Detecting androgen receptor (AR), AR variant 7 (AR-V7), prostate-specific membrane antigen (PSMA), and prostate-specific antigen (PSA) gene expression in CTCs and plasma exosome-derived cfRNA in patients with metastatic castration-resistant prostate cancer (mCRPC) by integrating the VTX-1 CTC isolation system with the QIAGEN AdnaTest.

BACKGROUND: Therapies for metastatic castration-resistant prostate cancer (mCRPC) include targeting the androgen receptor (AR) with androgen receptor inhibitors (ARIs) and prostate-specific membrane antigen (PSMA). Having the ability to detect AR, AR splice variant 7 (AR-V7), or PSMA in circulating tumor cells (CTCs) or circulating exosomal cell-free RNA (cfRNA) could be helpful to guide selection of the appropriate therapy for each individual patient. The Vortex Biosciences VTX-1 system is a label-free CTC isolation system that enables the detection of the expression of multiple genes in both CTCs and exosomal cfRNA from the same blood sample in patients with mCRPC. Detection of both AR-V7 and PSMA gene expression in both CTCs and cfRNA simultaneously has not yet been reported. METHODS: To characterize the combined VTX-1-AdnaDetect workflow, 22Rv1 cancer cells were spiked into blood from healthy donors and processed with the VTX-1 to mimic patient samples and assess performances (capture efficiency, purity, AR and AR-V7 expression). Then, we collected 19 blood samples from 16 patients with mCRPC and therapeutic resistance to androgen receptor inhibitors (ARIs). Plasma was separated and the plasma-depleted blood was processed further with the VTX-1 to collect CTCs. Both plasma exosomal cfRNA and CTCs were subsequently analyzed for AR, AR-V7, PSMA, and prostate-specific antigen (PSA) mRNA expression using the AdnaTest ProstateCancerPanel AR-V7 assay. RESULTS: AR-V7 expression could be detected in 22Rv1 cells spiked into blood from healthy volunteers as well as in CTCs and plasma-derived exosomal cfRNA from patients with mCRPC by processing blood with the VTX-1 CTC isolation system followed by the AdnaTest ProstateCancerPanel AR-V7 assay. 94.7% of patient blood samples (18/19) had detectable AR expression in either CTCs or exosomal cfRNA (16 in CTCs, 12 in cfRNA). 15.8% of the 19 patient blood samples (3/19) were found to have AR-V7-positive (AR-V7+) CTCs, one of which was also AR-V7+ in the exosomal cfRNA analysis. 42.1% of patient blood samples (8/19) were found to be PSMA positive (PSMA+): 26.3% (5/19) were PSMA+ in the CTC analysis and 31.6% (6/19) were PSMA+ in the exosomal cfRNA analysis. Of those 8 PSMA+ samples, 2 had detectable PSMA only in CTCs, and 3 had detectable PSMA only in exosomal cfRNA. CONCLUSION: VTX-1 enables isolation of CTCs and plasma exosomes from a single blood draw and can be used for detecting AR-V7 and PSMA mRNA in both CTCs and cfRNA in patients with mCRPC and resistance to ARIs. This technology facilitates combining RNA measurements in CTCs and exosomal cfRNA for future studies to develop potentially clinically relevant cancer biomarker detection in blood.

Engineering a sustainable future for point-of-care diagnostics and single-use microfluidic devices.

Single-use, disposable, point-of-care diagnostic devices carry great promise for global health, including meeting urgent needs for testing and diagnosis in places with limited laboratory facilities. Unfortunately, the production and disposal of single-use devices, whether in lateral flow assay, cartridges, cassettes, or lab-on-chip microfluidic format, also poses significant challenges for environmental and human health. Point-of-care devices are commonly manufactured from unsustainable polymeric materials derived from fossil sources. Their disposal often necessitates incineration to reduce infection risk, thereby creating additional release of CO2. Many devices also contain toxic chemicals, such as cyanide derivatives, that are damaging to environmental and human health if not disposed of safely. Yet, in the absence of government regulatory frameworks, safe and sustainable waste management for these novel medical devices is often left unaddressed. There is an urgent need to find novel solutions to avert environmental and human harm from these devices, especially in low- and middle-income countries where waste management infrastructure is often weak and where the use of point-of-care tests is projected to rise in coming years. We review here common materials used in the manufacture of single-use point-of-care diagnostic tests, examine the risks they pose to environmental and human health, and investigate replacement materials that can potentially reduce the impact of microfluidic devices on the production of harmful waste. We propose solutions available to point-of-care test developers to start embedding sustainability at an early stage in their design, and to reduce their non-renewable plastic consumption in research and product development.

Detection of EGFR Mutations in cfDNA and CTCs, and Comparison to Tumor Tissue in Non-Small-Cell-Lung-Cancer (NSCLC) Patients.

Lung cancer is the leading cause of cancer-related mortality worldwide. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) therapies, based on the evaluation of EGFR mutations, have shown dramatic clinical benefits. EGFR mutation assays are mainly performed on tumor biopsies, which carry risks, are not always successful and give results relevant to the timepoint of the assay. To detect secondary EGFR mutations, which cause resistance to 1st and 2nd generation TKIs and lead to the administration of a 3rd generation drug, effective and non-invasive monitoring of EGFR mutation status is needed. Liquid biopsy analytes, such as circulating tumor cells (CTCs) and circulating tumor DNA (cfDNA), allow such monitoring over the course of the therapy. The aim of this study was to develop and optimize a workflow for the evaluation of cfDNA and CTCs in NSCLC patients all from one blood sample. Using Vortex technology and EntroGen ctEGFR assay, EGFR mutations were identified at 0.5 ng of DNA (∼83 cells), with a sensitivity ranging from 0.1 to 2.0% for a total DNA varying from 25 ng (∼4 CTCs among 4000 white blood cells, WBCs) to 1 ng (∼4 CTCs among 200 WBCs). The processing of plasma-depleted-blood provided comparable capture recovery as whole blood, confirming the possibility of a multimodality liquid biopsy analysis (cfDNA and CTC DNA) from a single tube of blood. Different anticoagulants were evaluated and compared in terms of respective performance. Blood samples from 24 NSCLC patients and 6 age-matched healthy donors were analyzed with this combined workflow to minimize blood volume needed and sample-to-sample bias, and the EGFR mutation profile detected from CTCs and cfDNA was compared to matched tumor tissues. Despite the limited size of the patient cohort, results from this non-invasive EGFR mutation analysis are encouraging and this combined workflow represents a valuable means for informing therapy selection and for monitoring treatment of patients with NSCLC.

Tumor shedding and metastatic progression after tumor excision in patient-derived orthotopic xenograft models of triple-negative breast cancer.

Patient-derived orthotopic xenograft (PDOX) models have been verified as a useful method for studying human cancers in mice. Previous studies on the extent of metastases in these models have been limited by the necessity of welfare euthanasia (primary tumors reaching threshold size), at which point metastases may only be micrometers in diameter, few in number, and solely identified by step-sectioning of formalin-fixed paraffin-embedded tissue. These small micro-metastases are less suitable for many downstream molecular analyses than macro-metastases. Resection of the primary tumor by survival surgery has been proven to allow further time for metastases to grow. Although PDOX models of triple-negative breast cancer (TNBC) shed circulating tumor cells (CTCs) into the bloodstream and metastasize, similar to human TNBC, little data has been collected in these TNBC PDOX models regarding the association between CTC characteristics and distant metastasis following excision of the primary tumor xenograft. This study assembles a timeline of PDOX tumor shedding and metastatic tumor progression before and after tumor excision surgery. We report the ability to use tumorectomies to increase the lifespan of TNBC PDOX models with the potential to obtain larger metastases. CTC clusters and CTCs expressing a mesenchymal marker (vimentin) were associated with metastatic burden in lung and liver. The data collected through these experiments will guide the further use of PDOX models in studying metastatic TNBC.

Toward Microfluidic Label-Free Isolation and Enumeration of Circulating Tumor Cells from Blood Samples.

The isolation, analysis, and enumeration of circulating tumor cells (CTCs) from cancer patient blood samples are a paradigm shift for cancer patient diagnosis, prognosis, and treatment monitoring. Most methods used to isolate and enumerate these target cells rely on the expression of cell surface markers, which varies between patients, cancer types, tumors, and stages. Here, we propose a label-free high-throughput platform to isolate, enumerate, and size CTCs on two coupled microfluidic devices. Cancer cells were purified through a Vortex chip and subsequently flowed in-line to an impedance chip, where a pair of electrodes measured fluctuations of an applied electric field generated by cells passing through. A proof-of-concept of the coupling of those two devices was demonstrated with beads and cells. First, the impedance chip was tested as a stand-alone device: (1) with beads (mean counting error of 1.0%, sizing information clearly separated three clusters for 8, 15, and 20 um beads, respectively) as well as (2) with cancer cells (mean counting error of 3.5%). Second, the combined setup was tested with beads, then with cells in phosphate-buffered saline, and finally with cancer cells spiked in healthy blood. Experiments demonstrated that the Vortex HT chip enriched the cancer cells, which then could be counted and differentiated from smaller blood cells by the impedance chip based on size information. Further discrimination was shown with dual high-frequency measurements using electric opacity, highlighting the potential application of this combined setup for a fully integrated label-free isolation and enumeration of CTCs from cancer patient samples. © 2019 International Society for Advancement of Cytometry.

Investigating circulating tumor cells and distant metastases in patient-derived orthotopic xenograft models of triple-negative breast cancer.

BACKGROUND: Circulating tumor cells (CTCs) represent a temporal "snapshot" of a patient's cancer and changes that occur during disease evolution. There is an extensive literature studying CTCs in breast cancer patients, and particularly in those with metastatic disease. In parallel, there is an increasing use of patient-derived models in preclinical investigations of human cancers. Yet studies are still limited demonstrating CTC shedding and metastasis formation in patient-derived models of breast cancer. METHODS: We used seven patient-derived orthotopic xenograft (PDOX) models generated from triple-negative breast cancer (TNBC) patients to study CTCs and distant metastases. Tumor fragments from PDOX tissue from each of the seven models were implanted into 57 NOD scid gamma (NSG) mice, and tumor growth and volume were monitored. Human CTC capture from mouse blood was first optimized on the marker-agnostic Vortex CTC isolation platform, and whole blood was processed from 37 PDOX tumor-bearing mice. RESULTS: Staining and imaging revealed the presence of CTCs in 32/37 (86%). The total number of CTCs varied between different PDOX tumor models and between individual mice bearing the same PDOX tumors. CTCs were heterogeneous and showed cytokeratin (CK) positive, vimentin (VIM) positive, and mixed CK/VIM phenotypes. Metastases were detected in the lung (20/57, 35%), liver (7/57, 12%), and brain (1/57, less than 2%). The seven different PDOX tumor models displayed varying degrees of metastatic potential, including one TNBC PDOX tumor model that failed to generate any detectable metastases (0/8 mice) despite having CTCs present in the blood of 5/5 tested, suggesting that CTCs from this particular PDOX tumor model may typify metastatic inefficiency. CONCLUSION: PDOX tumor models that shed CTCs and develop distant metastases represent an important tool for investigating TNBC.

Classification of large circulating tumor cells isolated with ultra-high throughput microfluidic Vortex technology.

Circulating tumor cells (CTCs) are emerging as rare but clinically significant non-invasive cellular biomarkers for cancer patient prognosis, treatment selection, and treatment monitoring. Current CTC isolation approaches, such as immunoaffinity, filtration, or size-based techniques, are often limited by throughput, purity, large output volumes, or inability to obtain viable cells for downstream analysis. For all technologies, traditional immunofluorescent staining alone has been employed to distinguish and confirm the presence of isolated CTCs among contaminating blood cells, although cells isolated by size may express vastly different phenotypes. Consequently, CTC definitions have been non-trivial, researcher-dependent, and evolving. Here we describe a complete set of objective criteria, leveraging well-established cytomorphological features of malignancy, by which we identify large CTCs. We apply the criteria to CTCs enriched from stage IV lung and breast cancer patient blood samples using the High Throughput Vortex Chip (Vortex HT), an improved microfluidic technology for the label-free, size-based enrichment and concentration of rare cells. We achieve improved capture efficiency (up to 83%), high speed of processing (8 mL/min of 10x diluted blood, or 800 µL/min of whole blood), and high purity (avg. background of 28.8±23.6 white blood cells per mL of whole blood). We show markedly improved performance of CTC capture (84% positive test rate) in comparison to previous Vortex designs and the current FDA-approved gold standard CellSearch assay. The results demonstrate the ability to quickly collect viable and pure populations of abnormal large circulating cells unbiased by molecular characteristics, which helps uncover further heterogeneity in these cells.

High efficiency vortex trapping of circulating tumor cells.

Circulating tumor cells (CTCs) are important biomarkers for monitoring tumor dynamics and efficacy of cancer therapy. Several technologies have been demonstrated to isolate CTCs with high efficiency but achieve a low purity from a large background of blood cells. We have previously shown the ability to enrich CTCs with high purity from large volumes of blood through selective capture in microvortices using the Vortex Chip. The device consists of a narrow channel followed by a series of expansion regions called reservoirs. Fast flow in the narrow entry channel gives rise to inertial forces, which direct larger cells into trapping vortices in the reservoirs where they remain circulating in orbits. By studying the entry and stability of particles following entry into reservoirs, we discover that channel cross sectional area plays an important role in controlling the size of trapped particles, not just the orbital trajectories. Using these design modifications, we demonstrate a new device that is able to capture a wider size range of CTCs from clinical samples, uncovering further heterogeneity. This simple biophysical method opens doors for a range of downstream interventions, including genetic analysis, cell culture, and ultimately personalized cancer therapy.

Size-selective collection of circulating tumor cells using Vortex technology.

A blood-based, low cost alternative to radiation intensive CT and PET imaging is critically needed for cancer prognosis and management of its treatment. "Liquid biopsies" of circulating tumor cells (CTCs) from a relatively non-invasive blood draw are particularly ideal, as they can be repeated regularly to provide up to date molecular information about the cancer, which would also open up key opportunities for personalized therapies. Beyond solely diagnostic applications, CTCs are also a subject of interest for drug development and cancer research. In this paper, we adapt a technology previously introduced, combining the use of micro-scale vortices and inertial focusing, specifically for the high-purity extraction of CTCs from blood samples. First, we systematically varied parameters including channel dimensions and flow rates to arrive at an optimal device for maximum trapping efficiency and purity. Second, we validated the final device for capture of cancer cell lines in blood, considering several factors, including the effect of blood dilution, red blood cell lysis and cell deformability, while demonstrating cell viability and independence on EpCAM expression. Finally, as a proof-of-concept, CTCs were successfully extracted and enumerated from the blood of patients with breast (N = 4, 25-51 CTCs per 7.5 mL) and lung cancer (N = 8, 23-317 CTCs per 7.5 mL). Importantly, samples were highly pure with limited leukocyte contamination (purity 57-94%). This Vortex approach offers significant advantages over existing technologies, especially in terms of processing time (20 min for 7.5 mL of whole blood), sample concentration (collecting cells in a small volume down to 300 µL), applicability to various cancer types, cell integrity and purity. We anticipate that its simplicity will aid widespread adoption by clinicians and biologists who desire to not only enumerate CTCs, but also uncover new CTC biology, such as unique gene mutations, vesicle secretion and roles in metastatic processes.

Micro-scale blood plasma separation: from acoustophoresis to egg-beaters.

Plasma is a rich mine of various biomarkers including proteins, metabolites and circulating nucleic acids. The diagnostic and therapeutic potential of these analytes has been quite recently uncovered, and the number of plasma biomarkers will still be growing in the coming years. A significant part of the blood plasma preparation is still handled manually, off-chip, via centrifugation or filtration. These batch methods have variable waiting times, and are often performed under non-reproducible conditions that may impair the collection of analytes of interest, with variable degradation. The development of miniaturised modules capable of automated and reproducible blood plasma separation would aid in the translation of lab-on-a-chip devices to the clinical market. Here we propose a systematic review of major plasma analytes and target applications, alongside existing solutions for micro-scale blood plasma extraction, focusing on the approaches that have been biologically validated for specific applications.

Engineering fluid flow using sequenced microstructures.

Controlling the shape of fluid streams is important across scales: from industrial processing to control of biomolecular interactions. Previous approaches to control fluid streams have focused mainly on creating chaotic flows to enhance mixing. Here we develop an approach to apply order using sequences of fluid transformations rather than enhancing chaos. We investigate the inertial flow deformations around a library of single cylindrical pillars within a microfluidic channel and assemble these net fluid transformations to engineer fluid streams. As these transformations provide a deterministic mapping of fluid elements from upstream to downstream of a pillar, we can sequentially arrange pillars to apply the associated nested maps and, therefore, create complex fluid structures without additional numerical simulation. To show the range of capabilities, we present sequences that sculpt the cross-sectional shape of a stream into complex geometries, move and split a fluid stream, perform solution exchange and achieve particle separation. A general strategy to engineer fluid streams into a broad class of defined configurations in which the complexity of the nonlinear equations of fluid motion are abstracted from the user is a first step to programming streams of any desired shape, which would be useful for biological, chemical and materials automation.

High-throughput single-microparticle imaging flow analyzer.

Optical microscopy is one of the most widely used diagnostic methods in scientific, industrial, and biomedical applications. However, while useful for detailed examination of a small number (< 10,000) of microscopic entities, conventional optical microscopy is incapable of statistically relevant screening of large populations (> 100,000,000) with high precision due to its low throughput and limited digital memory size. We present an automated flow-through single-particle optical microscope that overcomes this limitation by performing sensitive blur-free image acquisition and nonstop real-time image-recording and classification of microparticles during high-speed flow. This is made possible by integrating ultrafast optical imaging technology, self-focusing microfluidic technology, optoelectronic communication technology, and information technology. To show the system's utility, we demonstrate high-throughput image-based screening of budding yeast and rare breast cancer cells in blood with an unprecedented throughput of 100,000 particles/s and a record false positive rate of one in a million.

Intrinsic particle-induced lateral transport in microchannels.

In microfluidic systems at low Reynolds number, the flow field around a particle is assumed to maintain fore-aft symmetry, with fluid diverted by the presence of a particle, returning to its original streamline downstream. This current model considers particles as passive components of the system. However, we demonstrate that at finite Reynolds number, when inertia is taken into consideration, particles are not passive elements in the flow but significantly disturb and modify it. In response to the flow field, particles translate downstream while rotating. The combined effect of the flow of fluid around particles, particle rotation, channel confinement (i.e., particle dimensions approaching those of the channel), and finite fluid inertia creates a net recirculating flow perpendicular to the primary flow direction within straight channels that resembles the well-known Dean flow in curved channels. Significantly, the particle generating this flow remains laterally fixed as it translates downstream and only the fluid is laterally transferred. Therefore, as the particles remain inertially focused, operations can be performed around the particles in a way that is compatible with downstream assays such as flow cytometry. We apply this particle-induced transfer to perform fluid switching and mixing around rigid microparticles as well as deformable cells. This transport phenomenon, requiring only a simple channel geometry with no external forces to operate, offers a practical approach for fluid transfer at high flow rates with a wide range of applications, including sample preparation, flow reaction, and heat transfer.

Rapid prototyping polymers for microfluidic devices and high pressure injections.

Multiple methods of fabrication exist for microfluidic devices, with different advantages depending on the end goal of industrial mass production or rapid prototyping for the research laboratory. Polydimethylsiloxane (PDMS) has been the mainstay for rapid prototyping in the academic microfluidics community, because of its low cost, robustness and straightforward fabrication, which are particularly advantageous in the exploratory stages of research. However, despite its many advantages and its broad use in academic laboratories, its low elastic modulus becomes a significant issue for high pressure operation as it leads to a large alteration of channel geometry. Among other consequences, such deformation makes it difficult to accurately predict the flow rates in complex microfluidic networks, change flow speed quickly for applications in stop-flow lithography, or to have predictable inertial focusing positions for cytometry applications where an accurate alignment of the optical system is critical. Recently, other polymers have been identified as complementary to PDMS, with similar fabrication procedures being characteristic of rapid prototyping but with higher rigidity and better resistance to solvents; Thermoset Polyester (TPE), Polyurethane Methacrylate (PUMA) and Norland Adhesive 81 (NOA81). In this review, we assess these different polymer alternatives to PDMS for rapid prototyping, especially in view of high pressure injections with the specific example of inertial flow conditions. These materials are compared to PDMS, for which magnitudes of deformation and dynamic characteristics are also characterized. We provide a complete and systematic analysis of these materials with side-by-side experiments conducted in our lab that also evaluate other properties, such as biocompatibility, solvent compatibility, and ease of fabrication. We emphasize that these polymer alternatives, TPE, PUMA and NOA, have some considerable strengths for rapid prototyping when bond strength, predictable operation at high pressure, or transitioning to commercialization are considered important for the application.

Fast and continuous plasma extraction from whole human blood based on expanding cell-free layer devices.

This paper presents promising microfluidic devices designed for continuous and passive extraction of plasma from whole human blood. These designs are based on red cells lateral migration and the resulting cell-free layer locally expanded by geometric singularities such as an enlargement of the channel or a cavity adjacent to the channel. After an explanation of flow patterns, different tests are described that confirm the advantages of both proposed singularities, providing a 1.5 and 2X increase in extraction yield compared to a reference device, for 1:20 diluted blood at 100 microL/min. Devices have also been successively optimized, with extraction yields up to 17.8%, and biologically validated for plasma extraction, with no protein loss or denaturation, no hemolysis and with excellent cell purity. Finally, the dilution effect has been experimentally investigated.

Real-time detection of lymphocytes binding on an antibody chip using SPR imaging.

We demonstrate the use of SPR imaging for the detection of site-specific binding of either B or T lymphocyte populations on an electrochemically-grafted antibody array.