Enhanced Biomechanically Mediated “Phagocytosis” in Detached Tumor Cells

Uptake of particles by cells involves various natural mechanisms that are essential for their biological functions. The same mechanisms are used in the engulfment of synthetic colloidal drug carriers, while the extent of the uptake affects the biological performance and selectivity. Thus far, little is known regarding the effect of external biomechanical stimuli on the capacity of the cells to uptake nano and micro carriers. This is relevant for anchorage-dependent cells that have detached from surfaces or for cells that travel in the body such as tumor cells, immune cells and various circulating stem cells. In this study, we hypothesize that cellular deformability is a crucial physical effector for the successful execution of the phagocytosis-like uptake in cancer cells. To test this assumption, we develop a well-controlled tunable method to compare the uptake of inert particles by cancer cells in adherent and non-adherent conditions. We introduce a self-designed 3D-printed apparatus, which enables constant stirring while facilitating a floating environment for cell incubation. We reveal a mechanically mediated phagocytosis-like behavior in various cancer cells, that was dramatically enhance in the detached cell state. Our findings emphasize the importance of including proper biomechanical cues to reliably mimic certain physiological scenarios. Beyond that, we offer a cost-effective accessible research tool to study mixed cultures for both adherent and non-adherent cells.

A Review of Single-Cell Adhesion Force Kinetics and Applications

Cells exert, sense, and respond to the different physical forces through diverse mechanisms and translating them into biochemical signals. The adhesion of cells is crucial in various developmental functions, such as to maintain tissue morphogenesis and homeostasis and activate critical signaling pathways regulating survival, migration, gene expression, and differentiation. More importantly, any mutations of adhesion receptors can lead to developmental disorders and diseases. Thus, it is essential to understand the regulation of cell adhesion during development and its contribution to various conditions with the help of quantitative methods. The techniques involved in offering different functionalities such as surface imaging to detect forces present at the cell-matrix and deliver quantitative parameters will help characterize the changes for various diseases. Here, we have briefly reviewed single-cell mechanical properties for mechanotransduction studies using standard and recently developed techniques. This is used to functionalize from the measurement of cellular deformability to the quantification of the interaction forces generated by a cell and exerted on its surroundings at single-cell with attachment and detachment events. The adhesive force measurement for single-cell microorganisms and single-molecules is emphasized as well. This focused review should be useful in laying out experiments which would bring the method to a broader range of research in the future.

Sub-Fractions of Red Blood Cells Respond Differently to Shear Exposure Following Superoxide Treatment

Red blood cell (RBC) deformability is an essential component of microcirculatory function that appears to be enhanced by physiological shear stress, while being negatively affected by supraphysiological shears and/or free radical exposure. Given that blood contains RBCs with non-uniform physical properties, whether all cells equivalently tolerate mechanical and oxidative stresses remains poorly understood. We thus partitioned blood into old and young RBCs which were exposed to phenazine methosulfate (PMS) that generates intracellular superoxide and/or specific mechanical stress. Measured RBC deformability was lower in old compared to young RBCs. PMS increased total free radicals in both sub-populations, and RBC deformability decreased accordingly. Shear exposure did not affect reactive species in the sub-populations but reduced RBC nitric oxide synthase (NOS) activation and intriguingly increased RBC deformability in old RBCs. The co-application of PMS and shear exposure also improved cellular deformability in older cells previously exposed to reactive oxygen species (ROS), but not in younger cells. Outputs of NO generation appeared dependent on cell age; in general, stressors applied to younger RBCs tended to induce S-nitrosylation of RBC cytoskeletal proteins, while older RBCs tended to reflect markers of nitrosative stress. We thus present novel findings pertaining to the interplay of mechanical stress and redox metabolism in circulating RBC sub-populations.

Single Cell Hydrodynamic Stretching and Microsieve Filtration Reveal Genetic, Phenotypic and Treatment-Related Links to Cellular Deformability

Deformability is shown to correlate with the invasiveness and metastasis of cancer cells. Recent studies suggest epithelial-to-mesenchymal transition (EMT) might enable cancer metastasis. However, the correlation of EMT with cancer cell deformability has not been well elucidated. Cellular deformability could also help evaluate the drug response of cancer cells. Here, we combine hydrodynamic stretching and microsieve filtration to study cellular deformability in several cellular models. Hydrodynamic stretching uses extensional flow to rapidly quantify cellular deformability and size with high throughput at the single cell level. Microsieve filtration can rapidly estimate relative deformability in cellular populations. We show that colorectal cancer cell line RKO with the mesenchymal-like feature is more flexible than the epithelial-like HCT116. In another model, the breast epithelial cells MCF10A with deletion of the TP53 gene are also significantly more deformable compared to their isogenic wildtype counterpart, indicating a potential genetic link to cellular deformability. We also find that the drug docetaxel leads to an increase in the size of A549 lung cancer cells. The ability to associate mechanical properties of cancer cells with their phenotypes and genetics using single cell hydrodynamic stretching or the microsieve may help to deepen our understanding of the basic properties of cancer progression.

Sensing of red blood cells with decreased membrane deformability by the human spleen

The current paradigm in the pathogenesis of several hemolytic red blood cell disorders is that reduced cellular deformability is a key determinant of splenic sequestration of affected red cells. Three distinct features regulate cellular deformability: membrane deformability, surface area-to-volume ratio (cell sphericity), and cytoplasmic viscosity. By perfusing normal human spleens ex vivo, we had previously showed that red cells with increased sphericity are rapidly sequestered by the spleen. Here, we assessed the retention kinetics of red cells with decreased membrane deformability but without marked shape changes. A controlled decrease in membrane deformability (increased membrane rigidity) was induced by treating normal red cells with increasing concentrations of diamide. Following perfusion, diamide-treated red blood cells (RBCs) were rapidly retained in the spleen with a mean clearance half-time of 5.9 minutes (range, 4.0-13.0). Splenic clearance correlated positively with increased membrane rigidity (r = 0.93; P < .0001). To determine to what extent this increased retention was related to mechanical blockade in the spleen, diamide-treated red cells were filtered through microsphere layers that mimic the mechanical sensing of red cells by the spleen. Diamide-treated red cells were retained in the microsphilters (median, 7.5%; range, 0%-38.6%), although to a lesser extent compared with the spleen (median, 44.1%; range, 7.3%-64.0%; P < .0001). Taken together, these results have implications for understanding the sensitivity of the human spleen to sequester red cells with altered cellular deformability due to various cellular alterations and for explaining clinical heterogeneity of RBC membrane disorders.

Nuclear deformability and telomere dynamics are regulated by cell geometric constraints

Forces generated by the cytoskeleton can be transmitted to the nucleus and chromatin via physical links on the nuclear envelope and the lamin meshwork. Although the role of these active forces in modulating prestressed nuclear morphology has been well studied, the effect on nuclear and chromatin dynamics remains to be explored. To understand the regulation of nuclear deformability by these active forces, we created different cytoskeletal states in mouse fibroblasts using micropatterned substrates. We observed that constrained and isotropic cells, which lack long actin stress fibers, have more deformable nuclei than elongated and polarized cells. This nuclear deformability altered in response to actin, myosin, formin perturbations, or a transcriptional down-regulation of lamin A/C levels in the constrained and isotropic geometry. Furthermore, to probe the effect of active cytoskeletal forces on chromatin dynamics, we tracked the spatiotemporal dynamics of heterochromatin foci and telomeres. We observed increased dynamics and decreased correlation of the heterochromatin foci and telomere trajectories in constrained and isotropic cell geometry. The observed enhanced dynamics upon treatment with actin depolymerizing reagents in elongated and polarized geometry were regained once the reagent was washed off, suggesting an inherent structural memory in chromatin organization. We conclude that active forces from the cytoskeleton and rigidity from lamin A/C nucleoskeleton can together regulate nuclear and chromatin dynamics. Because chromatin remodeling is a necessary step in transcription control and its memory, genome integrity, and cellular deformability during migration, our results highlight the importance of cell geometric constraints as critical regulators in cell behavior.

An optofluidic constriction chip for monitoring metastatic potential and drug response of cancer cells

We present a new optofluidic device with a calibrated microconstriction for the analysis of cellular deformability.

Hierarchical Determination of Nuclear Deformability by Lamin Isoforms During Adult Hematopoiesis: Implications in Blood Cell Trafficking

Abstract 1200 Over the last few decades, it has become clear that different blood lineages have distinct deformability, and that some blood cell types become softer in maturation perhaps to facilitate trafficking from marrow through the endothelial barrier and into the circulation (Lichtman, NEJM, 1970; 283:943–8). However, any common molecular basis of this phenomenon remains unclear. Cellular deformability is determined by elasticity of the cortex and the nucleus, and the latter is dynamically regulated by changes in expression and organization of the lamins (Pajerowski… Discher, PNAS, 2007; 104:15619–24). While gene regulation of hematopoiesis has been extensively studied, roles of lamins in blood lineages are less well understood. To address this, we developed a novel protein isoform expression analysis algorithm, “mass spectrometry calibrated intracellular flow cytometry”, to quantify lamin stoichiometries in human hematopoietic stem cells and progenitors through different mature blood lineages. This approach reveals the hematopoietic lineage map of lamins, showing that lamin A varies by 4-fold, while the normally ‘constitutive’ lamin B varies by 30-fold. During differentiation, lymphoid and myeloid lineages show decreased total lamin intensity and pliable nuclei as measured by micropipette aspiration, consistent with their ability to transmigrate into circulation. In contrast, megakaryocytes (MKs) remain in marrow because their polyploid nuclei are too large and rigid, as indicated by high lamin levels; this nuclear anchorage allows MKs to extend membrane projections into blood, where shear generates circulating platelets. Maturation of MKs is further regulated by serine phosphorylation of lamin A, since overexpression of a phospho-inactive mutant leads to increased polyploidization. Erythroid lineages share the same progenitor with MKs and migrate into blood as enucleated RBCs, because of high lamin A intensity relative to B in the progenitors and stiff chromatin. Consistent with this observation, microarray analysis of primary CD34+-derived cells indicates that some key erythroid genes are strongly correlated with lamin isoforms. Functional studies indicate that lamin A overexpression increases MK and erythroid differentiation by 2-fold, while the knockdown increases migration through pores by 2-fold. Surprisingly, increasing the lamin A to B ratio by lamin B1 knockdown decreases nuclear deformability by up to 50%, highlighting the importance of lamin isoform ratios, rather than absolute expression levels, in specifying nuclear rheology and hence traffickability of blood lineages. Finally, lamin A can be transcriptionally downregulated by retinoic acid by 2-fold, consistent with its well-established role in driving myeloid differentiation from progenitors. Together, the study suggests that nuclear deformability is hierarchically programmed by differential expression of the nucleoskeletal lamin A and B isoforms during hematopoietic differentiation, which in turn influence the ability of blood cells to migrate through marrow. Disclosures: No relevant conflicts of interest to declare.