-up revealed well preservation of macular function in PDMS-PmL, but not PDMS, transplanted porcine eyes. Trophic PEDF secretion of macular retina in PDMS-PmL group was also maintained to preserve retinal microenvironment in PDMS-PmL eyes at 2 year. Taken together, these data indicated that PDMS-PmL is able to sustain the physiological morphology and functions of polarized RPE monolayer, suggesting its potential of rescuing macular degeneration in vivo.

A High-Throughput Automated Microfluidic Platform for Calcium Imaging of Taste Sensing.

The human enteroendocrine L cell line NCI-H716, expressing taste receptors and taste signaling elements, constitutes a unique model for the studies of cellular responses to glucose, appetite regulation, gastrointestinal motility, and insulin secretion. Targeting these gut taste receptors may provide novel treatments for diabetes and obesity. However, NCI-H716 cells are cultured in suspension and tend to form multicellular aggregates, preventing high-throughput calcium imaging due to interferences caused by laborious immobilization and stimulus delivery procedures. Here, we have developed an automated microfluidic platform that is capable of trapping more than 500 single cells into microwells with a loading efficiency of 77% within two minutes, delivering multiple chemical stimuli and performing calcium imaging with enhanced spatial and temporal resolutions when compared to bath perfusion systems. Results revealed the presence of heterogeneity in cellular responses to the type, concentration, and order of applied sweet and bitter stimuli. Sucralose and denatonium benzoate elicited robust increases in the intracellular Ca(2+) concentration. However, glucose evoked a rapid elevation of intracellular Ca(2+) followed by reduced responses to subsequent glucose stimulation. Using Gymnema sylvestre as a blocking agent for the sweet taste receptor confirmed that different taste receptors were utilized for sweet and bitter tastes. This automated microfluidic platform is cost-effective, easy to fabricate and operate, and may be generally applicable for high-throughput and high-content single-cell analysis and drug screening.

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Towards an Endpoint Cell Motility Assay by a Microfluidic Platform.

In vitro cell motility assays are frequently used in the study of cell migration in response to anti-cancer drug treatment. Microfluidic systems represent a unique tool for the in vitro analysis of cell motility. However, they usually rely on using time-lapse microscopy to record the spatial temporal locations of the individual cells being tested. This has created a bottleneck for microfluidic systems to perform high-throughput experiments due to requirement of a costly time-lapse microscopy system. Here, we describe the development of a portable microfluidic device for endpoint analysis of cell motility. The reported device incorporates a cell alignment feature to position the seeded cells on the same initial location, so that the cells' motilities can be analyzed based on their locations at the end of the experiment after the cells have migrated. We show that the device was able to assess cancer cell motility after treatment with a migration inhibitory drug Indole-3-carbinol on MDA-MB-231 breast cancer cells, demonstrating the applicability of our device in screening anti-cancer drug compounds on cancer cells.

A microfluidic dual-well device for high-throughput single-cell capture and culture.

In vitro culture of single cells facilitates biological studies by deconvoluting complications from cell population heterogeneity. However, there is still a lack of simple yet high-throughput methods to perform single cell culture experiments. In this paper, we report the development and application of a microfluidic device with a dual-well (DW) design concept for high-yield single-cell loading (~77%) in large microwells (285 and 485 µm in diameter) which allowed for cell spreading, proliferation and differentiation. The increased single-cell loading yield is achieved by using sets of small microwells termed "capture-wells" and big microwells termed "culture-wells" according to their utilities for single-cell capture and culture, respectively. This novel device architecture allows the size of the "culture" microwells to be flexibly adjusted without affecting the single-cell loading efficiency making it useful for cell culture applications as demonstrated by our experiments of KT98 mouse neural stem cell differentiation, A549 and MDA-MB-435 cancer cell proliferation, and single-cell colony formation assay with A549 cells in this paper.

AlGaN/GaN high electron mobility transistors for protein-peptide binding affinity study.

Antibody-immobilized AlGaN/GaN high electron mobility transistors (HEMTs) were used to detect a short peptide consisting of 20 amino acids. One-binding-site model and two-binding-site model were used for the analysis of the electrical signals, revealing the number of binding sites on an antibody and the dissociation constants between the antibody and the short peptide. In the binding-site models, the surface coverage ratio of the short peptide on the sensor surface is relevant to the electrical signals resulted from the peptide-antibody binding on the HEMTs. Two binding sites on an antibody were observed and two dissociation constants, 4.404×10(-11) M and 1.596×10(-9) M, were extracted from the binding-site model through the analysis of the surface coverage ratio of the short peptide on the sensor surface. We have also shown that the conventional method to extract the dissociation constant from the linear regression of curve-fitting with Langmuir isotherm equation may lead to an incorrect information if the receptor has more than one binding site for the ligand. The limit of detection (LOD) of the sensor observed in the experimental result (~10 pM of the short peptide) is very close to the LOD (around 2.7-3.4 pM) predicted from the value of the smallest dissociation constants. The sensitivity of the sensor is not only dependent on the transistors, but also highly relies on the affinity of the ligand-receptor pair. The results demonstrate that the AlGaN/GaN HEMTs cannot only be used for biosensors, but also for the biological affinity study.

A fluorescent reporter of AMPK activity and cellular energy stress.

AMP-activated protein kinase (AMPK) is activated when the AMP/ATP ratio in cells is elevated due to energy stress. Here, we describe a biosensor, AMPKAR, that exhibits enhanced fluorescence resonance energy transfer (FRET) in response to phosphorylation by AMPK, allowing spatiotemporal monitoring of AMPK activity in single cells. We show that this reporter responds to a variety of stimuli that are known to induce energy stress and that the response is dependent on AMPK α1 and α2 and on the upstream kinase LKB1. Interestingly, we found that AMPK activation is confined to the cytosol in response to energy stress but can be observed in both the cytosol and nucleus in response to calcium elevation. Finally, using this probe with U2OS cells in a microfluidic device, we observed a very high cell-to-cell variability in the amplitude and time course of AMPK activation and recovery in response to pulses of glucose deprivation.

Isolation of circulating tumor cells using a microvortex-generating herringbone-chip.

Rare circulating tumor cells (CTCs) present in the bloodstream of patients with cancer provide a potentially accessible source for detection, characterization, and monitoring of nonhematological cancers. We previously demonstrated the effectiveness of a microfluidic device, the CTC-Chip, in capturing these epithelial cell adhesion molecule (EpCAM)-expressing cells using antibody-coated microposts. Here, we describe a high-throughput microfluidic mixing device, the herringbone-chip, or "HB-Chip," which provides an enhanced platform for CTC isolation. The HB-Chip design applies passive mixing of blood cells through the generation of microvortices to significantly increase the number of interactions between target CTCs and the antibody-coated chip surface. Efficient cell capture was validated using defined numbers of cancer cells spiked into control blood, and clinical utility was demonstrated in specimens from patients with prostate cancer. CTCs were detected in 14 of 15 (93%) patients with metastatic disease (median = 63 CTCs/mL, mean = 386 ± 238 CTCs/mL), and the tumor-specific TMPRSS2-ERG translocation was readily identified following RNA isolation and RT-PCR analysis. The use of transparent materials allowed for imaging of the captured CTCs using standard clinical histopathological stains, in addition to immunofluorescence-conjugated antibodies. In a subset of patient samples, the low shear design of the HB-Chip revealed microclusters of CTCs, previously unappreciated tumor cell aggregates that may contribute to the hematogenous dissemination of cancer.