Geotemporal Fluorophore Biodistribution Mapping of Colorectal Cancer: Micro and Macroscopic Insights.

Fluorescence-guided oncology promises to improve both the detection and treatment of malignancy. We sought to investigate the temporal distribution of indocyanine green (ICG), an exogenous fluorophore in human colorectal cancer. This analysis aims to enhance our understanding of ICG's effectiveness in current tumour detection and inform potential future diagnostic and therapeutic enhancements. METHODS: Fifty consenting patients undergoing treatment for suspected/confirmed colorectal neoplasia provided near infrared (NIR) video and imagery of transanally recorded and ex vivo resected rectal lesions following intravenous ICG administration (0.25 mg/kg), with a subgroup providing tissue samples for microscopic (including near infrared) analysis. Computer vision techniques detailed macroscopic 'early' (<15 min post ICG administration) and 'late' (>2 h) tissue fluorescence appearances from surgical imagery with digital NIR scanning (Licor, Lincoln, NE, USA) and from microscopic analysis (Nikon, Tokyo, Japan) undertaken by a consultant pathologist detailing tissue-level fluorescence distribution over the same time. RESULTS: Significant intra-tumoural fluorescence heterogeneity was seen 'early' in malignant versus benign lesions. In all 'early' samples, fluorescence was predominantly within the tissue stroma, with uptake within plasma cells, blood vessels and lymphatics, but not within malignant or healthy glands. At 'late' stage observation, fluorescence was visualised non-uniformly within the intracellular cytoplasm of malignant tissue but not retained in benign glands. Fluorescence also accumulated within any present peritumoural inflammatory tissue. CONCLUSION: This study demonstrates the time course diffusion patterns of ICG through both benign and malignant tumours in vivo in human patients at both macroscopic and microscopic levels, demonstrating important cellular drivers and features of geolocalisation and how they differ longitudinally after exposure to ICG.

Retinoic acid receptor ß modulates mechanosensing and invasion in pancreatic cancer cells via myosin light chain 2.

Pancreatic ductal adenocarcinoma (PDAC) is the most common and lethal form of pancreatic cancer, characterised by stromal remodelling, elevated matrix stiffness and high metastatic rate. Retinoids, compounds derived from vitamin A, have a history of clinical use in cancer for their anti-proliferative and differentiation effects, and more recently have been explored as anti-stromal therapies in PDAC for their ability to induce mechanical quiescence in cancer associated fibroblasts. Here, we demonstrate that retinoic acid receptor ß (RAR-ß) transcriptionally represses myosin light chain 2 (MLC-2) expression in pancreatic cancer cells. As a key regulatory component of the contractile actomyosin machinery, MLC-2 downregulation results in decreased cytoskeletal stiffness and traction force generation, impaired response to mechanical stimuli via mechanosensing and reduced ability to invade through the basement membrane. This work highlights the potential of retinoids to target the mechanical drivers of pancreatic cancer.

Modulation of sirtuins during monolayer chondrocyte culture influences cartilage regeneration upon transfer to a 3D culture environment.

This study examined the role of sirtuins in the regenerative potential of articular chondrocytes. Sirtuins (SIRT1-7) play a key role in regulating cartilage homeostasis. By inhibiting pro-inflammatory pathways responsible for cartilage degradation and promoting the expression of key matrix components, sirtuins have the potential to drive a favourable balance between anabolic and catabolic processes critical to regenerative medicine. When subjected to osmolarity and glucose concentrations representative of the in vivo niche, freshly isolated bovine chondrocytes exhibited increases in SIRT1 but not SIRT3 gene expression. Replicating methods adopted for the in vitro monolayer expansion of chondrocytes for cartilage regenerative therapies, we found that SIRT1 gene expression declined during expansion. Manipulation of sirtuin activity during in vitro expansion by supplementation with the SIRT1-specific activator SRT1720, nicotinamide mononucleotide, or the pan-sirtuin inhibitor nicotinamide, significantly influenced cartilage regeneration in subsequent 3D culture. Tissue mass, cellularity and extracellular matrix content were reduced in response to sirtuin inhibition during expansion, whilst sirtuin activation enhanced these measures of cartilage tissue regeneration. Modulation of sirtuin activity during monolayer expansion influenced H3K27me3, a heterochromatin mark with an important role in development and differentiation. Unexpectedly, treatment of primary chondrocytes with sirtuin activators in 3D culture reduced their matrix synthesis. Thus, modulating sirtuin activity during the in vitro monolayer expansion phase may represent a distinct opportunity to enhance the outcome of cartilage regenerative medicine techniques.

Covariation of Pluripotency Markers and Biomechanical Properties in Mouse Embryonic Stem Cells.

Pluripotent cells are subject to much interest as a source of differentiated cellular material for research models, regenerative medical therapies and novel applications such as lab-cultured meat. Greater understanding of the pluripotent state and control over its differentiation is therefore desirable. The role of biomechanical properties in directing cell fate and cell behavior has been increasingly well described in recent years. However, many of the mechanisms which control cell morphology and mechanical properties in somatic cells are absent from pluripotent cells. We leveraged naturally occurring variation in biomechanical properties and expression of pluripotency genes in murine ESCs to investigate the relationship between these parameters. We observed considerable variation in a Rex1-GFP expression reporter line and found that this variation showed no apparent correlation to cell spreading morphology as determined by circularity, Feret ratio, phase contrast brightness or cell spread area, either on a parameter-by-parameter basis, or when evaluated using a combined metric derived by principal component analysis from the four individual criteria. We further confirmed that cell volume does not co-vary with Rex1-GFP expression. Interestingly, we did find that a subpopulation of cells that were readily detached by gentle agitation collectively exhibited higher expression of Nanog, and reduced LmnA expression, suggesting that elevated pluripotency gene expression may correlate with reduced adhesion to the substrate. Furthermore, atomic force microscopy and quantitative fluorescent imaging revealed a connection between cell stiffness and Rex1-GFP reporter expression. Cells expressing high levels of Rex1-GFP are consistently of a relatively low stiffness, while cells with low levels of Rex1-GFP tend toward higher stiffness values. These observations indicate some interaction between pluripotency gene expression and biomechanical properties, but also support a strong role for other interactions between the cell culture regime and cellular biomechanical properties, occurring independently of the core transcriptional network that supports pluripotency.

A Biomimetic High Throughput Model of Cancer Cell Spheroid Dissemination onto Aligned Fibrillar Collagen.

Cell dissemination during tumor development is a characteristic of cancer metastasis. Dissemination from three-dimensional spheroid models on extracellular matrices designed to mimic tissue-specific physiological microenvironments may allow us to better elucidate the mechanism behind cancer metastasis and the response to therapeutic agents. The orientation of fibrillar collagen plays a key role in cellular processes and mediates metastasis through contact-guidance. Understanding how cells migrate on aligned collagen fibrils requires in vitro assays with reproducible and standardized orientation of collagen fibrils on the macro-to-nanoscale. Herein, we implement a spheroid-based migration assay, integrated with a fibrillar type I collagen matrix, in a manner compatible with high throughput image acquisition and quantitative analysis. The migration of highly proliferating U2OS osteosarcoma cell spheroids onto an aligned fibrillar type I collagen matrix was quantified. Cell dissemination from the spheroid was polarized with increased invasion in the direction of fibril alignment. The resulting area of cell dissemination had an aspect ratio of 1.2 ± 0.1 and an angle of maximum invasion distance of 5° ± 44° relative to the direction of collagen fibril alignment. The assay described here can be applied to a fully automated imaging and analysis pipeline for the assessment of tumor cell migration with high throughput screening.

Micromirror Total Internal Reflection Microscopy for High-Performance Single Particle Tracking at Interfaces.

Single particle tracking has found broad applications in the life and physical sciences, enabling the observation and characterization of nano- and microscopic motion. Fluorescence-based approaches are ideally suited for high-background environments, such as tracking lipids or proteins in or on cells, due to superior background rejection. Scattering-based detection is preferable when localization precision and imaging speed are paramount due to the in principle infinite photon budget. Here, we show that micromirror-based total internal reflection dark field microscopy enables background suppression previously only reported for interferometric scattering microscopy, resulting in nanometer localization precision at 6 µs exposure time for 20 nm gold nanoparticles with a 25 × 25 µm2 field of view. We demonstrate the capabilities of our implementation by characterizing sub-nanometer deterministic flows of 20 nm gold nanoparticles at liquid-liquid interfaces. Our results approach the optimal combination of background suppression, localization precision, and temporal resolution achievable with pure scattering-based imaging and tracking of nanoparticles at interfaces.

Pancreatic Ductal Adenocarcinoma: Relating Biomechanics and Prognosis.

Pancreatic ductal adenocarcinoma (PDAC) is the most common form of pancreatic cancer and carries a dismal prognosis. Resectable patients are treated predominantly with surgery while borderline resectable patients may receive neoadjuvant treatment (NAT) to downstage their disease prior to possible resection. PDAC tissue is stiffer than healthy pancreas, and tissue stiffness is associated with cancer progression. Another feature of PDAC is increased tissue heterogeneity. We postulate that tumour stiffness and heterogeneity may be used alongside currently employed diagnostics to better predict prognosis and response to treatment. In this review we summarise the biomechanical changes observed in PDAC, explore the factors behind these changes and describe the clinical consequences. We identify methods available for assessing PDAC biomechanics ex vivo and in vivo, outlining the relative merits of each. Finally, we discuss the potential use of radiological imaging for prognostic use.

Corrigendum: Mechanical Stimulation: A Crucial Element of Organ-on-Chip Models.

[This corrects the article DOI: 10.3389/fbioe.2020.602646.].

Mechanical Stimulation: A Crucial Element of Organ-on-Chip Models.

Organ-on-chip (OOC) systems recapitulate key biological processes and responses in vitro exhibited by cells, tissues, and organs in vivo. Accordingly, these models of both health and disease hold great promise for improving fundamental research, drug development, personalized medicine, and testing of pharmaceuticals, food substances, pollutants etc. Cells within the body are exposed to biomechanical stimuli, the nature of which is tissue specific and may change with disease or injury. These biomechanical stimuli regulate cell behavior and can amplify, annul, or even reverse the response to a given biochemical cue or drug candidate. As such, the application of an appropriate physiological or pathological biomechanical environment is essential for the successful recapitulation of in vivo behavior in OOC models. Here we review the current range of commercially available OOC platforms which incorporate active biomechanical stimulation. We highlight recent findings demonstrating the importance of including mechanical stimuli in models used for drug development and outline emerging factors which regulate the cellular response to the biomechanical environment. We explore the incorporation of mechanical stimuli in different organ models and identify areas where further research and development is required. Challenges associated with the integration of mechanics alongside other OOC requirements including scaling to increase throughput and diagnostic imaging are discussed. In summary, compelling evidence demonstrates that the incorporation of biomechanical stimuli in these OOC or microphysiological systems is key to fully replicating in vivo physiology in health and disease.

G Protein-Coupled Estrogen Receptor Regulates Actin Cytoskeleton Dynamics to Impair Cell Polarization.

Mechanical forces regulate cell functions through multiple pathways. G protein-coupled estrogen receptor (GPER) is a seven-transmembrane receptor that is ubiquitously expressed across tissues and mediates the acute cellular response to estrogens. Here, we demonstrate an unidentified role of GPER as a cellular mechanoregulator. G protein-coupled estrogen receptor signaling controls the assembly of stress fibers, the dynamics of the associated focal adhesions, and cell polarization via RhoA GTPase (RhoA). G protein-coupled estrogen receptor activation inhibits F-actin polymerization and subsequently triggers a negative feedback that transcriptionally suppresses the expression of monomeric G-actin. Given the broad expression of GPER and the range of cytoskeletal changes modulated by this receptor, our findings position GPER as a key player in mechanotransduction.

Attachment and antibiotic response of early-stage biofilms studied using resonant hyperspectral imaging.

Many bacterial species readily develop biofilms that act as a protective matrix against external challenge, e.g., from antimicrobial treatment. Therefore, biofilms are often responsible for persistent and recurring infections. Established methods for studying biofilms are either destructive or focus on the biofilm's surface. A non-destructive method that is sensitive to the underside of the biofilm is highly desirable, as it allows studying the penetration of antibiotics through the film. Here, we demonstrate that the high surface sensitivity of resonant hyperspectral imaging provides this capability. The method allows us to monitor the early stages of Escherichia coli biofilm formation, cell attachment and microcolony formation, in-situ and in real-time. We study the response of the biofilm to a number of different antibiotics and verify our observations using confocal microscopy. Based on this ability to closely monitor the surface-bound cells, resonant hyperspectral imaging gives new insights into the antimicrobial resistance of biofilms.

Simulating the evolutionary trajectories of metabolic pathways for insect symbionts in the genus Sodalis.

Insect-bacterial symbioses are ubiquitous, but there is still much to uncover about how these relationships establish, persist and evolve. The tsetse endosymbiont Sodalis glossinidius displays intriguing metabolic adaptations to its microenvironment, but the process by which this relationship evolved remains to be elucidated. The recent chance discovery of the free-living species of the genus Sodalis, Sodalis praecaptivus, provides a serendipitous starting point from which to investigate the evolution of this symbiosis. Here, we present a flux balance model for S. praecaptivus and empirically verify its predictions. Metabolic modelling is used in combination with a multi-objective evolutionary algorithm to explore the trajectories that S. glossinidius may have undertaken from this starting point after becoming internalized. The order in which key genes are lost is shown to influence the evolved populations, providing possible targets for future in vitro genetic manipulation. This method provides a detailed perspective on possible evolutionary trajectories for S. glossinidius in this fundamental process of evolutionary and ecological change.

GPER Activation Inhibits Cancer Cell Mechanotransduction and Basement Membrane Invasion via RhoA.

The invasive properties of cancer cells are intimately linked to their mechanical phenotype, which can be regulated by intracellular biochemical signalling. Cell contractility, induced by mechanotransduction of a stiff fibrotic matrix, and the epithelial-mesenchymal transition (EMT) promote invasion. Metastasis involves cells pushing through the basement membrane into the stroma-both of which are altered in composition with cancer progression. Agonists of the G protein-coupled oestrogen receptor (GPER), such as tamoxifen, have been largely used in the clinic, and interest in GPER, which is abundantly expressed in tissues, has greatly increased despite a lack of understanding regarding the mechanisms which promote its multiple effects. Here, we show that specific activation of GPER inhibits EMT, mechanotransduction and cell contractility in cancer cells via the GTPase Ras homolog family member A (RhoA). We further show that GPER activation inhibits invasion through an in vitro basement membrane mimic, similar in structure to the pancreatic basement membrane that we reveal as an asymmetric bilayer, which differs in composition between healthy and cancer patients.

Syndecan-4 tunes cell mechanics by activating the kindlin-integrin-RhoA pathway.

Extensive research over the past decades has identified integrins to be the primary transmembrane receptors that enable cells to respond to external mechanical cues. We reveal here a mechanism whereby syndecan-4 tunes cell mechanics in response to localized tension via a coordinated mechanochemical signalling response that involves activation of two other receptors: epidermal growth factor receptor and ß1 integrin. Tension on syndecan-4 induces cell-wide activation of the kindlin-2/ß1 integrin/RhoA axis in a PI3K-dependent manner. Furthermore, syndecan-4-mediated tension at the cell-extracellular matrix interface is required for yes-associated protein activation. Extracellular tension on syndecan-4 triggers a conformational change in the cytoplasmic domain, the variable region of which is indispensable for the mechanical adaptation to force, facilitating the assembly of a syndecan-4/α-actinin/F-actin molecular scaffold at the bead adhesion. This mechanotransduction pathway for syndecan-4 should have immediate implications for the broader field of mechanobiology.

Large-scale urban underground hydro-thermal modelling - A case study of the Royal Borough of Kensington and Chelsea, London.

The shallow subsurface of dense cities is increasingly exploited for various purposes due to the significant rise in urban populations. Past research has shown that underground activities have a significant impact on local subsurface temperatures. However, the resulting spatial variability of ground temperature elevations on a city-scale is not well understood due to the lack of sufficient information and modelling complexity at such large scales. Resilient and sustainable planning of underground developments and geothermal exploitation in the short and long-term necessitate more detailed, more reliable knowledge of subsurface thermal status. This paper investigates the impact of some common underground heat sources such as train tunnels and residential basements on subsurface temperature elevation on a large scale and highlights the influence of local geology, hydrogeology, density, and type and arrangement of the heat sources on ground thermal disturbance. To tackle the size issues and computational expenses of such a large-scale problem, a semi-3D hydro-thermal numerical approach is presented to capture the combined influence of underground built environment characteristics coupled with ground properties on ground temperature elevation within the Royals Borough of Kensington and Chelsea (RBKC), London. Numerical results show that the extent of ground thermal disturbance is mostly affected by geological and hydrogeological characteristics in permeable ground (River Terrace Deposits). Density and spatial distribution of heat sources, however, are critical parameters in ground temperature evaluation in highly impermeable ground such as London Clay Formation. The locality of temperature rise and potential ground energy within immediate impermeable ground surrounding heat sources versus significantly large extent of ground thermal disturbance in permeable ground, highlights the significant dependency of ground thermal state and geothermal potential at the studied site to the ground and underground built environment characteristics and necessitates a better understanding of shallow subsurface thermal state for a sustainable and resilient urban underground development.

Multiparameter antibiotic resistance detection based on hydrodynamic trapping of individual E. coli.

There is an urgent need to develop novel methods for assessing the response of bacteria to antibiotics in a timely manner. Antibiotics are traditionally assessed via their effect on bacteria in a culture medium, which takes 24-48 h and exploits only a single parameter, i.e. growth. Here, we present a multiparameter approach at the single-cell level that takes approximately an hour from spiking the culture to correctly classify susceptible and resistant strains. By hydrodynamically trapping hundreds of bacteria, we simultaneously monitor the evolution of motility and morphology of individual bacteria upon drug administration. We show how this combined detection method provides insights into the activity of antimicrobials at the onset of their action which single parameter and traditional tests cannot offer. Our observations complement the current growth-based methods and highlight the need for future antimicrobial susceptibility tests to take multiple parameters into account.

An in vitro investigation of the inflammatory response to the strain amplitudes which occur during high frequency oscillation ventilation and conventional mechanical ventilation.

Children randomised in the neonatal period to high frequency oscillatory ventilation (HFOV) or conventional mechanical ventilation (CMV) in the United Kingdom Oscillation study (UKOS) had superior lung function at 11 to 14 years of age. During HFOV, much smaller tidal volumes, but a higher mean airway distending pressure is delivered, hence, a possible explanation for a volume dependent effect on long term lung function could be an increase in inflammation in response to higher tidal volumes and strains. We tested that hypothesis by assessing interleukin-6 (IL-6) and -8 (IL-8) release from A549 alveolar analogue cells following biaxial mechanical strain applied at 0.5 Hz occurring during conditions mimicking strain during CMV (5-20% strain) and conditions mimicking strain during HFOV (17.5% ±â€¯2.5% strain) for up to 4 h. Cyclic strain of 5-20%, occurring during CMV, increased levels of both IL-6 and IL-8 compared to unstrained controls, while 17.5% ±â€¯2.5% strain, occurring during HFOV, was associated with significantly lower levels of IL-6 (46.31 ±â€¯2.66 versus 56.79 ±â€¯3.73 pg/mL) and IL-8 (1340.2 ±â€¯74.9 versus 2522 ±â€¯248 pg/mL) secretion compared to conditions occurring during CMV at four hours. These results may provide a possible explanation for the superior lung function in 11-14-year-old children who had been supported in the neonatal period by HFOV.

A Tale of Three Species: Adaptation of Sodalis glossinidius to Tsetse Biology, Wigglesworthia Metabolism, and Host Diet.

The tsetse fly is the insect vector for the Trypanosoma brucei parasite, the causative agent of human African trypanosomiasis. The colonization and spread of the trypanosome correlate positively with the presence of a secondary symbiotic bacterium, Sodalis glossinidius The metabolic requirements and interactions of the bacterium with its host are poorly understood, and herein we describe a metabolic model of S. glossinidius metabolism. The model enabled the design and experimental verification of a defined medium that supports S. glossinidius growth ex vivo This has been used subsequently to analyze in vitro aspects of S. glossinidius metabolism, revealing multiple unique adaptations of the symbiont to its environment. Continued dependence on a sugar, and the importance of the chitin monomer N-acetyl-d-glucosamine as a carbon and energy source, suggests adaptation to host-derived molecules. Adaptation to the amino acid-rich blood diet is revealed by a strong dependence on l-glutamate as a source of carbon and nitrogen and by the ability to rescue a predicted l-arginine auxotrophy. Finally, the selective loss of thiamine biosynthesis, a vitamin provided to the host by the primary symbiont Wigglesworthia glossinidia, reveals an intersymbiont dependence. The reductive evolution of S. glossinidius to exploit environmentally derived metabolites has resulted in multiple weaknesses in the metabolic network. These weaknesses may become targets for reagents that inhibit S. glossinidius growth and aid the reduction of trypanosomal transmission.IMPORTANCE Human African trypanosomiasis is caused by the Trypanosoma brucei parasite. The tsetse fly vector is of interest for its potential to prevent disease spread, as it is essential for T. brucei life cycle progression and transmission. The tsetse's mutualistic endosymbiont Sodalis glossinidius has a link to trypanosome establishment, providing a disease control target. Here, we describe a new, experimentally verified model of S. glossinidius metabolism. This model has enabled the development of a defined growth medium that was used successfully to test aspects of S. glossinidius metabolism. We present S. glossinidius as uniquely adapted to life in the tsetse, through its reliance on the blood diet and host-derived sugars. Additionally, S. glossinidius has adapted to the tsetse's obligate symbiont Wigglesworthia glossinidia by scavenging a vitamin it produces for the insect. This work highlights the use of metabolic modeling to design defined growth media for symbiotic bacteria and may provide novel inhibitory targets to block trypanosome transmission.

Retinoic Acid Receptor-ß Is Downregulated in Hepatocellular Carcinoma and Cirrhosis and Its Expression Inhibits Myosin-Driven Activation and Durotaxis in Hepatic Stellate Cells.

Hepatic stellate cells (HSCs) are essential perisinusoidal cells in both healthy and diseased liver. HSCs modulate extracellular matrix (ECM) homeostasis when quiescent, but in liver fibrosis, HSCs become activated and promote excess deposition of ECM molecules and tissue stiffening via force generation and mechanosensing. In hepatocellular carcinoma (HCC), activated HSCs infiltrate the stroma and migrate to the tumor core to facilitate paracrine signaling with cancer cells. Because the function of HSCs is known to be modulated by retinoids, we investigated the expression profile of retinoic acid receptor beta (RAR-ß) in patients with cirrhosis and HCC, as well as the effects of RAR-ß activation in HSCs. We found that RAR-ß expression is significantly reduced in cirrhotic and HCC tissues. Using a comprehensive set of biophysical methods combined with cellular and molecular biology, we have elucidated the biomechanical mechanism by which all trans-retinoic acid promotes HSC deactivation via RAR-ß-dependent transcriptional downregulation of myosin light chain 2 expression. Furthermore, this also abrogated mechanically driven migration toward stiffer substrates. Conclusion: Targeting mechanotransduction in HSCs at the transcriptional level may offer therapeutic options for a range of liver diseases.