Oligo cyc-DEP: On-chip cyclic immunofluorescence profiling of cell-derived nanoparticles.

We present a follow-on technique for the cyclic-immunofluorescence profiling of suspension particles isolated using dielectrophoresis. The original lab-on-chip technique ("cyc-DEP" [cyclic immunofluorescent imaging on dielectrophoretic chip]) was designed for the multiplex surveillance of circulating biomarkers. Nanoparticles were collected from low-volume liquid biopsies using microfluidic dielectrophoretic chip technology. Subsequent rounds of cyclic immunofluorescent labeling and quenching were imaged and quantified with a custom algorithm to detect multiple proteins. While cyc-DEP improved assay multiplicity, long runtimes threatened its clinical adoption. Here, we modify the original cyc-DEP platform to reduce assay runtimes. Nanoparticles were formulated from human prostate adenocarcinoma cells and collected using dielectrophoresis. Three proteins were labeled on-chip with a mixture of short oligonucleotide-conjugated antibodies. The sample was then incubated with complementary fluorophore-conjugated oligonucleotides, which were dehybridized using an ethylene carbonate buffer after each round of imaging. Oligonucleotide removal exhibited an average quenching efficiency of 98 ± 3% (n = 12 quenching events), matching the original cyc-DEP platform. The presented "oligo cyc-DEP" platform achieved clinically relevant sample-to-answer times, reducing the duration for three rounds of cyclic immunolabeling from approximately 20 to 6.5 h-a 67% decrease attributed to rapid fluorophore removal and the consolidated co-incubation of antibodies.

Serum autoantibody reactivity in bullous pemphigoid is associated with neuropsychiatric disorders and the use of antidiabetics and antipsychotics: a large, prospective cohort study.

BACKGROUND: Bullous pemphigoid (BP), the by far most frequent autoimmune blistering skin disease (AIBD), is immunopathologically characterized by autoantibodies against the two hemidesmosomal proteins BP180 (collagen type XVII) and BP230 (BPAG1 or dystonin). Several comorbidities and potentially disease-inducing medication have been described in BP, yet a systematic analysis of these clinically relevant findings and autoantibody reactivities has not been performed. OBJECTIVE: To determine associations of autoantibody reactivities with comorbidities and concomitant medication. METHODS: In this prospective multicenter study, 499 patients diagnosed with BP in 16 European referral centers were included. The relation between anti-BP180 NC16A and anti-BP230 IgG ELISA values at the time of diagnosis as well as comorbidities and concomitant medication collected by a standardized form were analysed. RESULTS: An association between higher serum anti-BP180 reactivity and neuropsychiatric but not atopic and metabolic disorders was observed as well as with the use of insulin or antipsychotics but not with dipeptidyl peptidase-4 (DPP4) inhibitors, inhibitors of platelet aggregation and L-thyroxine. The use of DPP4 inhibitors was associated with less anti-BP180 and anti-BP230 reactivity compared with BP patients without these drugs. This finding was even more pronounced when compared with diabetic BP patients without DPP4 inhibitors. Associations between anti-BP180 and anti-BP230 reactivities were also found in patients using insulin and antipsychotics, respectively, compared with patients without this medication, but not for the use of inhibitors of platelet aggregation, and L-thyroxine. CONCLUSION: Taken together, these data imply a relation between autoantibody reactivities at the time of diagnosis and both neuropsychiatric comorbidities as well as distinct concomitant medication suggesting a link between the pathological immune mechanisms and clinical conditions that precede the clinically overt AIBD.

Mast cell-deficient mice Mcpt5Cre/Dicer fl/fl redefine the role of mast cells in experimental bullous pemphigoid.

Background: Bullous pemphigoid (BP) is the most frequent autoimmune blistering disease of the skin affecting the elderly. BP is immunopathologically characterized by autoantibodies against BP180 and BP230. With the growing evidence of cell-mediated autoimmunity in the pathogenesis of BP, it still remains unclear whether mast cells (MCs) are involved, due to conflicting data obtained from Kit-dependent MC-deficient mouse models. Objectives: To clarify the role of MCs in experimental BP; the dynamics in cutaneous MC numbers, associated immune cells and the development of disease in Kit-independent MC-deficient mouse model. Methods: Employing a recently established murine adult passive transfer model of BP induced by the transfer of pathogenic immunoglobulin G (IgG), lesional skin biopsies were investigated histologically and immunohistochemically for the time-dependent MC accumulation and dermal infiltration. Results: The numbers of cutaneous MCs increased following the induction of BP, in part, maintained by MC proliferation. Numbers of T cells, neutrophils and eosinophils in the skin also increased after BP induction, with eosinophils showing a preferential co-localization with MCs. Furthermore, clinical disease manifestation in MC-deficient Mcpt5Cre/Dicer fl/fl mice remained unchanged compared to MC-sufficient Dicer fl/fl mice. The composition of the immune cell infiltration including as T cells, neutrophils and eosinophils was largely unaffected by the absence of MCs. Conclusion: MCs do not play a pivotal role in the pathogenesis of passive IgG-transfer mediated BP model. Their increase in number may be a bystander effect following tissue injury. We therefore suggest caution regarding the selection of MCs as sole targets for the development of novel drugs for BP.

Hybrid Silica-Coated PLGA Nanoparticles for Enhanced Enzyme-Based Therapeutics.

Some cancer cells rely heavily on non-essential biomolecules for survival, growth, and proliferation. Enzyme based therapeutics can eliminate these biomolecules, thus specifically targeting neoplastic cells; however, enzyme therapeutics are susceptible to immune clearance, exhibit short half-lives, and require frequent administration. Encapsulation of therapeutic cargo within biocompatible and biodegradable poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) is a strategy for controlled release. Unfortunately, PLGA NPs exhibit burst release of cargo shortly after delivery or upon introduction to aqueous environments where they decompose via hydrolysis. Here, we show the generation of hybrid silica-coated PLGA (SiLGA) NPs as viable drug delivery vehicles exhibiting sub-200 nm diameters, a metastable Zeta potential, and high loading efficiency and content. Compared to uncoated PLGA NPs, SiLGA NPs offer greater retention of enzymatic activity and slow the burst release of cargo. Thus, SiLGA encapsulation of therapeutic enzymes, such as asparaginase, could reduce frequency of administration, increase half-life, and improve efficacy for patients with a range of diseases.

Coexistence of bullous pemphigoid with neuropsychiatric comorbidities is associated with anti-BP230 seropositivity.

BACKGROUND: While clustering of bullous pemphigoid (BP) with neuropsychiatric diseases is well-established, the clinical and immunological profile of BP patients with this comorbidity remains to be decisively determined. OBJECTIVES: To evaluate the burden of neurological and psychiatric comorbidities among patients with BP and to elucidate the clinical, immunological and immunopathological features of patients with BP and comorbid neuropsychiatric conditions. METHODS: We performed a retrospective study encompassing patients diagnosed with BP throughout the years 2009-2020 in a specialized tertiary referral centre. Multivariate logistic regression model was used to identify predictors of neuropsychiatric conditions among patients with BP. RESULTS: The study included 273 patients with BP, of whom 123 (45.1%) presented with comorbid neuropsychiatric disease. Compared to the remaining patients with BP (n = 150), those with pre-existing neuropsychiatric diseases demonstrated older mean [standard deviation (SD)] age [81.7 (9.1) vs. 76.9 (10.1); P < 0.001], female preponderance (65.0% vs. 49.3%; P = 0.009), higher seropositivity rate of anti-BP230 (67.7% vs. 36.5%; P = 0.006) and higher levels of anti-BP180 NC16A IgG [651.3 (1279.6) vs. 370.4 (818.6) U/mL; P = 0.039]. In multivariate analysis, anti-BP230 seropositivity was independently associated with coexistence of BP with neuropsychiatric conditions [adjusted odds ratio (OR), 3.43; 95% CI, 1.24-9.52; P = 0.018]. In a sensitivity analysis confined to patients with neurological diseases (n = 103), older age [82.1 (8.4) vs. 77.2 (10.3); P < 0.001] and increased anti-BP230 seropositivity (68.0% vs. 39.7%; P = 0.018) were identified. CONCLUSIONS: The coexistence of BP with neuropsychiatric diseases is independently associated with the generation of anti-BP230 antibodies.

Incidence of pemphigoid diseases in Northern Germany in 2016 - first data from the Schleswig-Holstein Registry of Autoimmune Bullous Diseases.

BACKGROUND: Autoimmune bullous diseases (AIBD) are rare disorders characterized by autoantibody formation against components of adhesion molecules; in pemphigoid diseases (PD), these are proteins of hemidesmosomes and basement membrane, important for cell-matrix adhesion in skin and/or mucous membranes. Incidences of these diseases vary considerably between different populations. OBJECTIVES: To establish a registry prospectively recruiting all AIBD patients in a geographically well-defined region in Northern Germany (Schleswig-Holstein). METHODS: Only patients with verified disease (by clinical presentation, histology, direct and/or indirect immunofluorescence and /or ELISA) living in Schleswig-Holstein were included. Incidences of PD were estimated based on the total number of inhabitants in Schleswig-Holstein, stratified by birth year and sex. RESULTS: Of 67 patients with PD [35 male, 32 female, mean age 75 (standard deviation 14.3 years)], 83% were patients with bullous pemphigoid [n = 56, 28 male, 28 female, mean age 78 (SD 9.9)]. The resulting crude incidences were 23.4 patients/million/year for all pemphigoid patients, 19.6 patients/million/year for bullous pemphigoid (age-standardized 16.9 patients/million/year) with a strong increase in bullous pemphigoid patients in the age group of 85-90 years with 262 patients/million/year. Incidences for bullous pemphigoid were higher in urban compared to rural areas. Other PD (mucous membrane pemphigoid, linear IgA disease, anti-p200 pemphigoid) were less frequent with crude incidences of 2.1, 1.0 and 0.7 patients/million/year, respectively. CONCLUSIONS: This study prospectively analyses the incidence of PD in a carefully defined geographical area. The highest incidence among PD patients was found for bullous pemphigoid. The incidence of bullous pemphigoid is considerably increased compared to previous reports and reveals regional differences. Further studies are needed in order to clarify these findings.

Expression of PD-1 and Tim-3 is increased in skin of patients with bullous pemphigoid and pemphigus vulgaris.

BACKGROUND: Bullous pemphigoid (BP) and pemphigus vulgaris (PV) are common autoimmune bullous dermatoses (AIBD) characterized by blisters and erosions. Treatment options are limited and often insufficient. Immune checkpoint receptors play critical roles in immune homoeostasis and self- tolerance. Targeting checkpoint receptors is highly efficient in treatment of various cancers, but often also associated with autoimmune side effects. OBJECTIVES: We therefore aimed to investigate the expression of immune checkpoint receptors in patients with BP and PV. METHODS: We analysed expression of the checkpoint receptors programmed cell death protein 1 (PD-1), T-cell immunoglobulin and mucin domain 3 (Tim-3) and lymphocyte activation gene 3 (Lag-3) in lesional skin of patients with BP and PV compared to healthy control skin as well as the expression patterns of PD-1 and Tim-3 on various infiltrating immune cells in skin sections of AIBD by immunohistochemistry and immunofluorescence. We also measured serum levels of soluble PD-1, Tim-3 and Lag-3 in AIBD patients by ELISA. RESULTS: We report on increased expression of PD-1 and Tim-3, but not Lag-3, in lesional skin of patients with BP and PV. Investigating the expression pattern of PD-1 and Tim-3 on different cutaneous immune cells, we observed significant upregulation of PD-1 predominantly on infiltrating CD8 T cells and upregulation of Tim-3 on CD8 T cells as well as macrophages. CONCLUSIONS: Our results suggest exploring immune checkpoint receptors as novel therapeutic targets using an agonistic approach in autoimmune bullous diseases.

Quantitative atomic force microscopy provides new insight into matrix vesicle mineralization.

Matrix vesicles (MVs) are a class of extracellular vesicles that initiate mineralization in cartilage, bone, and other vertebrate tissues by accumulating calcium ions (Ca2+) and inorganic phosphate (Pi) within their lumen and forming a nucleation core (NC). After further sequestration of Ca2+ and Pi, the NC transforms into crystalline complexes. Direct evidence of the existence of the NC and its maturation have been provided solely by analyses of dried samples. We isolated MVs from chicken embryo cartilage and used atomic force microscopy peak force quantitative nanomechanical property mapping (AFM-PFQNM) to measure the nanomechanical and morphological properties of individual MVs under both mineralizing (+Ca2+) and non-mineralizing (-Ca2+) fluid conditions. The elastic modulus of MVs significantly increased by 4-fold after incubation in mineralization buffer. From AFM mapping data, we inferred the morphological changes of MVs as mineralization progresses: prior to mineralization, a punctate feature, the NC, is present within MVs and this feature grows and stiffens during mineralization until it occupies most of the MV lumen. Dynamic light scattering showed a significant increase in hydrodynamic diameter and no change in the zeta potential of hydrated MVs after incubation with Ca2+. This validates that crystalline complexes, which are strongly negative relative to MVs, were forming within the lumen of MVs. These data were substantiated by transmission electron microscopy energy dispersive X-ray and Fourier transform infrared spectroscopic analyses of dried MVs, which provide evidence that the complexes increased in size, crystallinity, and Ca/P ratio within MVs during the mineralization process.

Silica cloaking of adenovirus enhances gene delivery while reducing immunogenicity.

Viral gene therapy is a means of delivering genes to replace malfunctioning ones, to kill cancer cells, or to correct genetic mutations. This technology is emerging as a powerful clinical tool; however, it is still limited by viral tropism, uptake and clearance by the liver, and most importantly an immune response. To overcome these challenges, we sought to merge the robustness of viral gene expression and the versatility of nanoparticle technology. Here, we describe a method for cloaking adenovirus (Ad) in silica (SiAd) as a nanoparticle formulation that significantly enhances transduction. Intratumoral injections in human glioma xenografts revealed SiAd expressing luciferase improved tumor transduction while reducing liver uptake. In immune-competent mice SiAd induced no inflammatory cytokines and reduced production of neutralizing antibodies. Finally, SiAd expressing TNF-related apoptosis-inducing ligand inhibited tumor growth of glioma xenografts. These results reveal that silica cloaking of Ad can enhance viral gene delivery while reducing immunogenicity.

Supramolecular self assembly of nanodrill-like structures for intracellular delivery.

Despite recent advances in the supramolecular assembly of cell-penetrating peptide (CPP) nanostructures, the tuning of size, shape, morphology and packaging of drugs in these materials still remain unexplored. Herein, through sequential ligation of peptide building blocks, we create cell-penetrating self-assembling peptide nanomaterials (CSPNs) with the capability to translocate inside cells. We devised a triblock array of Tat48-59 [HIV-1 derived transactivator of transcription48-59] based CPPs, conjugated to up to four Phenylalanine (Phe) residues through an amphiphilic linker, (RADA)2. We observed that the sequential addition of Phe leads to the transition of CSPN secondary structures from a random coil, to a distorted α-helix, a ß-sheet, or a pure α-helix. This transition occurs due to formation of a heptad by virtue of even number of Phe. Atomic force microscopy revealed that CSPNs form distinct shapes reminiscent of a "drill-bit". CSPNs containing two, three or four Phe, self-assemble into "nanodrill-like structures" with a coarse-twisted, non-twisted or fine-twisted morphology, respectively. These nanodrills had a high capacity to encapsulate hydrophobic guest molecules. In particular, the coarse-twisted nanodrills demonstrate higher internalization and are able to deliver rapamycin, a hydrophobic small molecule that induced autophagy and are capable of in vivo delivery. Molecular dynamics studies provide microscopic insights into the structure of the nanodrills that can contribute to its morphology and ability to interact with cellular membrane. CSPNs represent a new modular drug delivery platform that can be programmed into exquisite structures through sequence-specific fine tuning of amino acids.

Rapid Isolation and Detection of Exosomes and Associated Biomarkers from Plasma.

Exosomes found in the circulation are a primary source of important cancer-related RNA and protein biomarkers that are expected to lead to early detection, liquid biopsy, and point-of-care diagnostic applications. Unfortunately, due to their small size (50-150 nm) and low density, exosomes are extremely difficult to isolate from plasma. Current isolation methods are time-consuming multistep procedures that are unlikely to translate into diagnostic applications. To address this issue, we demonstrate the ability of an alternating current electrokinetic (ACE) microarray chip device to rapidly isolate and recover glioblastoma exosomes from undiluted human plasma samples. The ACE device requires a small plasma sample (30-50 µL) and is able to concentrate the exosomes into high-field regions around the ACE microelectrodes within 15 min. A simple buffer wash removes bulk plasma materials, leaving the exosomes concentrated on the microelectrodes. The entire isolation process and on-chip fluorescence analysis is completed in less than 30 min which enables subsequent on-chip immunofluorescence detection of exosomal proteins, and provides viable mRNA for RT-PCR analysis. These results demonstrate the ability of the ACE device to streamline the process for isolation and recovery of exosomes, significantly reducing the number of processing steps and time required.

Severe bullous pemphigoid associated with pembrolizumab therapy for metastatic melanoma with complete regression.

Bullous pemphigoid (BP) is considered to be a humorally mediated autoimmune disease, but autoreactive T-cells and T-regulatory cells (Tregs) have also been implicated in this disease. Tregs and the programmed death-1 (PD-1) : programmed death ligand (PD-L) pathway are both critical in terminating immune response, and elimination of either can result in breakdown of tolerance and development of autoimmunity. We report a patient with metastatic malignant melanoma (MM), who underwent pembrolizumab (anti-PD-1) therapy following unsuccessful treatment with ipilimumab [anti-cytotoxic T-lymphocyte-associated protein (CTLA)-4]. The patient developed BP with increasing serum titres of anti-BP180 IgG autoantibodies and increasing disease severity during pembrolizumab therapy. High doses of corticosteroids and methotrexate were needed to control the BP. Following the termination of pembrolizumab therapy, imaging showed complete regression of all metastatic sites. This result may indicate a crucial role for T-cell suppressive activity in controlling and preventing BP.

Selection of DNA nanoparticles with preferential binding to aggregated protein target.

High affinity and specificity are considered essential for affinity reagents and molecularly-targeted therapeutics, such as monoclonal antibodies. However, life's own molecular and cellular machinery consists of lower affinity, highly multivalent interactions that are metastable, but easily reversible or displaceable. With this inspiration, we have developed a DNA-based reagent platform that uses massive avidity to achieve stable, but reversible specific recognition of polyvalent targets. We have previously selected these DNA reagents, termed DeNAno, against various cells and now we demonstrate that DeNAno specific for protein targets can also be selected. DeNAno were selected against streptavidin-, rituximab- and bevacizumab-coated beads. Binding was stable for weeks and unaffected by the presence of soluble target proteins, yet readily competed by natural or synthetic ligands of the target proteins. Thus DeNAno particles are a novel biomolecular recognition agent whose orthogonal use of avidity over affinity results in uniquely stable yet reversible binding interactions.

Acoustic Microcannons: Toward Advanced Microballistics.

Acoustically triggered microcannons, capable of loading and firing nanobullets (Nbs), are presented as powerful microballistic tools. Hollow conically shaped microcannon structures have been synthesized electrochemically and fully loaded with nanobullets made of silica or fluorescent microspheres, and perfluorocarbon emulsions, embedded in a gel matrix stabilizer. Application of a focused ultrasound pulse leads to the spontaneous vaporization of the perfluorocarbon emulsions within the microcannon and results in the rapid ejection of the nanobullets. Such Nbs "firing" at remarkably high speeds (on the magnitude of meters per second) has been modeled theoretically and demonstrated experimentally. Arrays of microcannons anchored in a template membrane were used to demonstrate the efficient Nbs loading and the high penetration capabilities of the ejected Nbs in a tissue phantom gel. This acoustic-microcannon approach could be translated into advanced microscale ballistic tools, capable of efficient loading and firing of multiple cargoes, and offer improved accessibility to target locations and enhanced tissue penetration properties.

The influence of distance between microbubbles on the fluid flow produced during ultrasound exposure.

The collapse dynamics of lipid monolayer-coated microbubbles in the clinically-relevant size range under 6 µm in diameter have not been studied directly due to their small size obscuring the collapse visualization. This study investigates the influence of inter-microbubble distance on the shape of lipid debris clouds created by the collapse of the microbubble destroying the microbubble lipid monolayer. The shape was highly influenced by the fluid motion that occurred as the microbubbles collapsed. It was observed that at inter-microbubble distances smaller than 37 µm the microbubbles began to interact with one another resulting in distorted and ellipsoid-shaped debris clouds. At inter-microbubble distances less than 10 µm, significantly elongated debris clouds were observed that extended out from the original microbubble location in a single direction. These distortions show a significant distance-dependent interaction between microbubbles. It was observed that microbubbles in physical contact with one another behaved in the same manner as separate microbubbles less than 10 µm apart creating significantly elongated debris clouds. It can be hypothesized that small inter-microbubble distances influence the microbubble to collapse asymmetrically resulting in the creation of fluid jets that contribute to the formation of debris fields that are elongated in a single direction.

Manipulating nanoscale features on the surface of dye-loaded microbubbles to increase their ultrasound-modulated fluorescence output.

The nanoscale surface features of lipid-coated microbubbles can dramatically affect how the lipids interact with one another as the microbubble diameter expands and contracts under the influence of ultrasound. During microbubble manufacturing, the different lipid shell species naturally partition forming concentrated lipid islands. In this study the dynamics of how these nanoscale islands accommodate the expansion of the microbubbles are monitored by measuring the fluorescence intensity changes that occur as self-quenching lipophilic dye molecules embedded in the lipid layer change their distance from one another. It was found that when the dye molecules were concentrated in islands, less than 5% of the microbubbles displayed measurable fluorescence intensity modulation indicating the islands were not able to expand sufficiently for the dye molecules to separate from one another. When the microbubbles were heated and cooled rapidly through the lipid transition temperature the islands were melted creating an even distribution of dye about the surface. This resulted in over 50% of the microbubbles displaying the fluorescence-modulated signal indicating that the dye molecules could now separate sufficiently to change their self-quenching efficiency. The separation of the surface lipids in these different formations has significant implications for microbubble development as ultrasound and optical contrast agents.

Molecular epidemiology of Legionnaires' disease in Israel.

National surveillance of Legionnaires' disease (LD) is important to inform control measures and facilitate international networking for timely reporting. This study is the first to describe the molecular epidemiology of LD in Israel. Case notifications for 2006-2011, collated through mandatory reporting, were identified and demographic, clinical and laboratory data were extracted. Unrelated clinical and environmental Legionella pneumophila strains were characterized using standard procedures, Dresden panel of monoclonal antibodies and the ESCMID Study Group for Legionella Infections (ESGLI) Sequence-Based Typing scheme. In all, 294 cases were reported (crude incidence 0.67 cases/100 000; age-standardized incidence 1/100 000). LD epidemiological trends and features largely resembled those of the EU, except for a larger proportion of nosocomial cases. Of 28 clinical and 23 environmental strains analysed, 71.4% and 21.7% were serogroup (sg) 1 and the most common immunological subgroup was OLDA/Oxford (64%). Of the clinical strains, OLDA/Oxford, ST1 was the most common (43%) followed by Allentown/France, ST40 (14%). The unusual sg 3 ST338 was found in 17.4% of environmental strains. Novel STs were detected amongst 23.5% of strains. These findings warrant further molecular investigation. Molecular epidemiology data generated from neighbouring countries newly adopting the ESGLI typing scheme for L. pneumophila contribute to understanding of regional strain diversity.

In vivo ultrasound visualization of non-occlusive blood clots with thrombin-sensitive contrast agents.

The use of microbubbles as ultrasound contrast agents is one of the primary methods to diagnose deep venous thrombosis. However, current microbubble imaging strategies require either a clot sufficiently large to produce a circulation filling defect or a clot with sufficient vascularization to allow for targeted accumulation of contrast agents. Previously, we reported the design of a microbubble formulation that modulated its ability to generate ultrasound contrast from interaction with thrombin through incorporation of aptamer-containing DNA crosslinks in the encapsulating shell, enabling the measurement of a local chemical environment by changes in acoustic activity. However, this contrast agent lacked sufficient stability and lifetime in blood to be used as a diagnostic tool. Here we describe a PEG-stabilized, thrombin-activated microbubble (PSTA-MB) with sufficient stability to be used in vivo in circulation with no change in biomarker sensitivity. In the presence of actively clotting blood, PSTA-MBs showed a 5-fold increase in acoustic activity. Specificity for the presence of thrombin and stability under constant shear flow were demonstrated in a home-built in vitro model. Finally, PSTA-MBs were able to detect the presence of an active clot within the vena cava of a rabbit sufficiently small as to not be visible by current non-specific contrast agents. By activating in non-occlusive environments, these contrast agents will be able to detect clots not diagnosable by current contrast agents.

Isolation of rare tumor cells from blood cells with buoyant immuno-microbubbles.

Circulating tumor cells (CTCs) are exfoliated at various stages of cancer, and could provide invaluable information for the diagnosis and prognosis of cancers. There is an urgent need for the development of cost-efficient and scalable technologies for rare CTC enrichment from blood. Here we report a novel method for isolation of rare tumor cells from excess of blood cells using gas-filled buoyant immuno-microbubbles (MBs). MBs were prepared by emulsification of perfluorocarbon gas in phospholipids and decorated with anti-epithelial cell adhesion molecule (EpCAM) antibody. EpCAM-targeted MBs efficiently (85%) and rapidly (within 15 minutes) bound to various epithelial tumor cells suspended in cell medium. EpCAM-targeted MBs efficiently (88%) isolated frequent tumor cells that were spiked at 100,000 cells/ml into plasma-depleted blood. Anti-EpCAM MBs efficiently (>77%) isolated rare mouse breast 4T1, human prostate PC-3 and pancreatic cancer BxPC-3 cells spiked into 1, 3 and 7 ml (respectively) of plasma-depleted blood. Using EpCAM targeted MBs CTCs from metastatic cancer patients were isolated, suggesting that this technique could be developed into a valuable clinical tool for isolation, enumeration and analysis of rare cells.