Immune cells and inflammatory mediators cause endothelial dysfunction in a vascular microphysiological system.

Functional assessment of endothelium serves as an important indicator of vascular health and is compromised in vascular disorders including hypertension, atherosclerosis, and preeclampsia. Endothelial dysfunction in these cases is linked to dysregulation of the immune system involving both changes to immune cells and increased secretion of inflammatory cytokines. Herein, we utilize a well-established microfluidic device to generate a 3-dimensional vascular microphysiological system (MPS) consisting of a tubular blood vessel lined with human umbilical vein endothelial cells (HUVECs) to evaluate endothelial function measured via endothelial permeability and Ca2+ signaling. We evaluated the effect of a mixture of factors associated with inflammation and cardiovascular disease (TNFα, VEGF-A, IL-6 at 10 ng ml-1 each) on vascular MPS and inferred that inflammatory mediators contribute to endothelial dysfunction by disrupting the endothelial barrier over a 48 hour treatment and by diminishing coordinated Ca2+ activity over a 1 hour treatment. We also evaluated the effect of peripheral blood mononuclear cells (PBMCs) on endothelial permeability and Ca2+ signaling in the HUVEC MPS. HUVECs were co-cultured with PBMCs either directly wherein PBMCs passed through the lumen or indirectly with PBMCs embedded in the supporting collagen hydrogel. We revealed that phytohemagglutinin (PHA)-M activated PBMCs cause endothelial dysfunction in MPS both through increased permeability and decreased coordinated Ca2+ activity compared to non-activated PBMCs. Our MPS has potential applications in modeling cardiovascular disorders and screening for potential treatments using measures of endothelial function.

Visualization-enhanced under-oil open microfluidic system for in situ characterization of multi-phase chemical reactions.

Under-oil open microfluidic system, utilizing liquid-liquid boundaries for confinements, offers inherent advantages including clogging-free flow channels, flexible access to samples, and adjustable gas permeation, making it well-suited for studying multi-phase chemical reactions that are challenging for closed microfluidics. However, reports on the novel system have primarily focused on device fabrication and functionality demonstrations within biology, leaving their application in broader chemical analysis underexplored. Here, we present a visualization-enhanced under-oil open microfluidic system for in situ characterization of multi-phase chemical reactions with Raman spectroscopy. The enhanced system utilizes a semi-transparent silicon (Si) nanolayer over the substrate to enhance visualization in both inverted and upright microscope setups while reducing Raman noise from the substrate. We validated the system's chemical stability and capability to monitor gas evolution and gas-liquid reactions in situ. The enhanced under-oil open microfluidic system, integrating Raman spectroscopy, offers a robust open-microfluidic platform for label-free molecular sensing and real-time chemical/biochemical process monitoring in multi-phase systems.

Surface colonization by Flavobacterium johnsoniae promotes its survival in a model microbial community.

Flavobacterium johnsoniae is a ubiquitous soil and rhizosphere bacterium, but despite its abundance, the factors contributing to its success in communities are poorly understood. Using a model microbial community, The Hitchhikers of the Rhizosphere (THOR), we determined the effects of colonization on the fitness of F. johnsoniae in the community. Insertion sequencing, a massively parallel transposon mutant screen, on sterile sand identified 25 genes likely to be important for surface colonization. We constructed in-frame deletions of candidate genes predicted to be involved in cell membrane biogenesis, motility, signal transduction, and transport of amino acids and lipids. All mutants poorly colonized sand, glass, and polystyrene and produced less biofilm than the wild type, indicating the importance of the targeted genes in surface colonization. Eight of the nine colonization-defective mutants were also unable to form motile biofilms or zorbs, thereby suggesting that the affected genes play a role in group movement and linking stationary and motile biofilm formation genetically. Furthermore, we showed that the deletion of colonization genes in F. johnsoniae affected its behavior and survival in THOR on surfaces, suggesting that the same traits are required for success in a multispecies microbial community. Our results provide insight into the mechanisms of surface colonization by F. johnsoniae and form the basis for further understanding its ecology in the rhizosphere. IMPORTANCE: Microbial communities direct key environmental processes through multispecies interactions. Understanding these interactions is vital for manipulating microbiomes to promote health in human, environmental, and agricultural systems. However, microbiome complexity can hinder our understanding of the underlying mechanisms in microbial community interactions. As a first step toward unraveling these interactions, we explored the role of surface colonization in microbial community interactions using The Hitchhikers Of the Rhizosphere (THOR), a genetically tractable model community of three bacterial species, Flavobacterium johnsoniae, Pseudomonas koreensis, and Bacillus cereus. We identified F. johnsoniae genes important for surface colonization in solitary conditions and in the THOR community. Understanding the mechanisms that promote the success of bacteria in microbial communities brings us closer to targeted manipulations to achieve outcomes that benefit agriculture, the environment, and human health.

PTP1B phosphatase dampens iPSC-derived neutrophil motility and antimicrobial function.

Neutrophils are rapidly recruited to sites of infection and are critical for pathogen clearance. Therapeutic use of primary neutrophils has been limited as they have a short lifespan and are not amenable to genetic manipulation. Human induced pluripotent stem cells (iPSCs) can provide a robust source of neutrophils for infusion and are genetically tractable. However, current work has indicated that dampened intracellular signaling limits iPSC-derived neutrophil (iNeutrophil) cellular activation and antimicrobial response. Here, we show that protein tyrosine phosphatase 1B (PTP1B) inhibits intracellular signaling and dampens iNeutrophil effector function. Deletion of the PTP1B phosphatase increased PI3K and ERK signaling and was associated with increased F-actin polymerization, cell migration and phagocytosis. In contrast, other effector functions like NETosis and ROS production were reduced. PTP1B-deficient neutrophils were more responsive to A. fumigatus and displayed rapid recruitment and control of hyphal growth. Accordingly, depletion of PTP1B increased production of inflammatory factors including the neutrophil chemokine IL-8. Taken together, these findings suggest that PTP1B limits iNeutrophil motility and antimicrobial function.

Neutrophil motility is regulated by both cell intrinsic and endothelial cell ARPC1B.

Neutrophil-directed motility is necessary for host defense, but its dysregulation can also cause collateral tissue damage. Actinopathies are monogenic disorders that affect the actin cytoskeleton and lead to immune dysregulation. Deficiency in ARPC1B, a component of the Arp2/3 complex, results in vascular neutrophilic inflammation; however, the mechanism remains unclear. Here, we generated human induced pluripotent stem cell (iPSC)-derived neutrophils (denoted iNeutrophils) that are deficient in ARPC1B and show impaired migration and a switch from forming pseudopodia to forming elongated filopodia. We show, using a blood vessel on a chip model, that primary human neutrophils have impaired movement across an endothelium deficient in APRC1B. We also show that the combined deficiency of ARPC1B in iNeutrophils and endothelium results in further reduction in neutrophil migration. Taken together, these results suggest that ARPC1B in endothelium is sufficient to drive neutrophil behavior. Furthermore, the findings provide support for using the iPSC system to understand human neutrophil biology and model disease in a genetically tractable system.

The effect of whole blood logistics on neutrophil non-specific activation and kinetics ex vivo.

While the exquisite sensitivity of neutrophils enables their rapid response to infection in vivo; this same sensitivity complicates the ex vivo study of neutrophils. Handling of neutrophils ex vivo is fraught with unwanted heterogeneity and alterations that can diminish the reproducibility of assays and limit what biological conclusions can be drawn. There is a need to better understand the influence of ex vivo procedures on neutrophil behavior to guide improved protocols for ex vivo neutrophil assessment to improve inter/intra-experimental variability. Here, we investigate how whole blood logistics (i.e., the procedure taken from whole blood collection to delivery of the samples to analytical labs and storage before neutrophil interrogation) affects neutrophil non-specific activation (i.e., baseline apoptosis and NETosis) and kinetics (i.e., activation over time). All the experiments (60+ whole blood neutrophil isolations across 36 blood donors) are performed by a single operator with optimized isolation and culture conditions, and automated image analysis, which together increase rigor and consistency. Our results reveal: (i) Short-term storage (< 8 h) of whole blood does not significantly affect neutrophil kinetics in subsequent two-dimensional (2D) cell culture; (ii) Neutrophils from long-term storage (> 24 h) in whole blood show significantly higher stability (i.e., less non-specific activation) compared to the control group with the isolated cells in 2D culture. (iii) Neutrophils have greater non-specific activation and accelerated kinetic profiles when stored in whole blood beyond 48 h.

Naive primary neutrophils play a dual role in the tumor microenvironment.

The tumor microenvironment (TME) is characterized by a network of cancer cells, recruited immune cells and extracellular matrix (ECM) in a hypoxic microenvironment. However, the specific role of neutrophils during tumor development, and their interactions with other immune cells is still not well understood. Thus, there is a need to investigate the interaction between primary neutrophils and natural killer cells and the resulting effects on tumor development. Here we use both standard well plate culture and an under oil microfluidic (UOM) assay with an integrated extracellular cell matrix (ECM) bridge to elucidate how naive primary neutrophils respond to both patient derived tumor cells and tumor cell lines. Our data demonstrated that both patient derived head and neck squamous cell carcinoma (HNSCC) tumor cells and MDA-MB-231 breast cancer cells trigger cluster formation in neutrophils, and the swarm of neutrophils restricts tumor invasion through the generation of reactive oxygen species (ROS) and neutrophil extracellular trap (NETs) release within the neutrophil cluster. However, we also observed that the presence of neutrophils downregulates granzyme B in NK-92 cells and the resulting NETs can obstruct NK cells from penetrating the tumor mass in vitro suggesting a dual role for neutrophils in the TME. Further, using label-free optical metabolic imaging (OMI) we observed changes in the metabolic activities of primary neutrophils during the different swarming phases when challenged with tumor cells. Finally, our data demonstrates that neutrophils in direct contact, or in close proximity, with tumor cells exhibit greater metabolic activities (lower nicotinamide adenine dinucleotide phosphate (NAD(P)H) mean lifetime) compared to non-contact neutrophils.

Microphysiological systems for solid tumor immunotherapy: opportunities and challenges.

Immunotherapy remains more effective for hematologic tumors than for solid tumors. One of the main challenges to immunotherapy of solid tumors is the immunosuppressive microenvironment these tumors generate, which limits the cytotoxic capabilities of immune effector cells (e.g., cytotoxic T and natural killer cells). This microenvironment is characterized by hypoxia, nutrient starvation, accumulated waste products, and acidic pH. Tumor-hijacked cells, such as fibroblasts, macrophages, and T regulatory cells, also contribute to this inhospitable microenvironment for immune cells by secreting immunosuppressive cytokines that suppress the antitumor immune response and lead to immune evasion. Thus, there is a strong interest in developing new drugs and cell formulations that modulate the tumor microenvironment and reduce tumor cell immune evasion. Microphysiological systems (MPSs) are versatile tools that may accelerate the development and evaluation of these therapies, although specific examples showcasing the potential of MPSs remain rare. Advances in microtechnologies have led to the development of sophisticated microfluidic devices used to recapitulate tumor complexity. The resulting models, also known as microphysiological systems (MPSs), are versatile tools with which to decipher the molecular mechanisms driving immune cell antitumor cytotoxicity, immune cell exhaustion, and immune cell exclusion and to evaluate new targeted immunotherapies. Here, we review existing microphysiological platforms to study immuno-oncological applications and discuss challenges and opportunities in the field.

Liquid-liquid interfaces enable tunable cell confinement to recapitulate surrounding tissue deformations during neutrophil interstitial migration in vivo.

Cell migration is regulated by an interplay between both chemical and mechanical cues. Immune cells navigate through interstitial spaces and generate forces to deform surrounding cells, which in turn exert opposing pressures that regulate cell morphology and motility mechanisms. Current in vitro systems to study confined cell migration largely utilize rigid materials orders of magnitude stiffer than surrounding cells, limiting insights into how these local physical interactions regulate interstitial cell motility. Here, we first characterize mechanical interactions between neutrophils and surrounding cells in larval zebrafish and subsequently engineer in vitro migration channels bound by a deformable liquid-liquid interface that responds to cell generated pressures yielding a gradient of confinement across the length of a single cell. Tuning confining pressure gradients replicates mechanical interactions with surrounding cells during interstitial migration in vivo . We find that neutrophils favor a bleb-based mechanism of force generation to deform a barrier applying cell-scale confining forces. This work introduces a biomimetic material interface that enables new avenues of exploring the influence of mechanical forces on cell migration.

Modeling Sepsis-Associated ARDS Using a Lung Endothelial Microphysiological System.

Acute respiratory distress syndrome due to non-pulmonary causes exhibits prominent endothelial activation which is challenging to assess in critically ill patients. Preclinical in vivo models of sepsis and ARDS have failed to yield useful therapies in humans, perhaps due to interspecies differences in inflammatory responses. Use of microphysiological systems (MPS) offer improved fidelity to human biological responses and better predict pharmacological responses than traditional culture. We adapted a lung endothelial MPS based on the LumeNEXT platform to evaluate the effect of plasma from critically ill sepsis patients on endothelial permeability, adhesion molecule expression and inflammatory cytokine production. Lumens incubated with sepsis plasma exhibited areas of contraction, loss of cellular coverage, and luminal defects. Sepsis plasma-incubated lumens had significantly increased permeability compared to lumens incubated with healthy donor plasma. ICAM-1 expression increased significantly in lumens incubated with sepsis plasma compared with those incubated with healthy control plasma, while concentrations of IL-6, IL-18, and soluble VEGF-R1 increased in sepsis plasma before and after incubation in the MPS compared with healthy control plasma. Use of the lung endothelial MPS may enable interrogation of specific mechanisms of endothelial dysfunction that promote ARDS in sepsis patients.

Microphysiological model reveals the promise of memory-like natural killer cell immunotherapy for HIV± cancer.

Numerous studies are exploring the use of cell adoptive therapies to treat hematological malignancies as well as solid tumors. However, there are numerous factors that dampen the immune response, including viruses like human immunodeficiency virus. In this study, we leverage human-derived microphysiological models to reverse-engineer the HIV-immune system interaction and evaluate the potential of memory-like natural killer cells for HIV+ head and neck cancer, one of the most common tumors in patients living with human immunodeficiency virus. Here, we evaluate multiple aspects of the memory-like natural killer cell response in human-derived bioengineered environments, including immune cell extravasation, tumor penetration, tumor killing, T cell dependence, virus suppression, and compatibility with retroviral medication. Overall, these results suggest that memory-like natural killer cells are capable of operating without T cell assistance and could simultaneously destroy head and neck cancer cells as well as reduce viral latency.

Griddient: a microfluidic array to generate reconfigurable gradients on-demand for spatial biology applications.

Biological tissues are highly organized structures where spatial-temporal gradients (e.g., nutrients, hypoxia, cytokines) modulate multiple physiological and pathological processes including inflammation, tissue regeneration, embryogenesis, and cancer progression. Current in vitro technologies struggle to capture the complexity of these transient microenvironmental gradients, do not provide dynamic control over the gradient profile, are complex and poorly suited for high throughput applications. Therefore, we have designed Griddent, a user-friendly platform with the capability of generating controllable and reversible gradients in a 3D microenvironment. Our platform consists of an array of 32 microfluidic chambers connected to a 384 well-array through a diffusion port at the bottom of each reservoir well. The diffusion ports are optimized to ensure gradient stability and facilitate manual micropipette loading. This platform is compatible with molecular and functional spatial biology as well as optical and fluorescence microscopy. In this work, we have used this platform to study cancer progression.

A high-throughput sensory assay for parasitic and free-living nematodes.

Sensory pathways first elucidated in Caenorhabditis elegans are conserved across free-living and parasitic nematodes, even though each species responds to a diverse array of compounds. Most nematode sensory assays are performed by tallying observations of worm behavior on two-dimensional planes using agarose plates. These assays have been successful in the study of volatile sensation but are poorly suited for investigation of water-soluble gustation or parasitic nematodes without a free-living stage. In contrast, gustatory assays tend to be tedious, often limited to the manipulation of a single individual at a time. We have designed a nematode sensory assay using a microfluidics device that allows for the study of gustation in a 96-well, three-dimensional environment. This device is suited for free-living worms and parasitic worms that spend their lives in an aqueous environment, and we have used it to show that ivermectin inhibits the gustatory ability of vector-borne parasitic nematodes. Insight box Nematodes are powerful model organisms for understanding the sensory biology of multicellular eukaryotes, and many parasitic species cause disease in humans. Simple sensory assays performed on agarose plates have been the bedrock for establishing the neuronal, genetic, and developmental foundations for many sensory modalities in nematodes. However, these classical assays are poorly suited for translational movement of many parasitic nematodes and the sensation of water-soluble molecules (gustation). We have designed a device for high-throughput nematode sensory assays in a gel matrix. This 'gustatory microplate' is amenable to several species and reveals novel responses by free-living and parasitic nematodes to cues and drugs.

A high-throughput nematode sensory assay reveals an inhibitory effect of ivermectin on parasite gustation.

Sensory pathways first elucidated in Caenorhabditis elegans are conserved across free-living and parasitic nematodes, even though each species responds to a diverse array of compounds. Most nematode sensory assays are performed by tallying observations of worm behavior on two-dimensional planes using agarose plates. These assays have been successful in the study of volatile sensation but are poorly suited for investigation of water-soluble gustation or parasitic nematodes without a free-living stage. In contrast, gustatory assays tend to be tedious, often limited to the manipulation of a single individual at a time. We have designed a nematode sensory assay using a microfluidics device that allows for the study of gustation in a 96-well, three-dimensional environment. This device is suited for free-living worms and parasitic worms that spend their lives in an aqueous environment, and we have used it to show that ivermectin inhibits the gustatory ability of vector-borne parasitic nematodes.

Microphysiological head and neck cancer model identifies novel role of lymphatically secreted monocyte migration inhibitory factor in cancer cell migration and metabolism.

Regional metastasis of head and neck cancer (HNC) is prevalent (approximately 50% of patients at diagnosis), yet the underlying drivers and mechanisms of lymphatic spread remain unclear. The complex tumor microenvironment (TME) of HNC plays a crucial role in disease maintenance and progression; however, the contribution of the lymphatics remains underexplored. We created a primary patient cell derived microphysiological system that incorporates cancer-associated-fibroblasts from patients with HNC alongside a HNC tumor spheroid and a lymphatic microvessel to create an in vitro TME platform to investigate metastasis. Screening of soluble factor signaling identified novel secretion of macrophage migration inhibitory factor (MIF) by lymphatic endothelial cells conditioned in the TME. Importantly, we also observed patient-to-patient heterogeneity in cancer cell migration similar to the heterogeneity observed in clinical disease. Optical metabolic imaging at the single cell level identified a distinct metabolic profile of migratory versus non-migratory HNC cells in a microenvironment dependent manner. Additionally, we report a unique role of MIF in increasing HNC reliance on glycolysis over oxidative phosphorylation. This multicellular, microfluidic platform expands the tools available to explore HNC biology in vitro through multiple orthogonal outputs and establishes a system with enough resolution to visualize and quantify patient-to-patient heterogeneity.

The effect of whole blood logistics on neutrophil non-specific activation and kinetics ex vivo.

While the exquisite sensitivity of neutrophils enables their rapid response to infection in vivo; this same sensitivity complicates the ex vivo study of neutrophils. Handling of neutrophils ex vivo is fraught with unwanted heterogeneity and alterations that can diminish the reproducibility of assays and limit what biological conclusions can be drawn. There is a need to better understand the influence of ex vivo procedures on neutrophil behavior to guide improved protocols for ex vivo neutrophil assessment to improve inter/intra-experimental variability. Here, we investigate how whole blood logistics (i.e., the procedure taken from whole blood collection to delivery of the samples to analytical labs and storage before neutrophil interrogation) affects neutrophil non-specific activation (i.e., baseline apoptosis and NETosis) and kinetics (i.e., activation over time). All the experiments (60+ whole blood neutrophil isolations across 36 blood donors) are performed by a single operator with optimized isolation and culture conditions, and automated image analysis, which together increase rigor and consistency. Our results reveal: i) Short-term storage (<8 h) of whole blood does not significantly affect neutrophil kinetics in subsequent two-dimensional (2D) cell culture; ii) Neutrophils from long-term storage (>24 h) in whole blood show significantly higher stability (i.e., less non-specific activation) compared to the control group with the isolated cells in 2D culture. iii) Neutrophils have greater non-specific activation and accelerated kinetic profiles when stored in whole blood beyond 48 h.

Under-oil open microfluidic systems for rapid phenotypic antimicrobial susceptibility testing.

Antimicrobial susceptibility testing (AST) remains the cornerstone of effective antimicrobial selection and optimization in patients. Despite recent advances in rapid pathogen identification and resistance marker detection with molecular diagnostics (e.g., qPCR, MALDI-TOF MS), phenotypic (i.e., microbial culture-based) AST methods - the gold standard in hospitals/clinics - remain relatively unchanged over the last few decades. Microfluidics-based phenotypic AST has been growing fast in recent years, aiming for rapid (i.e., turnaround time <8 h), high-throughput, and automated species identification, resistance detection, and antibiotics screening. In this pilot study, we describe the application of a multi-liquid-phase open microfluidic system, named under-oil open microfluidic systems (UOMS), to achieve a rapid phenotypic AST. UOMS provides an open microfluidics-based solution for rapid phenotypic AST (UOMS-AST) by implementing and recording a pathogen's antimicrobial activity in micro-volume testing units under an oil overlay. UOMS-AST allows free physical access (e.g., by standard pipetting) to the system and label-free, single-cell resolution optical access. UOMS-AST can accurately and rapidly determine antimicrobial activities [including susceptibility/resistance breakpoint and minimum inhibitory concentration (MIC)] from nominal sample/bacterial cells in a system aligned with clinical laboratory standards where open systems and optical microscopy are predominantly adopted. Further, we combine UOMS-AST with a cloud lab data analytic technique for real-time image analysis and report generation to provide a rapid (<4 h) sample-to-report turnaround time, shedding light on its utility as a versatile (e.g., low-resource setting and manual laboratory operation, or high-throughput automated system) phenotypic AST platform for hospital/clinic use.

The lymphatic endothelium-derived follistatin: activin A axis regulates neutrophil motility in response to Pseudomonas aeruginosa.

The lymphatic system plays an active role during infection, however the role of lymphatic-neutrophil interactions in host-defense responses is not well understood. During infection with pathogens such as Pseudomonas aeruginosa, Staphylococcus aureus and Yersinia pestis, neutrophils traffic from sites of infection through the lymphatic vasculature, to draining lymph nodes to interact with resident lymphocytes. This process is poorly understood, in part, due to the lack of in vitro models of the lymphatic system. Here we use a 3D microscale lymphatic vessel model to examine neutrophil-lymphatic cell interactions during host defense responses to pathogens. In previous work, we have shown that follistatin is secreted at high concentrations by lymphatic endothelial cells during inflammation. Follistatin inhibits activin A, a member of the TGF-ß superfamily, and, together, these molecules form a signaling pathway that plays a role in regulating both innate and adaptive immune responses. Although follistatin and activin A are constitutively produced in the pituitary, gonads and skin, their major source in the serum and their effects on neutrophils are poorly understood. Here we report a microfluidic model that includes both blood and lymphatic endothelial vessels, and neutrophils to investigate neutrophil-lymphatic trafficking during infection with P. aeruginosa. We found that lymphatic endothelial cells produce secreted factors that increase neutrophil migration toward P. aeruginosa, and are a significant source of both follistatin and activin A during Pseudomonas infection. We determined that follistatin produced by lymphatic endothelial cells inhibits activin A, resulting in increased neutrophil migration. These data suggest that the follistatin:activin A ratio influences neutrophil trafficking during infection with higher ratios increasing neutrophil migration.

A clinical-grade liquid biomarker detects neuroendocrine differentiation in prostate cancer.

BackgroundNeuroendocrine prostate cancer (NEPC) is an aggressive subtype, the presence of which changes the prognosis and management of metastatic prostate cancer.MethodsWe performed analytical validation of a Circulating Tumor Cell (CTC) multiplex RNA qPCR assay to identify the limit of quantification (LOQ) in cell lines, synthetic cDNA, and patient samples. We next profiled 116 longitudinal samples from a prospectively collected institutional cohort of 17 patients with metastatic prostate cancer (7 NEPC, 10 adenocarcinoma) as well as 265 samples from 139 patients enrolled in 3 adenocarcinoma phase II trials of androgen receptor signaling inhibitors (ARSIs). We assessed a NEPC liquid biomarker via the presence of neuroendocrine markers and the absence of androgen receptor (AR) target genes.ResultsUsing the analytical validation LOQ, liquid biomarker NEPC detection in the longitudinal cohort had a per-sample sensitivity of 51.35% and a specificity of 91.14%. However, when we incorporated the serial information from multiple liquid biopsies per patient, a unique aspect of this study, the per-patient predictions were 100% accurate, with a receiver-operating-curve (ROC) AUC of 1. In the adenocarcinoma ARSI trials, the presence of neuroendocrine markers, even while AR target gene expression was retained, was a strong negative prognostic factor.ConclusionOur analytically validated CTC biomarker can detect NEPC with high diagnostic accuracy when leveraging serial samples that are only feasible using liquid biopsies. Patients with expression of NE genes while retaining AR-target gene expression may indicate the transition to neuroendocrine differentiation, with clinical characteristics consistent with this phenotype.FundingNIH (DP2 OD030734, 1UH2CA260389, R01CA247479, and P30 CA014520), Department of Defense (PC190039 and PC200334), and Prostate Cancer Foundation (Movember Foundation - PCF Challenge Award).