Microfluidic Devices for Precise Measurements of Cell Directionality Reveal a Role for Glutamine during Cell Migration.

Cancer cells that migrate from tumors into surrounding tissues are responsible for cancer dissemination through the body. Microfluidic devices have been instrumental in discovering unexpected features of cancer cell migration, including the migration in self-generated gradients and the contributions of cell-cell contact during collective migration. Here, we design microfluidic channels with five successive bifurcations to characterize the directionality of cancer cell migration with high precision. We find that the directional decisions of cancer cells moving through bifurcating channels in response to self-generated epidermal growth factor (EGF) gradients require the presence of glutamine in the culture media. A biophysical model helps quantify the contribution of glucose and glutamine to cancer cell orientation during migration in self-generated gradients. Our study uncovers an unexpected interplay between cancer cell metabolism and cancer cell migration studies and may eventually lead to new ways to delay cancer cell invasion.

Microfluidic devices for precise measurements of cell directionality reveal a role for glutamine during cell migration.

Cancer cells that migrate from tumors into surrounding tissues are responsible for cancer dissemination through the body. Microfluidic devices have been instrumental in discovering unexpected features of cancer cell migration, including the migration in self-generated gradients and the contributions of cell-cell contact during collective migration. Here, we design microfluidic channels with five successive bifurcations to characterize the directionality of cancer cell migration with high precision. We uncover an unexpected role for glutamine in epithelial cancer cell orientation, which could be replaced by alfa-keto glutarate but not glucose.

A single-cell liver atlas of Plasmodium vivax infection.

Malaria-causing Plasmodium vivax parasites can linger in the human liver for weeks to years and reactivate to cause recurrent blood-stage infection. Although they are an important target for malaria eradication, little is known about the molecular features of replicative and non-replicative intracellular liver-stage parasites and their host cell dependence. Here, we leverage a bioengineered human microliver platform to culture patient-derived P. vivax parasites for transcriptional profiling. Coupling enrichment strategies with bulk and single-cell analyses, we capture both parasite and host transcripts in individual hepatocytes throughout the course of infection. We define host- and state-dependent transcriptional signatures and identify unappreciated populations of replicative and non-replicative parasites that share features with sexual transmissive forms. We find that infection suppresses the transcription of key hepatocyte function genes and elicits an anti-parasite innate immune response. Our work provides a foundation for understanding host-parasite interactions and reveals insights into the biology of P. vivax dormancy and transmission.

Peptide-based urinary monitoring of fibrotic nonalcoholic steatohepatitis by mass-barcoded activity-based sensors.

Noninvasive detection of nonalcoholic steatohepatitis (NASH), the progressive form of nonalcoholic fatty liver disease, promises to improve patient screening, accelerate drug trials, and reduce health care costs. On the basis of protease dysregulation of the biological pathways of fibrotic NASH, we developed the Glympse Bio Test System (GBTS) for multiplexed quantification of liver protease activity. GBTS-NASH comprises a mixture of 19 mass-barcoded PEGylated peptides that is administered intravenously and senses liver protease activity by releasing mass-barcoded reporters into urine for analysis by mass spectrometry. To identify a protease signature of NASH, transcriptomic analysis of 355 human liver biopsies identified a 13-protease panel that discriminated clinically relevant NASH ≥F2 fibrosis from F0-F1 with high classification accuracy across two independent patient datasets. We screened 159 candidate substrates to identify a panel of 19 peptides that exhibited high activity for our 13-protease panel. In the choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) mouse model, binary classifiers trained on urine samples discriminated fibrotic NASH from simple steatosis and healthy controls across a range of nondisease conditions and indicated disease regression upon diet change [area under receiver operating characteristics (AUROCs) > 0.97]. Using a hepatoprotective triple combination treatment (FXR agonist, ACC and ASK1 inhibitors) in a rat model of NASH, urinary classification distinguished F0-F1 from ≥F2 animals and indicated therapeutic response as early as 1 week on treatment (AUROCs >0.91). Our results support GBTS-NASH to diagnose fibrotic NASH via an infusion of peptides, monitor changes in disease severity, and indicate early treatment response.

Engineered Livers for Infectious Diseases.

Engineered liver systems come in a variety of platform models, from 2-dimensional cocultures of primary human hepatocytes and stem cell-derived progeny, to 3-dimensional organoids and humanized mice. Because of the species-specificity of many human hepatropic pathogens, these engineered systems have been essential tools for biologic discovery and therapeutic agent development in the context of liver-dependent infectious diseases. Although improvement of existing models is always beneficial, and the addition of a robust immune component is a particular need, at present, considerable progress has been made using this combination of research platforms. We highlight advances in the study of hepatitis B and C viruses and malaria-causing Plasmodium falciparum and Plasmodium vivax parasites, and underscore the importance of pairing the most appropriate model system and readout modality with the particular experimental question at hand, without always requiring a platform that recapitulates human physiology in its entirety.

In Vitro Culture, Drug Sensitivity, and Transcriptome of Plasmodium Vivax Hypnozoites.

The unique relapsing nature of Plasmodium vivax infection is a major barrier to malaria eradication. Upon infection, dormant liver-stage forms, hypnozoites, linger for weeks to months and then relapse to cause recurrent blood-stage infection. Very little is known about hypnozoite biology; definitive biomarkers are lacking and in vitro platforms that support phenotypic studies are needed. Here, we recapitulate the entire liver stage of P. vivax in vitro, using a multiwell format that incorporates micropatterned primary human hepatocyte co-cultures (MPCCs). MPCCs feature key aspects of P. vivax biology, including establishment of persistent small forms and growing schizonts, merosome release, and subsequent infection of reticulocytes. We find that the small forms exhibit previously described hallmarks of hypnozoites, and we pilot MPCCs as a tool for testing candidate anti-hypnozoite drugs. Finally, we employ a hybrid capture strategy and RNA sequencing to describe the hypnozoite transcriptome and gain insight into its biology.

Towards a Humanized Mouse Model of Liver Stage Malaria Using Ectopic Artificial Livers.

The malaria liver stage is an attractive target for antimalarial development, and preclinical malaria models are essential for testing such candidates. Given ethical concerns and costs associated with non-human primate models, humanized mouse models containing chimeric human livers offer a valuable alternative as small animal models of liver stage human malaria. The best available human liver chimeric mice rely on cellular transplantation into mice with genetically engineered liver injury, but these systems involve a long and variable humanization process, are expensive, and require the use of breeding-challenged mouse strains which are not widely accessible. We previously incorporated primary human hepatocytes into engineered polyethylene glycol (PEG)-based nanoporous human ectopic artificial livers (HEALs), implanted them in mice without liver injury, and rapidly generated human liver chimeric mice in a reproducible and scalable fashion. By re-designing the PEG scaffold to be macroporous, we demonstrate the facile fabrication of implantable porous HEALs that support liver stage human malaria (P. falciparum) infection in vitro, and also after implantation in mice with normal liver function, 60% of the time. This proof-of-concept study demonstrates the feasibility of applying a tissue engineering strategy towards the development of scalable preclinical models of liver stage malaria infection for future applications.

Host AMPK Is a Modulator of Plasmodium Liver Infection.

Manipulation of the master regulator of energy homeostasis AMP-activated protein kinase (AMPK) activity is a strategy used by many intracellular pathogens for successful replication. Infection by most pathogens leads to an activation of host AMPK activity due to the energetic demands placed on the infected cell. Here, we demonstrate that the opposite is observed in cells infected with rodent malaria parasites. Indeed, AMPK activity upon the infection of hepatic cells is suppressed and dispensable for successful infection. By contrast, an overactive AMPK is deleterious to intracellular growth and replication of different Plasmodium spp., including the human malaria parasite, P. falciparum. The negative impact of host AMPK activity on infection was further confirmed in mice under conditions that activate its function. Overall, this work establishes the role of host AMPK signaling as a suppressive pathway of Plasmodium hepatic infection and as a potential target for host-based antimalarial interventions.

Human iPSC-derived hepatocyte-like cells support Plasmodium liver-stage infection in vitro.

Malaria eradication is a major goal in public health but is challenged by relapsing malaria species, expanding drug resistance, and the influence of host genetics on antimalarial drug efficacy. To overcome these hurdles, it is imperative to establish in vitro assays of liver-stage malaria for drug testing. Induced pluripotent stem cells (iPSC) potentially allow the assessment of donor-specific drug responses, and iPSC-derived hepatocyte-like cells (iHLCs) can facilitate the study of host genetics on host-pathogen interactions and the discovery of novel targets for antimalarial drug development. We establish in vitro liver-stage malaria infections in iHLCs using P. berghei, P. yoelii, P. falciparum, and P. vivax and show that differentiating cells acquire permissiveness to malaria infection at the hepatoblast stage. We also characterize antimalarial drug metabolism capabilities of iHLCs using prototypical antimalarial drugs and demonstrate that chemical maturation of iHLCs can improve their potential for antimalarial drug testing applications.

Micropatterned coculture of primary human hepatocytes and supportive cells for the study of hepatotropic pathogens.

The development of therapies and vaccines for human hepatropic pathogens requires robust model systems that enable the study of host-pathogen interactions. However, in vitro liver models of infection typically use either hepatoma cell lines that exhibit aberrant physiology or primary human hepatocytes in culture conditions in which they rapidly lose their hepatic phenotype. To achieve stable and robust in vitro primary human hepatocyte models, we developed micropatterned cocultures (MPCCs), which consist of primary human hepatocytes organized into 2D islands that are surrounded by supportive fibroblast cells. By using this system, which can be established over a period of days, and maintained over multiple weeks, we demonstrate how to recapitulate in vitro hepatic life cycles for the hepatitis B and C viruses and the Plasmodium pathogens P. falciparum and P. vivax. The MPCC platform can be used to uncover aspects of host-pathogen interactions, and it has the potential to be used for drug and vaccine development.

Magnetic barcode assay for genetic detection of pathogens.

The task of rapidly identifying patients infected with Mycobacterium tuberculosis in resource-constrained environments remains a challenge. A sensitive and robust platform that does not require bacterial isolation or culture is critical in making informed diagnostic and therapeutic decisions. Here we introduce a platform for the detection of nucleic acids based on a magnetic barcoding strategy. PCR-amplified mycobacterial genes are sequence-specifically captured on microspheres, labelled by magnetic nanoprobes and detected by nuclear magnetic resonance. All components are integrated into a single, small fluidic cartridge for streamlined on-chip operation. We use this platform to detect M. tuberculosis and identify drug-resistance strains from mechanically processed sputum samples within 2.5 h. The specificity of the assay is confirmed by detecting a panel of clinically relevant non-M. tuberculosis bacteria, and the clinical utility is demonstrated by the measurements in M. tuberculosis-positive patient specimens. Combined with portable systems, the magnetic barcode assay holds promise to become a sensitive, high-throughput and low-cost platform for point-of-care diagnostics.