Cell-free DNA and circulating tumor cell kinetics in a pre-clinical head and neck Cancer model undergoing radiation therapy.

BACKGROUND: Monitoring circulating tumor DNA (ctDNA) and circulating tumor cells (CTCs), known as liquid biopsies, continue to be developed as diagnostic and prognostic markers for a wide variety of cancer indications, mainly due to their minimally invasive nature and ability to offer a wide range of phenotypic and genetic information. While liquid biopsies maintain significant promising benefits, there is still limited information regarding the kinetics of ctDNA and CTCs following radiation therapy which remains a vital treatment modality in head and neck cancers. This study aims to describe the kinetics of ctDNA and CTCs following radiation exposure in a preclinical rabbit model with VX2 induced buccal carcinoma. METHODS: Seven rabbits were inoculated with VX2 cells in the buccal mucosa and subjected to radiation. At selected time points, blood sampling was performed to monitor differing levels of ctDNA and CTC. Plasma ctDNA was measured with quantitative PCR for papillomavirus E6 while CTCs were quantified using an immunomagnetic nanoparticles within a microfluidic device. Comparisons of CTC detection with EpCAM compared to multiple surface markers (EGFR, HER2 and PSMA) was evaluated and correlated with the tumor size. RESULTS: Plasma ctDNA reflects the overall tumor burden within the animal model. Analysis of correlations between ctDNA with tumor and lymph node volumes showed a positive correlation (R = 0.452 and R = 0.433 [p < 0.05]), respectively. Over the course of treatment, ctDNA levels declined and quickly becomes undetectable following tumor eradication. While during the course of treatment, ctDNA levels were noted to rise particularly upon initiation of radiation following scheduled treatment breaks. Levels of CTCs were observed to increase 1 week following inoculation of tumor to the primary site. For CTC detection, the use of multiple surface markers showed a greater sensitivity when compared to detection using only EpCAM. Plasma CTC levels remained elevated following radiation therapy which may account for an increased shedding of CTCs following radiation. CONCLUSION: This study demonstrates the utility of ctDNA and CTCs detection in response to radiation treatment in a preclinical head and neck model, allowing for better understanding of liquid biopsy applications in both clinical practice and research development.

Tracking the expression of therapeutic protein targets in rare cells by antibody-mediated nanoparticle labelling and magnetic sorting.

Molecular-level features of tumours can be tracked using single-cell analyses of circulating tumour cells (CTCs). However, single-cell measurements of protein expression for rare CTCs are hampered by the presence of a large number of non-target cells. Here, we show that antibody-mediated labelling of intracellular proteins in the nucleus, mitochondria and cytoplasm of human cells with magnetic nanoparticles enables analysis of target proteins at the single-cell level by sorting the cells according to their nanoparticle content in a microfluidic device with cell-capture zones sandwiched between arrays of magnets. We used the magnetic labelling and cell-sorting approach to track the expression of therapeutic protein targets in CTCs isolated from blood samples of mice with orthotopic prostate xenografts and from patients with metastatic castration-resistant prostate cancer. We also show that mutated proteins that are drug targets or markers of therapeutic response can be directly identified in CTCs, analysed at the single-cell level and used to predict how mice with drug-susceptible and drug-resistant pancreatic tumour xenografts respond to therapy.

Ultrasensitive and rapid quantification of rare tumorigenic stem cells in hPSC-derived cardiomyocyte populations.

The ability to detect rare human pluripotent stem cells (hPSCs) in differentiated populations is critical for safeguarding the clinical translation of cell therapy, as these undifferentiated cells have the capacity to form teratomas in vivo. The detection of hPSCs must be performed using an approach compatible with traceable manufacturing of therapeutic cell products. Here, we report a novel microfluidic approach, stem cell quantitative cytometry (SCQC), for the quantification of rare hPSCs in hPSC-derived cardiomyocyte (CM) populations. This approach enables the ultrasensitive capture, profiling, and enumeration of trace levels of hPSCs labeled with magnetic nanoparticles in a low-cost, manufacturable microfluidic chip. We deploy SCQC to assess the tumorigenic risk of hPSC-derived CM populations in vivo. In addition, we isolate rare hPSCs from the differentiated populations using SCQC and characterize their pluripotency.

Phenotypic Profiling of Circulating Tumor Cells in Metastatic Prostate Cancer Patients Using Nanoparticle-Mediated Ranking.

The analysis of circulating tumor cells (CTCs) provides a means to collect information about the evolving properties of a tumor during cancer progression and treatment. For patients with metastatic prostate cancer, noninvasive serial measurements of bloodborne cells may provide a means to tailor therapeutic decisions based on an individual patient's response. Here, we used a high-sensitivity profiling approach to monitor CTCs in patients with metastatic castrate-resistant prostate cancer (mCRPC) undergoing treatment with abiraterone and enzalutamide, two drugs used to treat advanced prostate cancer. The capture and profiling approach uses antibody-functionalized magnetic nanoparticles to sort cells according to protein expression levels. CTCs are tagged with magnetic nanoparticles conjugated to an antibody specific for the epithelial cell adhesion molecule (EpCAM) and sorted into four zones of a microfluidic device based on EpCAM expression levels. Our approach was compared to the FDA-cleared CellSearch method, and we demonstrate significantly higher capture efficiency of low-EpCAM cells compared to the commercial method. The nanoparticle-based approach detected CTCs from 86% of patients at baseline, compared to CellSearch which only detected CTCs from 60% of patients. Patients were stratified as prostate specific antigen (PSA) progressive versus responsive based on clinically acceptable definitions, and it was observed that patients with a limited response to therapy had elevated levels of androgen receptor variant 7 (ARV7) and the mesenchymal marker, N-cadherin, expressed on their CTCs. In addition, these CTCs exhibited lower EpCAM expression. The results highlight features of CTCs associated with disease progression on abiraterone or enzalutamide, including mesenchymal phenotypes and increased expression levels of ARV7. The use of a high-sensitivity method to capture and profile CTCs provides more informative data concerning the phenotypic properties of these cells as patients undergo treatment relative to an FDA-cleared method.

Nanoparticle-Mediated Capture and Electrochemical Detection of Methicillin-Resistant Staphylococcus aureus.

The spread of antibiotic-resistant bacteria poses a global threat to public health. Conventional bacterial detection and identification methods often require pre-enrichment and/or sample preprocessing and purification steps that can prolong diagnosis by days. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most widespread antibiotic-resistant bacteria and is the leading cause of hospital-acquired infections. Here, we have developed a method to specifically capture and detect MRSA directly from patient nasal swabs with no prior culture and minimal processing steps using a microfluidic device and antibody-functionalized magnetic nanoparticles. Bacteria are captured based on antibody recognition of a membrane-bound protein marker that confers ß-lactam antibiotic resistance. MRSA identification is then achieved by the use of a strain-specific antibody functionalized with alkaline phosphatase for electrochemical detection. This approach ensures that only those bacteria of the target strain and resistance profile are measured. The method has a limit of detection of 845 CFU/mL and excellent discrimination against high concentrations of common nontarget nasal flora with a turnaround time of under 4.5 h. This detection method was successfully validated using clinical nasal swab specimens ( n = 30) and has the potential to be tailored to various bacterial targets.

Combining Desmopressin and Docetaxel for the Treatment of Castration-Resistant Prostate Cancer in an Orthotopic Model.

BACKGROUND/AIM: Desmopressin is a synthetic analogue of the antidiuretic hormone vasopressin. It has recently been demonstrated to inhibit tumor progression and metastasis in breast cancer models. Docetaxel is a chemotherapy agent for castrate-resistant prostate cancer (CRPC). In this study, the ability of CRPC cells to grow and develop in vivo tumors in an animal model was evaluated, in order to investigate the anti-tumor effect of desmopressin in combination with docetaxel. MATERIALS AND METHODS: The CRPC cell line PC3 was used for orthotopic inoculation in male athymic nude mice. The mice were randomly assigned to one of the four treatment groups: Control, docetaxel, desmopressin or combination therapy. Following the last treatment, tumors were excised and measured. Blood samples were processed for CTC analysis. RESULTS: Docetaxel treatment resulted in a significant reduction in tumor volume compared to control. The combination therapy resulted in even more significant reduction (31.2%) in tumor volume. There was a complete absence of CTCs in the combination group. CONCLUSION: Our pilot study demonstrated an enhanced efficacy of docetaxel-based therapy in combination with desmopressin.

Single-cell mRNA cytometry via sequence-specific nanoparticle clustering and trapping.

Cell-to-cell variation in gene expression creates a need for techniques that can characterize expression at the level of individual cells. This is particularly true for rare circulating tumour cells, in which subtyping and drug resistance are of intense interest. Here we describe a method for cell analysis-single-cell mRNA cytometry-that enables the isolation of rare cells from whole blood as a function of target mRNA sequences. This approach uses two classes of magnetic particles that are labelled to selectively hybridize with different regions of the target mRNA. Hybridization leads to the formation of large magnetic clusters that remain localized within the cells of interest, thereby enabling the cells to be magnetically separated. Targeting specific intracellular mRNAs enablescirculating tumour cells to be distinguished from normal haematopoietic cells. No polymerase chain reaction amplification is required to determine RNA expression levels and genotype at the single-cell level, and minimal cell manipulation is required. To demonstrate this approach we use single-cell mRNA cytometry to detect clinically important sequences in prostate cancer specimens.

Isolation of Phenotypically Distinct Cancer Cells Using Nanoparticle-Mediated Sorting.

Isolating subpopulations of heterogeneous cancer cells is an important capability for the meaningful characterization of circulating tumor cells at different stages of tumor progression and during the epithelial-to-mesenchymal transition. Here, we present a microfluidic device that can separate phenotypically distinct subpopulations of cancer cells. Magnetic nanoparticles coated with antibodies against the epithelial cell adhesion molecule (EpCAM) are used to separate breast cancer cells in the microfluidic platform. Cells are sorted into different zones on the basis of the levels of EpCAM expression, which enables the detection of cells that are losing epithelial character and becoming more mesenchymal. The phenotypic properties of the isolated cells with low and high EpCAM are then assessed using matrix-coated surfaces for collagen uptake analysis, and an NAD(P)H assay that assesses metabolic activity. We show that low-EpCAM expressing cells have higher collagen uptake and higher folate-induced NAD(P)H responses compared to those of high-EpCAM expressing cells. In addition, we tested SKBR3 cancer cells undergoing chemically induced hypoxia. The induced cells have reduced expression of EpCAM, and we find that these cells have higher collagen uptake and NAD(P)H metabolism relative to noninduced cells. This work demonstrates that nanoparticle-mediated binning facilitates the isolation of functionally distinct cell subpopulations and allows surface marker expression to be associated with invasiveness, including collagen uptake and metabolic activity.

Profiling Functional and Biochemical Phenotypes of Circulating Tumor Cells Using a Two-Dimensional Sorting Device.

During cancer progression, tumors shed circulating tumor cells (CTCs) into the bloodstream. CTCs that originate from the same primary tumor can have heterogeneous phenotypes and, while some CTCs possess benign properties, others have high metastatic potential. Deconstructing the heterogeneity of CTCs is challenging and new methods are needed that can sort small numbers of cancer cells according to their phenotypic properties. Here we describe a new microfluidic approach that profiles, along two independent phenotypic axes, the behavior of heterogeneous cell subpopulations. Cancer cells are first profiled according to expression of a surface marker using a nanoparticle-enabled approach. Along the second dimension, these subsets are further separated into subpopulations corresponding to migration profiles generated in response to a chemotactic agent. We deploy this new technique and find a strong correlation between the surface expression and migration potential of CTCs present in blood from mice with xenografted tumors. This system provides an important new means to characterize functional diversity in circulating tumor cells.

Tracking the dynamics of circulating tumour cell phenotypes using nanoparticle-mediated magnetic ranking.

Profiling the heterogeneous phenotypes of rare circulating tumour cells (CTCs) in whole blood is critical to unravelling the complex and dynamic properties of these potential clinical markers. This task is challenging because these cells are present at parts per billion levels among normal blood cells. Here we report a new nanoparticle-enabled method for CTC characterization, called magnetic ranking cytometry, which profiles CTCs on the basis of their surface expression phenotype. We achieve this using a microfluidic chip that successfully processes whole blood samples. The approach classifies CTCs with single-cell resolution in accordance with their expression of phenotypic surface markers, which is read out using magnetic nanoparticles. We deploy this new technique to reveal the dynamic phenotypes of CTCs in unprocessed blood from mice as a function of tumour growth and aggressiveness. We also test magnetic ranking cytometry using blood samples collected from cancer patients.

Rapid Detection of Cat Cystatin C (cCys-C) Using Immuno-Pillar Chips.

We demonstrated a rapid immunoassay for detection of cat cystatin C (cCys-C) which is an important marker for chronic kidney disease in cats, using immuno-pillar chips. The required amount of reagent solution is 200 times smaller than that for the conventional ELISA in the 96-well microplate (0.5 µL versus 100 µL). In addition, the total assay time in the proposed method is more than 12 times shorter than in the conventional method (20 min versus 240 min). The limit of detection in the new method of 3 ng mL-1 is comparable to that of the conventional method (1 ng mL-1) and it is in the clinically relevant range.

Novel concept of washing for microfluidic paper-based analytical devices based on capillary force of paper substrates.

A novel washing technique for microfluidic paper-based analytical devices (µPADs) that is based on the spontaneous capillary action of paper and eliminates unbound antigen and antibody in a sandwich immunoassay is reported. Liquids can flow through a porous medium (such as paper) in the absence of external pressure as a result of capillary action. Uniform results were achieved when washing a paper substrate in a PDMS holder which was integrated with a cartridge absorber acting as a porous medium. Our study demonstrated that applying this washing technique would allow µPADs to become the least expensive microfluidic device platform with high reproducibility and sensitivity. In a model µPAD assay that utilized this novel washing technique, C-reactive protein (CRP) was detected with a limit of detection (LOD) of 5 µg mL-1. Graphical Abstract A novel washing technique for microfluidic paper-based analytical devices (µPADs) that is based on the spontaneous capillary action of paper and eliminates unbound antigen and antibody in a sandwich immunoassay is reported.

Aptamer and Antisense-Mediated Two-Dimensional Isolation of Specific Cancer Cell Subpopulations.

Cancer cells, and in particular those found circulating in blood, can have widely varying phenotypes and molecular profiles despite a common origin. New methods are needed that can deconvolute the heterogeneity of cancer cells and sort small numbers of cells to aid in the characterization of cancer cell subpopulations. Here, we describe a new molecular approach to capturing cancer cells that isolates subpopulations using two-dimensional sorting. Using aptamer-mediated capture and antisense-triggered release, the new strategy sorts cells according to levels of two different markers and thereby separates them into their corresponding subpopulations. Using a phenotypic assay, we demonstrate that the subpopulations isolated have markedly different properties. This system provides an important new tool for identifying circulating tumor cell subtypes.

Beyond the Capture of Circulating Tumor Cells: Next-Generation Devices and Materials.

Over the last decade, significant progress has been made towards the development of approaches that enable the capture of rare circulating tumor cells (CTCs) from the blood of cancer patients, a critical capability for noninvasive tumor profiling. These advances have leveraged new insights in materials chemistry and microfluidics and allowed the capture and enumeration of CTCs with unprecedented sensitivity. However, it has become increasingly clear that simply capturing and counting tumor cells launched into the bloodstream may not provide the information needed to advance our understanding of the biology of these rare cells, or to allow us to better exploit them in medicine. A variety of advances have now emerged demonstrating that more information can be extracted from CTCs with next-generation devices and materials featuring tailored physical and chemical properties. In this Minireview, the last ten years of work in this area will be discussed, with an emphasis on the groundbreaking work of the last five years, during which the focus has moved beyond the simple capture of CTCs and gravitated towards approaches that enable in-depth analysis.

Interrogating Circulating Microsomes and Exosomes Using Metal Nanoparticles.

A chip-based approach for electrochemical characterization and detection of microsomes and exosomes based on direct electro-oxidation of metal nanoparticles (MNPs) that specifically recognize surface markers of these vesicles is reported. It is found that exosomes and microsomes derived from prostate cancer cells can be identified by their surface proteins EpCAM and PSMA, suggesting the potential of exosomes and microsomes for use as diagnostic biomarkers.

An integrated microfluidic chip for immunocapture, preconcentration and separation of ß-amyloid peptides.

We present an integrated microfluidic chip for detection of ß-amyloid (Aß) peptides. Aß peptides are major biomarkers for the diagnosis of Alzheimer's disease (AD) in its early stages. This microfluidic device consists of three main parts: (1) An immunocapture microcolumn based on self-assembled magnetic beads coated with antibodies specific to Aß peptides, (2) a nano-porous membrane made of photopolymerized hydrogel for preconcentration, and (3) a microchip electrophoresis (MCE) channel with fluorescent detection. Sub-milliliter sample volume is either mixed off-chip with antibody coated magnetic beads and injected into the device or is injected into an already self-assembled column of magnetic beads in the microchannel. The captured peptides on the beads are then electrokinetically eluted and re-concentrated onto the nano-membrane in a few nano-liters. By integrating the nano-membrane, total assay time was reduced and also off-chip re-concentration or buffer exchange steps were not needed. Finally, the concentrated peptides in the chip are separated by electrophoresis in a polymer-based matrix. The device was applied to the capture and MCE analysis of differently truncated peptides Aß (1-37, 1-39, 1-40, and 1-42) and was able to detect as low as 25 ng of synthetic Aß peptides spiked in undiluted cerebrospinal fluid (CSF). The device was also tested with CSF samples from healthy donors. CSF samples were fluorescently labelled and pre-mixed with the magnetic beads and injected into the device. The results indicated that Aß1-40, an important biomarker for distinguishing patients with frontotemporal lobe dementia from controls and AD patients, was detectable. Although the sensitivity of this device is not yet enough to detect all Aß subtypes in CSF, this is the first report on an integrated or semi-integrated device for capturing and analyzing of differently truncated Aß peptides. The method is less demanding and faster than the conventional Western blotting method currently used for research.

An instrument-free, screen-printed paper microfluidic device that enables bio and chemical sensing.

This paper describes a simple and instrument-free screen-printing method to fabricate hydrophilic channels by patterning polydimethylsiloxane (PDMS) onto chromatography paper. Clearly recognizable border lines were formed between hydrophilic and hydrophobic areas. The minimum width of the printed channel to deliver an aqueous sample was 600 µm, as obtained by this method. Fabricated microfluidic paper-based analytical devices (µPADs) were tested for several colorimetric assays of pH, glucose, and protein in both buffer and artificial urine samples and results were obtained in less than 30 min. The limits of detection (LODs) for glucose and bovine serum albumin (BSA) were 5 mM and 8 µM, respectively. Furthermore, the pH values of different solutions were visually recognised with the naked eye by using a sensitive ink. Ultimately, it is expected that this PDMS-screen-printing (PSP) methodology for µPADs can be readily translated to other colorimetric detection and hydrophilic channels surrounded by a hydrophobic polymer can be formed to transport fluids toward target zones.

Sample-to-Answer Isolation and mRNA Profiling of Circulating Tumor Cells.

The isolation and rapid molecular characterization of circulating tumor cells (CTCs) from a liquid biopsy could enable the convenient and effective characterization of the state and aggressiveness of cancerous tumors. Existing technologies enumerate CTCs using immunostaining; however, these approaches are slow, labor-intensive, and often fail to enable further genetic characterization of CTCs. Here, we report on an integrated circuit that combines the capture of CTCs with the profiling of their gene expression signatures. Specifically, we use a velocity valley chip to efficiently capture magnetic nanoparticle-bound CTCs, which are then directly analyzed for their gene expression profiles using nanostructured microelectrode biosensors. CTCs are captured with 97% efficiency from 2 mL of whole blood, yielding a 500-fold concentration within 1 h. We show efficient capture of as few as 2 cancer cells/(mL of blood) and demonstrate that the gene expression module accurately profiles the expression of prostate-specific genes in CTCs captured from whole blood. This advance provides the first sample-to-answer solution for gene-based testing of CTCs. The approach was successfully validated using samples collected from prostate cancer patients: both CTCs and prostate-specific antigen (PSA) mRNA sequences were detected in all cancer patient samples and not in the healthy controls.

Nanoparticle-based sorting of circulating tumor cells by epithelial antigen expression during disease progression in an animal model.

Circulating tumor cells (CTCs) can be used as markers for the detection, characterization, and targeted therapeutic management of cancer. We recently developed a nanoparticle-mediated approach for capture and sorting of CTCs based on their specific epithelial phenotype. In the current study, we investigate the phenotypic transition of tumor cells in an animal model and show the correlation of this transition with tumor progression. VX2 tumor cells were injected into rabbits, and CTCs were evaluated during tumor progression and correlated with computerized tomography (CT) measurements of tumor volume. The results showed a dramatic increase of CTCs during the four weeks of tumor growth. Following resection, CTC levels dropped but then rebounded, likely due to lymph node metastases. Additionally, CTCs showed a marked loss of the epithelial cell adhesion molecule (EpCAM) relative to precursor cells. In conclusion, the device accurately traces disease progression and CTC phenotypic shift in an animal model. FROM THE CLINICAL EDITOR: The detection of circulating tumor cells (CTCs) has been used to predict disease prognosis. In this study, the authors developed a nanoparticle-mediated platform based on microfluidics to analyze the differential expressions of epithelial cell adhesion molecule (EpCAM) on CTCs in an animal model. It was found that the loss of EpCAM correlated with disease progression. Hence, the use of this platform may be further applied in other cancer models in the future.

In Situ Electrochemical ELISA for Specific Identification of Captured Cancer Cells.

Circulating tumor cells (CTCs) are cancer cells disseminated from a tumor into the bloodstream. Their presence in patient blood samples has been associated with metastatic disease. Here, we report a simple system that enables the isolation and detection of these rare cancer cells. By developing a sensitive electrochemical ELISA method integrated within a microfluidic cell capture system, were we able to reliably detect very low levels of cancer cells in whole blood. Our results indicate that the new system provides the clinically relevant specificity and sensitivity needed for a convenient, point-of-need assay for cancer cell counting.