A comparison of methods to assess cell mechanical properties.

The mechanical properties of cells influence their cellular and subcellular functions, including cell adhesion, migration, polarization, and differentiation, as well as organelle organization and trafficking inside the cytoplasm. Yet reported values of cell stiffness and viscosity vary substantially, which suggests differences in how the results of different methods are obtained or analyzed by different groups. To address this issue and illustrate the complementarity of certain approaches, here we present, analyze, and critically compare measurements obtained by means of some of the most widely used methods for cell mechanics: atomic force microscopy, magnetic twisting cytometry, particle-tracking microrheology, parallel-plate rheometry, cell monolayer rheology, and optical stretching. These measurements highlight how elastic and viscous moduli of MCF-7 breast cancer cells can vary 1,000-fold and 100-fold, respectively. We discuss the sources of these variations, including the level of applied mechanical stress, the rate of deformation, the geometry of the probe, the location probed in the cell, and the extracellular microenvironment.

Novel Curcumin-Resveratrol Solid Nanoparticles Synergistically Inhibit Proliferation of Melanoma Cells.

Polyphenols such as curcumin (Cur) and resveratrol (Res) have been recently shown to have potential to inhibit proliferation of highly aggressive melanoma cells. This study was designed to investigate the feasibility of a topical delivery system, using a solid lipid nanoparticles (SLNs) loaded delivery systems, that can enhance the skin penetration and anti-cancer efficacy of combination of these polyphenols. Negatively charged Cur-Res SLNs with a mean diameter of 180.2 ± 7.7 nm were prepared using high shear homogenization method. Cur-Res SLNs were found to be stable up to 2 weeks under 4°C. The in vitro release study showed that Res was released five time more than curcumin. The permeability of resveratrol was about 1.67 times that of curcumin from the SLN-gel formulation which was significantly (p < 0.05) lower than from SLN suspension. More than 70% of Cur-Res SLNs were bound to skin locally in a skin binding study suggesting potentially utility of Cur-Res SLNs in the treatment of localized melanoma. In fact, the electrical cell-substrate impedance sensing (ECIS) measurements suggested that Cur-Res combination has potential to stop cell migration of B16F10 melanoma cells. Furthermore, both, Cur-Res SLNs and Cur-Res solution at the ratio of 3:1 demonstrated a strong synergistic inhibition of SK-MEL-28 melanoma cell proliferation. Further evaluation of Cur-Res SLNs in vivo melanoma models are warranted to establish the clinical utility of Cur-Res formulations in melanoma therapy.

Real-time deformability cytometry: on-the-fly cell mechanical phenotyping.

We introduce real-time deformability cytometry (RT-DC) for continuous cell mechanical characterization of large populations (>100,000 cells) with analysis rates greater than 100 cells/s. RT-DC is sensitive to cytoskeletal alterations and can distinguish cell-cycle phases, track stem cell differentiation into distinct lineages and identify cell populations in whole blood by their mechanical fingerprints. This technique adds a new marker-free dimension to flow cytometry with diverse applications in biology, biotechnology and medicine.

Actin polymerization as a key innate immune effector mechanism to control Salmonella infection.

Salmonellosis is one of the leading causes of food poisoning worldwide. Controlling bacterial burden is essential to surviving infection. Nucleotide-binding oligomerization domain-like receptors (NLRs), such as NLRC4, induce inflammasome effector functions and play a crucial role in controlling Salmonella infection. Inflammasome-dependent production of IL-1ß recruits additional immune cells to the site of infection, whereas inflammasome-mediated pyroptosis of macrophages releases bacteria for uptake by neutrophils. Neither of these functions is known to directly kill intracellular salmonellae within macrophages. The mechanism, therefore, governing how inflammasomes mediate intracellular bacterial-killing and clearance in host macrophages remains unknown. Here, we show that actin polymerization is required for NLRC4-dependent regulation of intracellular bacterial burden, inflammasome assembly, pyroptosis, and IL-1ß production. NLRC4-induced changes in actin polymerization are physically manifested as increased cellular stiffness, and leads to reduced bacterial uptake, production of antimicrobial molecules, and arrested cellular migration. These processes act in concert to limit bacterial replication in the cell and dissemination in tissues. We show, therefore, a functional link between innate immunity and actin turnover in macrophages that underpins a key host defense mechanism for the control of salmonellosis.

Mechanotransduction in neutrophil activation and deactivation.

Mechanotransduction refers to the processes through which cells sense mechanical stimuli by converting them to biochemical signals and, thus, eliciting specific cellular responses. Cells sense mechanical stimuli from their 3D environment, including the extracellular matrix, neighboring cells and other mechanical forces. Incidentally, the emerging concept of mechanical homeostasis,long term or chronic regulation of mechanical properties, seems to apply to neutrophils in a peculiar manner, owing to neutrophils' ability to dynamically switch between the activated/primed and deactivated/deprimed states. While neutrophil activation has been known for over a century, its deactivation is a relatively recent discovery. Even more intriguing is the reversibility of neutrophil activation and deactivation. We review and critically evaluate recent findings that suggest physiological roles for neutrophil activation and deactivation and discuss possible mechanisms by which mechanical stimuli can drive the oscillation of neutrophils between the activated and resting states. We highlight several molecules that have been identified in neutrophil mechanotransduction, including cell adhesion and transmembrane receptors, cytoskeletal and ion channel molecules. The physiological and pathophysiological implications of such mechanically induced signal transduction in neutrophils are highlighted as a basis for future work. This article is part of a Special Issue entitled: Mechanobiology.

Chemotherapy impedes in vitro microcirculation and promotes migration of leukemic cells with impact on metastasis.

Although most cancer drugs target the proliferation of cancer cells, it is metastasis, the complex process by which cancer cells spread from the primary tumor to other tissues and organs of the body where they form new tumors, that leads to over 90% of all cancer deaths. Thus, there is an urgent need for anti-metastasis therapy. Surprisingly, emerging evidence suggests that certain anti-cancer drugs such as paclitaxel and doxorubicin can actually promote metastasis, but the mechanism(s) behind their pro-metastatic effects are still unclear. Here, we use a microfluidic microcirculation mimetic (MMM) platform which mimics the capillary constrictions of the pulmonary and peripheral microcirculation, to determine if in-vivo-like mechanical stimuli can evoke different responses from cells subjected to various cancer drugs. In particular, we show that leukemic cancer cells treated with doxorubicin and daunorubicin, commonly used anti-cancer drugs, have over 100% longer transit times through the device, compared to untreated leukemic cells. Such delays in the microcirculation are known to promote extravasation of cells, a key step in the metastatic cascade. Furthermore, we report a significant (p < 0.01) increase in the chemotactic migration of the doxorubicin treated leukemic cells. Both enhanced retention in the microcirculation and enhanced migration following chemotherapy, are pro-metastatic effects which can serve as new targets for anti-metastatic drugs.

Myosin II Activity Softens Cells in Suspension.

The cellular cytoskeleton is crucial for many cellular functions such as cell motility and wound healing, as well as other processes that require shape change or force generation. Actin is one cytoskeleton component that regulates cell mechanics. Important properties driving this regulation include the amount of actin, its level of cross-linking, and its coordination with the activity of specific molecular motors like myosin. While studies investigating the contribution of myosin activity to cell mechanics have been performed on cells attached to a substrate, we investigated mechanical properties of cells in suspension. To do this, we used multiple probes for cell mechanics including a microfluidic optical stretcher, a microfluidic microcirculation mimetic, and real-time deformability cytometry. We found that nonadherent blood cells, cells arrested in mitosis, and naturally adherent cells brought into suspension, stiffen and become more solidlike upon myosin inhibition across multiple timescales (milliseconds to minutes). Our results hold across several pharmacological and genetic perturbations targeting myosin. Our findings suggest that myosin II activity contributes to increased whole-cell compliance and fluidity. This finding is contrary to what has been reported for cells attached to a substrate, which stiffen via active myosin driven prestress. Our results establish the importance of myosin II as an active component in modulating suspended cell mechanics, with a functional role distinctly different from that for substrate-adhered cells.

Simulated Microgravity-Induced Changes to Drug Response in Cancer Cells Quantified Using Fluorescence Morphometry.

Unlike plants that have special gravity-sensing cells, such special cells in animals are yet to be discovered. However, microgravity, the condition of apparent weightlessness, causes bone, muscular and immune system dysfunctions in astronauts following spaceflights. Decades of investigations show correlations between these organ and system-level dysfunctions with changes induced at the cellular level both by simulated microgravity as well as microgravity conditions in outer space. Changes in single bone, muscle and immune cells include morphological abnormalities, altered gene expression, protein expression, metabolic pathways and signaling pathways. These suggest that human cells mount some response to microgravity. However, the implications of such adjustments on many cellular functions and responses are not clear. Here, we addressed the question whether microgravity induces alterations to drug response in cancer cells. We used both adherent cancer cells (T98G) and cancer cells in suspension (K562) to confirm the known effects of simulated microgravity and then treated the K562 cells with common cancer drugs (hydroxyurea and paclitaxel) following 48 h of exposure to simulated microgravity via a NASA-developed rotary cell culture system. Through fluorescence-guided morphometry, we found that microgravity abolished a significant reduction (p < 0.01) in the nuclear-to-cytoplasm ratio of cancer cells treated with hydroxyurea. Our results call for more studies on the impact of microgravity on cellular drug response, in light of the growing need for space medicine, as space exploration grows.

Microfluidic Microcirculation Mimetic for Exploring Biophysical Mechanisms of Chemotherapy-Induced Metastasis.

There is rapidly emerging evidence from pre-clinical studies, patient samples and patient subpopulations that certain chemotherapeutics inadvertently produce prometastatic effects. Prior to this, we showed that doxorubicin and daunorubicin stiffen cells before causing cell death, predisposing the cells to clogging and extravasation, the latter being a step in metastasis. Here, we investigate which other anti-cancer drugs might have similar prometastatic effects by altering the biophysical properties of cells. We treated myelogenous (K562) leukemic cancer cells with the drugs nocodazole and hydroxyurea and then measured their mechanical properties using a microfluidic microcirculation mimetic (MMM) device, which mimics aspects of blood circulation and enables the measurement of cell mechanical properties via transit times through the device. We also quantified the morphological properties of cells to explore biophysical mechanisms underlying the MMM results. Results from MMM measurements show that nocodazole- and hydroxyurea-treated K562 cells exhibit significantly altered transit times. Nocodazole caused a significant (p < 0.01) increase in transit times, implying a stiffening of cells. This work shows the feasibility of using an MMM to explore possible biophysical mechanisms that might contribute to chemotherapy-induced metastasis. Our work also suggests cell mechanics as a therapeutic target for much needed antimetastatic strategies in general.

Chromatin decondensation and nuclear softening accompany Nanog downregulation in embryonic stem cells.

The interplay between epigenetic modification and chromatin compaction is implicated in the regulation of gene expression, and it comprises one of the most fascinating frontiers in cell biology. Although a complete picture is still lacking, it is generally accepted that the differentiation of embryonic stem (ES) cells is accompanied by a selective condensation into heterochromatin with concomitant gene silencing, leaving access only to lineage-specific genes in the euchromatin. ES cells have been reported to have less condensed chromatin, as they are capable of differentiating into any cell type. However, pluripotency itself-even prior to differentiation-is a split state comprising a naïve state and a state in which ES cells prime for differentiation. Here, we show that naïve ES cells decondense their chromatin in the course of downregulating the pluripotency marker Nanog before they initiate lineage commitment. We used fluorescence recovery after photobleaching, and histone modification analysis paired with a novel, to our knowledge, optical stretching method, to show that ES cells in the naïve state have a significantly stiffer nucleus that is coupled to a globally more condensed chromatin state. We link this biophysical phenotype to coinciding epigenetic differences, including histone methylation, and show a strong correlation of chromatin condensation and nuclear stiffness with the expression of Nanog. Besides having implications for transcriptional regulation and embryonic cell sorting and suggesting a putative mechanosensing mechanism, the physical differences point to a system-level regulatory role of chromatin in maintaining pluripotency in embryonic development.

Comparison of stresses on homogeneous spheroids in the optical stretcher computed with geometrical optics and generalized Lorenz-Mie theory.

We present two electromagnetic frameworks to compare the surface stresses on spheroidal particles in the optical stretcher (a dual-beam laser trap that can be used to capture and deform biological cells). The first model is based on geometrical optics (GO) and limited in its applicability to particles that are much greater than the incident wavelength. The second framework is more sophisticated and hinges on the generalized Lorenz-Mie theory (GLMT). Despite the difference in complexity between both theories, the stress profiles computed with GO and GLMT are in good agreement with each other (relative errors are on the order of 1-10%). Both models predict a diminishing of the stresses for larger wavelengths and a strong increase of the stresses for shorter laser-cell distances. Results indicate that surface stresses on a spheroid with an aspect ratio of 1.2 hardly differ from the stresses on a sphere of similar size. Knowledge of the surface stresses and whether or not they redistribute during the stretching process is of crucial importance in real-time applications of the stretcher that aim to discern the viscoelastic properties of cells for purposes of cell characterization, sorting, and medical diagnostics.

Development of In Vitro Assays for Advancing Radioimmunotherapy against Brain Tumors.

Glioblastoma (GBM) is the most common primary brain tumor. Due to high resistance to treatment, local invasion, and a high risk of recurrence, GBM patient prognoses are often dismal, with median survival around 15 months. The current standard of care is threefold: surgery, radiation therapy, and chemotherapy with temozolomide (TMZ). However, patient survival has only marginally improved. Radioimmunotherapy (RIT) is a fourth modality under clinical trials and aims at combining immunotherapeutic agents with radiotherapy. Here, we develop in vitro assays for the rapid evaluation of RIT strategies. Using a standard cell irradiator and an Electric Cell Impedance Sensor, we quantify cell migration following the combination of radiotherapy and chemotherapy with TMZ and RIT with durvalumab, a PD-L1 immune checkpoint inhibitor. We measure cell survival using a cloud-based clonogenic assay. Irradiated T98G and U87 GBM cells migrate significantly (p < 0.05) more than untreated cells in the first 20−40 h post-treatment. Addition of TMZ increases migration rates for T98G at 20 Gy (p < 0.01). Neither TMZ nor durvalumab significantly change cell survival in 21 days post-treatment. Interestingly, durvalumab abolishes the enhanced migration effect, indicating possible potency against local invasion. These results provide parameters for the rapid supplementary evaluation of RIT against brain tumors.

Private sector initiatives to tackle the burden of COVID-19: experiences from the Nigerian frontline.

Across Africa, there is some evidence of COVID-19 private sector activities to tackle COVID-19 which include the development of rapid diagnostic kits, deployment of e-health platforms for bespoke health workforce training, disease surveillance, reporting, auto-screening and advisories. Inequities in living and access to care by disadvantaged populations in the rural areas have been ameliorated by multi-pronged responses such as that mounted by the Joseph Ukpo Hospitals and Research Institute (JUHRI) in Nigeria. The provision, production and donation of personal protective equipment (PPE), the production of hand sanitizers and the engagement of the local community in the process represents an effective strategy to contain COVID-19, protect health workers and provide pathways for economic support for people whose sources of income have been upended during the pandemic. The JUHRI experience underpinned by Catholic medical ethics provides concrete evidence of the value of private sector participation in dealing with public health emergencies.

In vitro radiotherapy and chemotherapy alter migration of brain cancer cells before cell death.

Although radiotherapy and most cancer drugs target the proliferation of cancer cells, it is metastasis, the complex process by which cancer cells spread from the primary tumor to other tissues and organs of the body where they form new tumors, that leads to over 90% of all cancer deaths. Thus, there is an urgent need for anti-metastasis strategies alongside chemotherapy and radiotherapy. An important step in the metastatic cascade is migration. It is the first step in metastasis via local invasion. Here we address the question whether ionizing radiation and/or chemotherapy might inadvertently promote metastasis and/or invasiveness by enhancing cell migration. We used a standard laboratory irradiator, Faxitron CellRad, to irradiate both non-cancer (HCN2 neurons) and cancer cells (T98G glioblastoma) with 2 Gy, 10 Gy and 20 Gy of X-rays. Paclitaxel (5 µM) was used for chemotherapy. We then measured the attachment and migration of the cells using an electric cell substrate impedance sensing device. Both the irradiated HCN2 cells and T98G cells showed significantly (p < 0.01) enhanced migration compared to non-irradiated cells, within the first 20-40 h following irradiation with 20 Gy. Our results suggest that cell migration should be a therapeutic target in anti-metastasis/anti-invasion strategies for improved radiotherapy and chemotherapy outcomes.

Quantum Dots for Assessment of Reactive Oxygen Species Accumulation During Chemotherapy and Radiotherapy.

Quantum dots (QDs) are semiconductor nanoparticles ranging in size from 2 to 10 nm. QDs are increasingly being developed for biomedical imaging, targeted drug delivery, and green energy technology. Here we describe the novel utilization of biocompatible CdSe-ZnS core-shell semiconductor nanoparticles for assessment of reactive oxygen species (ROS) in the context of chemotherapy and radiotherapy, both of which are important modalities in the treatment of cancer.

Microgravity Modulates Effects of Chemotherapeutic Drugs on Cancer Cell Migration.

Microgravity or the condition of apparent weightlessness causes bone, muscular and immune system dysfunctions in astronauts following spaceflights. These organ and system-level dysfunctions correlate with changes induced at the single cell level both by simulated microgravity on earth as well as microgravity conditions in outer space (as in the international space station). Reported changes in single bone cells, muscle cells and white blood cells include structural/morphological abnormalities, changes in gene expression, protein expression, metabolic pathways and signaling pathways, suggesting that cells mount some response or adjustment to microgravity. However, the implications of such adjustments on many cellular functions and responses are not clear largely because the primary mechanism of gravity sensing in animal cells is unknown. Here, we used a rotary cell culture system developed by NASA to subject leukemic and erythroleukemic cancer cells to microgravity for 48 h and then quantified their innate immune response to common anti-cancer drugs using biophysical parameters and our recently developed quantum-dot-based fluorescence spectroscopy. We found that leukemic cancer cells treated with daunorubicin show increased chemotactic migration (p < 0.01) following simulated microgravity (µg) compared to normal gravity on earth (1 g). However, cells treated with doxorubicin showed enhanced migration both in 1 g and following µg. Our results show that microgravity modulates cancer cell response to chemotherapy in a drug-dependent manner. These results suggest using simulated microgravity as an immunomodulatory tool for the development of new immunotherapies for both space and terrestrial medicine.

Determination of cell elasticity through hybrid ray optics and continuum mechanics modeling of cell deformation in the optical stretcher.

The optical stretcher is a dual-beam trap capable of stretching individual cells. Previous studies have used either ray- or wave-optical models to compute the optical pressure on the surface of a spherical cell. We have extended the ray-optics model to account for focusing by the spherical interface and the effects of multiple internal reflections. Simulation results for red-blood cells (RBCs) show that internal reflections can lead to significant perturbation of the deformation, leading to a systematic error in the determination of cellular elasticity. Calibration studies show excellent agreement between the predicted and measured escape force, and RBC stiffness measurements are consistent with literature values. Measurements of the elasticity of murine osteogenic cells reveal that these cells are approximately 5.4 times stiffer than RBCs.

Fluorescence intensity modulation of CdSe/ZnS quantum dots assesses reactive oxygen species during chemotherapy and radiotherapy for cancer cells.

Quantum dots (QDs) are semiconductor nanoparticles ranging in size from 2 to 10 nm. QDs are increasingly being developed for biomedical imaging, targeted drug delivery and green energy technology. These have led to much research on QD interactions with various physical, chemical and biological systems. For biological systems, research has focused on the biocompatibility/cytotoxicity of QDs in the context of imaging/therapy. However, there is a paucity of work on how biological systems and bioactive molecules might be used to alter the optoelectronic properties of QDs. Here, it is shown that these properties can be altered by reactive oxygen species (ROS) from chemotherapeutic media and biological cells following controlled changes in cellular activities. Using CdSe/ZnS core-shell QDs, spectroscopic analysis of optically excited QDs with HL60, K562 and T98G cancer cell lines is performed. Our results show statistically significant (P < 0.0001) modulation of the fluorescence emission spectra of the QDs due to the ROS produced by common chemotherapeutic drugs, daunorubicin and doxorubicin and by cells following chemotherapy/radiotherapy. This optical modulation, in addition to assessing ROS generation, will possibly enhance applications of QDs in simultaneous diagnostic imaging and nanoparticle-mediated drug delivery as well as simultaneous ROS assessment and radiosensitization for improved outcomes in cancer treatments. Reactive molecular species produced by biological cells and chemotherapeutic drugs can create electric fields that alter the photophysical properties of QDs, and this can be used for concurrent monitoring of cellular activities, while inducing changes in those cellular activities.

The Value of Mobile Ultrasound Services in Rural Communities in South-South Nigeria.

AIM: This paper examines the activities of mobile services units including ultrasound services in rural and urban communities in the Calabar region of South-South, Nigeria. MATERIALS AND METHODS: Consenting individuals were invited and attended five medical outreach activities in rural and urban areas of the Calabar region between January and June 2016. Abdomino-pelvic scans were done. Subsequently the results were analyzed. RESULTS: Five hundred and seventy-four (574) individuals had Abdomino-pelvic scans done, using a curvilinear probe to assess the abdomino-pelvic organs. The female to male ratio was 1.46:1. The age ranged from 1-78 years with a mean of 40.63 (standard deviation of 17.5). The commonest sonographic finding was uterine fibroids, 21 (8.1%). Fifty-four percent of the scans were normal. The commonest sonographic finding in men was prostatic enlargement. CONCLUSION: Medical outreach activities provided by mobile units provide much needed ultrasound services in poor resource settings in Nigeria. Significant clinical pathologies were identified at fairly high rates.

Mechanical deformation induces depolarization of neutrophils.

The transition of neutrophils from a resting state to a primed state is an essential requirement for their function as competent immune cells. This transition can be caused not only by chemical signals but also by mechanical perturbation. After cessation of either, these cells gradually revert to a quiescent state over 40 to 120 min. We use two biophysical tools, an optical stretcher and a novel microcirculation mimetic, to effect physiologically relevant mechanical deformations of single nonadherent human neutrophils. We establish quantitative morphological analysis and mechanical phenotyping as label-free markers of neutrophil priming. We show that continued mechanical deformation of primed cells can cause active depolarization, which occurs two orders of magnitude faster than by spontaneous depriming. This work provides a cellular-level mechanism that potentially explains recent clinical studies demonstrating the potential importance, and physiological role, of neutrophil depriming in vivo and the pathophysiological implications when this deactivation is impaired, especially in disorders such as acute lung injury.