Fusobacterium nucleatum infection modulates the transcriptome and epigenome of HCT116 colorectal cancer cells in an oxygen-dependent manner.

Fusobacterium nucleatum, a gram-negative oral bacterium, has been consistently validated as a strong contributor to the progression of several types of cancer, including colorectal (CRC) and pancreatic cancer. While previous in vitro studies have shown that intracellular F. nucleatum enhances malignant phenotypes such as cell migration, the dependence of this regulation on features of the tumor microenvironment (TME) such as oxygen levels are wholly uncharacterized. Here we examine the influence of hypoxia in facilitating F. nucleatum invasion and its effects on host responses focusing on changes in the global epigenome and transcriptome. Using a multiomic approach, we analyze epigenomic alterations of H3K27ac and global transcriptomic alterations sustained within a hypoxia and normoxia conditioned CRC cell line HCT116 at 24 h following initial infection with F. nucleatum. Our findings reveal that intracellular F. nucleatum activates signaling pathways and biological processes in host cells similar to those induced upon hypoxia conditioning in the absence of infection. Furthermore, we show that a hypoxic TME favors F. nucleatum invasion and persistence and therefore infection under hypoxia may amplify malignant transformation by exacerbating the effects induced by hypoxia alone. These results motivate future studies to investigate host-microbe interactions in tumor tissue relevant conditions that more accurately define parameters for targeted cancer therapies.

Multiplexed microfluidic platform for stem-cell derived pancreatic islet ß cells.

Stem cell-derived ß cells offer an alternative to primary islets for biomedical discoveries as well as a potential surrogate for islet transplantation. The expense and challenge of obtaining and maintaining functional stem cell-derived ß cells calls for a need to develop better high-content and high-throughput culture systems. Microphysiological systems (MPS) are promising high-content in vitro platforms, but scaling for high-throughput screening and discoveries remain a challenge. Traditionally, simultaneous multiplexing of liquid handling and cell loading poses a challenge in the design of high-throughput MPS. Furthermore, although MPS for islet ß culture/testing have been developed, studies on multi-day culture of stem-cell derived ß cells in MPS have been limited. We present a scalable, multiplexed islet ß MPS device that incorporates microfluidic gradient generators to parallelize fluid handling for culture and test conditions. We demonstrated the viability and functionality of the stem cell-derived enriched ß clusters (eBCs) for a week, as assessed by the ∼2 fold insulin release by the clusters to glucose challenge. To show the scalable multiplexing for drug testing, we demonstrated the loss of stimulation index after long-term exposure to logarithmic concentration range of glybenclamide. The MPS cultured eBCs also confirmed a glycolytic bottleneck as inferred by insulin secretion responses to metabolites methyl succinate and glyceric acid. Thus, we present an innovative culture platform for eBCs with a balance of high-content and high-throughput characteristics.

Heart Muscle Microphysiological System for Cardiac Liability Prediction of Repurposed COVID-19 Therapeutics.

Despite global efforts, it took 7 months between the proclamation of global SARS-CoV-2 pandemic and the first FDA-approved treatment for COVID-19. During this timeframe, clinicians focused their efforts on repurposing drugs, such as hydroxychloroquine (HCQ) or azithromycin (AZM) to treat hospitalized COVID-19 patients. While clinical trials are time-consuming, the exponential increase in hospitalizations compelled the FDA to grant an emergency use authorization for HCQ and AZM as treatment for COVID-19, although there was limited evidence of their combined efficacy and safety. The authorization was revoked 4 months later, giving rise to controversial political and scientific debates illustrating important challenges such as premature authorization of potentially ineffective or unsafe therapeutics, while diverting resources from screening of effective drugs. Here we report on a preclinical drug screening platform, a cardiac microphysiological system (MPS), to rapidly identify clinically relevant cardiac liabilities associated with HCQ and AZM. The cardiac MPS is a microfabricated fluidic system in which cardiomyocytes derived from human induced pluripotent stem cells self-arrange into a uniaxially beating tissue. The drug response was measured using outputs that correlate with clinical measurements such as action potential duration (proxy for clinical QT interval) and drug-biomarker pairing. The cardiac MPS predicted clinical arrhythmias associated with QT prolongation and rhythm instabilities in tissues treated with HCQ. We found no change in QT interval upon acute exposure to AZM, while still observing a significant increase in arrhythmic events. These results suggest that this MPS can not only predict arrhythmias, but it can also identify arrhythmias even when QT prolongation is absent. When exposed to HCQ and AZM polytherapy, this MPS faithfully reflected clinical findings, in that the combination of drugs synergistically increased QT interval when compared to single drug exposure, while not worsening the overall frequency of arrhythmic events. The high content cardiac MPS can rapidly evaluate the cardiac safety of potential therapeutics, ultimately accelerating patients' access to safe and effective treatments.

Integrated Isogenic Human Induced Pluripotent Stem Cell-Based Liver and Heart Microphysiological Systems Predict Unsafe Drug-Drug Interaction.

Three-dimensional (3D) microphysiological systems (MPSs) mimicking human organ function in vitro are an emerging alternative to conventional monolayer cell culture and animal models for drug development. Human induced pluripotent stem cells (hiPSCs) have the potential to capture the diversity of human genetics and provide an unlimited supply of cells. Combining hiPSCs with microfluidics technology in MPSs offers new perspectives for drug development. Here, the integration of a newly developed liver MPS with a cardiac MPS-both created with the same hiPSC line-to study drug-drug interaction (DDI) is reported. As a prominent example of clinically relevant DDI, the interaction of the arrhythmogenic gastroprokinetic cisapride with the fungicide ketoconazole was investigated. As seen in patients, metabolic conversion of cisapride to non-arrhythmogenic norcisapride in the liver MPS by the cytochrome P450 enzyme CYP3A4 was inhibited by ketoconazole, leading to arrhythmia in the cardiac MPS. These results establish integration of hiPSC-based liver and cardiac MPSs to facilitate screening for DDI, and thus drug efficacy and toxicity, isogenic in the same genetic background.

A thermodynamic scaling law for electrically perturbed lipid membranes: Validation with steepest entropy ascent framework.

Experimental evidence has demonstrated the ability of transient pulses of electric fields to alter mammalian cell behavior. Strategies with these pulsed electric fields (PEFs) have been developed for clinical applications in cancer therapeutics, in-vivo decellularization, and tissue regeneration. Successful implementation of these strategies involve understanding how PEFs impact the cellular structures and, hence, cell behavior. The caveat, however, is that the PEF parameter space (i.e., comprising different pulse widths, amplitudes, number of pulses) is large, and design of experiments to explore all possible combinations of pulse parameters is prohibitive from a cost and time standpoint. In this study, a scaling law based on the Ising model is introduced to understand the impact of PEFs on the outer cell lipid membrane so that an understanding developed in one PEF pulse regime may be extended to another. Combining non-Markovian Monte Carlo techniques to determine density-of-states with a novel non-equilibrium thermodynamic framework based on the principle of steepest entropy ascent, the applicability of this scaling model to predict the behavior of both thermally quenched and electrically perturbed lipid membranes is demonstrated. A comparison of the predictions made by the steepest-entropy-ascent quantum thermodynamic (SEAQT) framework to experimental data is performed to validate the robustness of this computational methodology and the resulting scaling law.

Stem cell-based vascularization of microphysiological systems.

Microphysiological systems (MPSs) (i.e., tissue or organ chips) exploit microfluidics and 3D cell culture to mimic tissue and organ-level physiology. The advent of human induced pluripotent stem cell (hiPSC) technology has accelerated the use of MPSs to study human disease in a range of organ systems. However, in the reduction of system complexity, the intricacies of vasculature are an often-overlooked aspect of MPS design. The growing library of pluripotent stem cell-derived endothelial cell and perivascular cell protocols have great potential to improve the physiological relevance of vasculature within MPS, specifically for in vitro disease modeling. Three strategic categories of vascular MPS are outlined: self-assembled, interface focused, and 3D biofabricated. This review discusses key features and development of the native vasculature, linking that to how hiPSC-derived vascular cells have been generated, the state of the art in vascular MPSs, and opportunities arising from interdisciplinary thinking.

Electro-chemo-mechanical model to investigate multi-pulse electric-field-driven integrin clustering.

The effect of pulsed electric fields (PEFs) on transmembrane proteins is not fully understood; how do chemo-mechanical cues in the microenvironment mediate the electric field sensing by these proteins? To answer this key gap in knowledge, we have developed a kinetic Monte Carlo statistical model of the integrin proteins that integrates three components of the morphogenetic field (i.e., chemical, mechanical, and electrical cues). Specifically, the model incorporates the mechanical stiffness of the cell membrane, the ligand density of the extracellular environment, the glycocalyx stiffness, thermal Brownian motion, and electric field induced diffusion. The effects of both steady-state electric fields and transient PEF pulse trains on integrin clustering are studied. Our results reveal that electric-field-driven integrin clustering is mediated by membrane stiffness and ligand density. In addition, we explore the effects of PEF pulse-train parameters (amplitude, polarity, and pulse-width) on integrin clustering. In summary, we demonstrate a computational methodology to incorporate experimental data and simulate integrin clustering when exposed to PEFs for time-scales comparable to experiments (seconds-minutes). Thus, we propose a blueprint for understanding PEF/electric field effects on protein induced signaling and highlight key impediments to incorporating experimental values into computational models such as the kinetic Monte Carlo method.

Influence of Pulsed Electric Fields and Mitochondria-Cytoskeleton Interactions on Cell Respiration.

Pulsed electric fields with microsecond pulse width (µsPEFs) are used clinically; namely, irreversible electroporation/Nanoknife is used for soft tissue tumor ablation. The µsPEF pulse parameters used in irreversible electroporation (0.5-1 kV/cm, 80-100 pulses, ∼100 µs each, 1 Hz frequency) may cause an internal field to develop within the cell because of the disruption of the outer cell membrane and subsequent penetration of the electric field. An internal field may disrupt voltage-sensitive mitochondria, although the research literature has been relatively unclear regarding whether such disruptions occur with µsPEFs. This investigation reports the influence of clinically used µsPEF parameters on mitochondrial respiration in live cells. Using a high-throughput Agilent Seahorse machine, it was observed that µsPEF exposure comprising 80 pulses with amplitudes of 600 or 700 V/cm did not alter mitochondrial respiration in 4T1 cells measured after overnight postexposure recovery. To record alterations in mitochondrial function immediately after µsPEF exposure, high-resolution respirometry was used to measure the electron transport chain state via responses to glutamate-malate and ADP and mitochondrial membrane potential via response to carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. In addition to measuring immediate mitochondrial responses to µsPEF exposure, measurements were also made on cells permeabilized using digitonin and those with compromised cytoskeleton due to actin depolymerization via treatment with the drug latrunculin B. The former treatment was used as a control to tease out the effects of plasma membrane permeabilization, whereas the latter was used to investigate indirect effects on the mitochondria that may occur if µsPEFs impact the cytoskeleton on which the mitochondria are anchored. Based on the results, it was concluded that within the pulse parameters tested, µsPEFs alone do not hinder mitochondrial physiology but can be used to impact the mitochondria upon compromising the actin. Mitochondrial susceptibility to µsPEF after actin depolymerization provides, to our knowledge, a novel avenue for cancer therapeutics.

Irreversible electroporation inhibits pro-cancer inflammatory signaling in triple negative breast cancer cells.

Low-level electric fields have been demonstrated to induce spatial re-distribution of cell membrane receptors when applied for minutes or hours. However, there is limited literature on the influence on cell signaling with short transient high-amplitude pulses typically used in irreversible electroporation (IRE) for cancer treatment. Moreover, literature on signaling pertaining to immune cell trafficking after IRE is conflicting. We hypothesized that pulse parameters (field strength and exposure time) influence cell signaling and subsequently impact immune-cell trafficking. This hypothesis was tested in-vitro on triple negative breast cancer cells treated with IRE, where the effects of pulse parameters on key cell signaling factors were investigated. Importantly, real time PCR mRNA measurements and ELISA protein analyses revealed that thymic stromal lymphopoietin (TSLP) signaling was down regulated by electric field strengths above a critical threshold, irrespective of exposure times spanning those typically used clinically. Comparison with other treatments (thermal shock, chemical poration, kinase inhibitors) revealed that IRE has a unique effect on TSLP. Because TSLP signaling has been demonstrated to drive pro-cancerous immune cell phenotypes in breast and pancreatic cancers, our finding motivates further investigation into the potential use of IRE for induction of an anti-tumor immune response in vivo.

Influence of newly designed monorail pressure sensor catheter on coronary diagnostic parameters: an in vitro study.

The decision to perform intervention on a patient with coronary stenosis is often based on functional diagnostic parameters obtained from pressure and flow measurements using sensor-tipped guidewire at maximal vasodilation (hyperemia). Recently, a rapid exchange Monorail Pressure Sensor catheter of 0.022″ diameter (MPS22), with pressure sensor at distal end has been developed for improved assessment of stenosis severity. The hollow shaft of the MPS22 is designed to slide over any standard 0.014″ guidewire (G14). Hence, influence of MPS22 diameter on coronary diagnostic parameters needs investigation. An in vitro experiment was conducted to replicate physiologic flows in three representative area stenosis (AS): mild (64% AS), intermediate (80% AS), and severe (90% AS), for two arterial diameters, 3mm (N2; more common) and 2.5mm (N1). Influence of MPS22 on diagnostic parameters: fractional flow reserve (FFR) and pressure drop coefficient (CDP) was evaluated both at hyperemic and basal conditions, while comparing it with G14. The FFR values decreased for the MPS22 in comparison to G14, (Mild: 0.87 vs 0.88, Intermediate: 0.68 vs 0.73, Severe: 0.48 vs 0.56) and CDP values increased (Mild: 16 vs 14, Intermediate: 75 vs 56, Severe: 370 vs 182) for N2. Similar trend was observed in the case of N1. The FFR values were found to be well above (mild) and below (intermediate and severe) the diagnostic cut-off of 0.75. Therefore, MPS22 catheter can be used as a possible alternative to G14. Further, irrespective of the MPS22 or G14, basal FFR (FFRb) had overlapping ranges in close proximity for clinically relevant mild and intermediate stenoses that will lead to diagnostic uncertainty under both N1 and N2. However, CDPb had distinct ranges for different stenosis severities and could be a potential diagnostic parameter under basal conditions.

Influence of variable native arterial diameter and vasculature status on coronary diagnostic parameters.

In current practice, diagnostic parameters, such as fractional flow reserve (FFR) and coronary flow reserve (CFR), are used to determine the severity of a coronary artery stenosis. FFR is defined as the ratio of hyperemic pressures distal (p(rh)) and proximal (p(ah)) to a stenosis. CFR is the ratio of flow at hyperemic and basal condition. Another diagnostic parameter suggested by our group is the pressure drop coefficient (CDP). CDP is defined as the ratio of the pressure drop across the stenosis to the upstream dynamic pressure. These parameters are evaluated by invasively measuring flow (CFR), pressure (FFR), or both (CDP) in a diseased artery using guidewire tipped with a sensor. Pathologic state of artery is indicated by lower CFR (<2). Similarly, FFR lower than 0.75 leads to clinical intervention. Cutoff for CDP is under investigation. Diameter and vascular condition influence both flow and pressure drop, and thus, their effect on FFR and CDP was studied. In vitro experiment coupled with pressure-flow relationships from human clinical data was used to simulate pathophysiologic conditions in two representative arterial diameters, 2.5 mm (N1) and 3 mm (N2). With a 0.014 in. (0.35 mm) guidewire inserted, diagnostic parameters were evaluated for mild (∼64% area stenosis (AS)), intermediate (∼80% AS), and severe (∼90% AS) stenosis for both N1 and N2 arteries, and between two conditions, with and without myocardial infarction (MI). Arterial diameter did not influence FFR for clinically relevant cases of mild and intermediate stenosis (difference < 5%). Stenosis severity was underestimated due to higher FFR (mild: ∼9%, intermediate: ∼ 20%, severe: ∼ 30%) for MI condition because of lower pressure drops, and this may affect clinical decision making. CDP varied with diameter (mild: ∼20%, intermediate: ∼24%, severe: by 2.5 times), and vascular condition (mild: ∼35%, intermediate: ∼14%, severe: ∼ 9%). However, nonoverlapping range of CDP allowed better delineation of stenosis severities irrespective of diameter and vascular condition.