Single-cell mechanical assay unveils viscoelastic similarities in normal and neoplastic brain cells.

Understanding cancer cell mechanics allows for the identification of novel disease mechanisms, diagnostic biomarkers, and targeted therapies. In this study, we utilized our previously established fluid shear stress assay to investigate and compare the viscoelastic properties of normal immortalized human astrocytes and invasive human glioblastoma (GBM) cells when subjected to physiological levels of shear stress that are present in the brain microenvironment. We used a parallel-flow microfluidic shear system and a camera-coupled optical microscope to expose single cells to fluid shear stress and monitor the resulting deformation in real time, respectively. From the video-rate imaging, we fed cell deformation information from digital image correlation into a three-parameter generalized Maxwell model to quantify the nuclear and cytoplasmic viscoelastic properties of single cells. We further quantified actin cytoskeleton density and alignment in immortalized human astrocytes and GBM cells via fluorescence microscopy and image analysis techniques. Results from our study show that contrary to the behavior of many extracranial cells, normal and cancerous brain cells do not exhibit significant differences in their viscoelastic properties. Moreover, we also found that the viscoelastic properties of the nucleus and cytoplasm as well as the actin cytoskeletal densities of both brain cell types are similar. Our work suggests that malignant GBM cells exhibit unique mechanical behaviors not seen in other cancer cell types. These results warrant future studies to elucidate the distinct biophysical characteristics of the brain and reveal novel mechanical attributes of GBM and other primary brain tumors.

Single-cell mechanical analysis reveals viscoelastic similarities between normal and neoplastic brain cells.

Understanding cancer cell mechanics allows for the identification of novel disease mechanisms, diagnostic biomarkers, and targeted therapies. In this study, we utilized our previously established fluid shear stress assay to investigate and compare the viscoelastic properties of normal immortalized human astrocytes (IHAs) and invasive human glioblastoma (GBM) cells when subjected to physiological levels of shear stress that are present in the brain microenvironment. We used a parallel-flow microfluidic shear system and a camera-coupled optical microscope to expose single cells to fluid shear stress and monitor the resulting deformation in real-time, respectively. From the video-rate imaging, we fed cell deformation information from digital image correlation into a three-parameter generalized Maxwell model to quantify the nuclear and cytoplasmic viscoelastic properties of single cells. We further quantified actin cytoskeleton density and alignment in IHAs and GBM cells via immunofluorescence microscopy and image analysis techniques. Results from our study show that contrary to the behavior of many extracranial cells, normal and cancerous brain cells do not exhibit significant differences in their viscoelastic behavior. Moreover, we also found that the viscoelastic properties of the nucleus and cytoplasm as well as the actin cytoskeletal densities of both brain cell types are similar. Our work suggests that malignant GBM cells exhibit unique mechanical behaviors not seen in other cancer cell types. These results warrant future study to elucidate the distinct biophysical characteristics of the brain and reveal novel mechanical attributes of GBM and other primary brain tumors.

Targeted drug-loaded PLGA-PCL microspheres for specific and localized treatment of triple negative breast cancer.

The paper presents the results of the experimental and analytical study of targeted drug-loaded polymer-based microspheres made from blend polymer of polylactic-co-glycolic acid and polycaprolactone (PLGA-PCL) for targeted and localized cancer drug delivery. In vitro sustained release with detailed thermodynamically driven drug release kinetics, over a period of three months using encapsulated targeted drugs (prodigiosin-EphA2 or paclitaxel-EphA2) and control drugs [Prodigiosin (PGS), and paclitaxel (PTX)] were studied. Results from in vitro study showed a sustained and localized drug release that is well-characterized by non-Fickian Korsmeyer-Peppas kinetics model over the range of temperatures of 37 °C (body temperature), 41 °C, and 44 °C (hyperthermic temperatures). The in vitro alamar blue, and flow cytometry assays in the presence of the different drug-loaded polymer formulations resulted to cell death and cytotoxicity that was evidence through cell inhibition and late apoptosis on triple negative breast cancer (TNBC) cells (MDA-MB 231). In vivo studies carried out on groups of 4-week-old athymic nude mice that were induced with subcutaneous TNBC, showed that the localized release of the EphA2-conjugated drugs was effective in complete elimination of residual tumor after local surgical resection. Finally, ex vivo histopathological analysis carried out on the euthanized mice revealed no cytotoxicity and absence of breast cancer metastases in the liver, kidney, and lungs 12 weeks after treatment. The implications of the results are then discussed for the development of encapsulated EphA2-conjugated drugs formulation in the specific targeting, localized, and sustain drug release for the elimination of local recurred TNBC tumors after surgical resection.

Shear Assay Protocol for the Determination of Single-Cell Material Properties.

Irregular biomechanics are a hallmark of cancer biology subject to extensive study. The mechanical properties of a cell are similar to those of a material. A cell's resistance to stress and strain, its relaxation time, and its elasticity are all properties that can be derived and compared to other types of cells. Quantifying the mechanical properties of cancerous (malignant) versus normal (non-malignant) cells allows researchers to further uncover the biophysical fundamentals of this disease. While the mechanical properties of cancer cells are known to consistently differ from the mechanical properties of normal cells, a standard experimental procedure to deduce these properties from cells in culture is lacking. This paper outlines a procedure to quantify the mechanical properties of single cells in vitro using a fluid shear assay. The principle behind this assay involves applying fluid shear stress onto a single cell and optically monitoring the resulting cellular deformation over time. Cell mechanical properties are subsequently characterized using digital image correlation (DIC) analysis and fitting an appropriate viscoelastic model to the experimental data generated from the DIC analysis. Overall, the protocol outlined here aims to provide a more effective and targeted method for the diagnosis of difficult-to-treat cancers.

Sustained release of alpha-methylacyl-CoA racemase (AMACR) antibody-conjugated and free doxorubicin from silica nanoparticles for prostate cancer cell growth inhibition.

This article presents silica nanoparticles for the sustained release of AMACR antibody-conjugated and free doxorubicin (DOX) for the inhibition of prostate cancer cell growth. Inorganic MCM-41 silica nanoparticles were synthesized, functionalized with phenylboronic acid groups (MCM-B), and capped with dextran (MCM-B-D). The nanoparticles were then characterized using Fourier-transform infrared spectroscopy, scanning electron microscopy, transmission electron microscopy, zeta potential analysis, nitrogen sorption, X-ray diffraction, and thermogravimetric analysis, before exploring their potential for drug loading and controlled drug release. This was done using a model prostate cancer drug, DOX, and a targeted prostate cancer drug, α-Methyl Acyl-CoA racemase (AMACR) antibody-conjugated DOX, which attaches specifically to AMACR proteins that are overexpressed on the surfaces of prostate cancer cells. The kinetics of sustained drug release over 30 days was then studied using zeroth order, first order, second order, Higuchi, and the Korsmeyer-Peppas models, while the thermodynamics of drug release was elucidated by determining the entropy and enthalpy changes. The flux of the released DOX was also simulated using the COMSOL Multiphysics software package. Generally, the AMACR antibody-conjugated DOX drug-loaded nanoparticles were more effective than the free DOX drug-loaded formulations in inhibiting the growth of prostate cancer cells in vitro over a 96 h period. The implications of the results are then discussed for the development of drug-eluting structures for the localized and targeted treatment of prostate cancer.

Do Tumor Mechanical Stresses Promote Cancer Immune Escape?

Immune evasion-a well-established cancer hallmark-is a major barrier to immunotherapy efficacy. While the molecular mechanisms and biological consequences underpinning immune evasion are largely known, the role of tissue mechanical stresses in these processes warrants further investigation. The tumor microenvironment (TME) features physical abnormalities (notably, increased fluid and solid pressures applied both inside and outside the TME) that drive cancer mechanopathologies. Strikingly, in response to these mechanical stresses, cancer cells upregulate canonical immune evasion mechanisms, including epithelial-mesenchymal transition (EMT) and autophagy. Consideration and characterization of the origins and consequences of tumor mechanical stresses in the TME may yield novel strategies to combat immunotherapy resistance. In this Perspective, we posit that tumor mechanical stresses-namely fluid shear and solid stresses-induce immune evasion by upregulating EMT and autophagy. In addition to exploring the basis for our hypothesis, we also identify explicit gaps in the field that need to be addressed in order to directly demonstrate the existence and importance of this biophysical relationship. Finally, we propose that reducing or neutralizing fluid shear stress and solid stress-induced cancer immune escape may improve immunotherapy outcomes.

Mechanical and metabolic interplay in the brain metastatic microenvironment.

In this Perspective, we provide our insights and opinions about the contribution-and potential co-regulation-of mechanics and metabolism in incurable breast cancer brain metastasis. Altered metabolic activity can affect cancer metastasis as high glucose supply and demand in the brain microenvironment favors aerobic glycolysis. Similarly, the altered mechanical properties of disseminating cancer cells facilitate migration to and metastatic seeding of the brain, where local metabolites support their progression. Cancer cells in the brain and the brain tumor microenvironment often possess opposing mechanical and metabolic properties compared to extracranial cancer cells and their microenvironment, which inhibit the ease of extravasation and metastasis of these cells outside the central nervous system. We posit that the brain provides a metabolic microenvironment that mechanically reinforces the cellular structure of cancer cells and supports their metastatic growth while restricting their spread from the brain to external organs.

Laser-induced heating of polydimethylsiloxane-magnetite nanocomposites for hyperthermic inhibition of triple-negative breast cancer cell proliferation.

This paper presents the results of an experimental and computational study of the effects of laser-induced heating provided by magnetite nanocomposite structures that are being developed for the localized hyperthermic treatment of triple-negative breast cancer. Magnetite nanoparticle-reinforced polydimethylsiloxane (PDMS) nanocomposites were fabricated with weight percentages of 1%, 5%, and 10% magnetite nanoparticles. The nanocomposites were exposed to incident Near Infrared (NIR) laser beams with well-controlled powers. The laser-induced heating is explored in: (i) heating liquid media (deionized water and cell growth media [Leibovitz L15+]) to characterize the photothermal properties of the nanocomposites, (ii) in vitro experiments that explore the effects of localized heating on triple-negative breast cancer cells, and (iii) experiments in which the laser beams penetrate through chicken tissue to heat up nanocomposite samples embedded at different depths beneath the chicken skin. The resulting plasmonic laser-induced heating is explained using composite theories and heat transport models. The results show that the laser/nanocomposite interactions decrease the viability of triple-negative breast cancer cells (MDA-MB-231) at temperatures in the hyperthermia domain between 41 and 44°C. Laser irradiation did not cause any observed physical damage to the chicken tissue. The potential in vivo performance of the PDMS nanocomposites was also investigated using computational finite element models of the effects of laser/magnetite nanocomposite interactions on the temperatures and thermal doses experienced by tissues that surround the nanocomposite devices. The implications of the results are then discussed for the development of implantable nanocomposite devices for localized treatment of triple-negative breast cancer tissue via hyperthermia.

Investigation of creep properties and the cytoskeletal structures of non-tumorigenic breast cells and triple-negative breast cancer cells.

This article presents the correlation of creep and viscoelastic properties to the cytoskeletal structure of both tumorigenic and non-tumorigenic cells. Unique shear assay and strain mapping techniques were used to study the creep and viscoelastic properties of single non-tumorigenic and tumorigenic cells. At least 20 individual cells, three locations per cell, were studied. From the results, lower densities in the volume of actin, and keratin 18 structures were observed with the progression of cancer and were correlated to the increased creep rates and reduced mechanical properties (Young's moduli and viscosities) of tumorigenic (MDA-MB-231) cells. The study reveals significant differences between the creep and viscoelastic properties of non-tumorigenic breast cells versus tumorigenic cells. The variations in the creep strain rates are shown to be well characterized by lognormal distributions, while the statistical variations in the viscoelastic properties are well-described by normal distributions. The implications of the results are discussed for the study of discrete cell behaviors, strain and viscoelastic responses of the cell, and the role of cell cytoskeleton in the onset and progression of cancers.

Actin cytoskeletal structure and the statistical variations of the mechanical properties of non-tumorigenic breast and triple-negative breast cancer cells.

This paper presents the results of a study of the actin cytoskeletal structures and the statistical variations in the actin fluorescence intensities and viscoelastic properties of non-tumorigenic breast cells and triple-negative breast cancer cells at different stages of tumor progression. The variation in the actin content of the cell cytoskeletal structures is shown to be consistent with the viscoelastic properties of the cell as it progresses from non-tumorigenic to more metastatic states. The corresponding viscoelastic properties of the nuclei and the cytoplasm (Young's moduli, viscosities, and relaxation times) of the cells are also measured using Digital Image Correlation (DIC) and shear assay techniques. These properties are shown to exhibit statistical variations that are well characterized by normal distributions. The changes in the mean properties of individual cancer cells are tested using Fisher pairwise comparisons and the analysis of variance (ANOVA). The implications of the results are then discussed for the development of shear assay techniques and mechanical biomarkers for the detection of triple-negative breast cancer at different stages of tumor progression.