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.

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.

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.