Breast cancer stem cells: mechanobiology reveals highly invasive cancer cell subpopulations.

Cancer stem-like cells (CSCs) are a typically small subpopulation of highly tumorigenic cells that can self-renew, differentiate, drive tumor progression, and may mediate drug resistance and metastasis. Metastasis driving CSCs are expected to be highly invasive. To determine the relative invasiveness of CSCs, we isolate distinct subpopulations in the metastatic, MDA-MB-231 breast-cancer cell line, identified by the stem-cell markers aldehyde dehydrogenase (ALDH) and CD44. We determine CSC-subpopulation invasiveness levels using our rapid (2 h) mechanobiology-based assay. Specifically, invasive cells forcefully push and indent the surface of physiological-stiffness synthetic gels to cell-scale depths, where the percentage of indenting cells and their attained depths have previously provided clinically relevant predictions of the metastatic risk in different cancer types. We observe that the small (3.2%) CD44+ALDH+ cell-subpopulation indents more and attains significantly deeper depths (65% indenting to 6 ± 0.3 µm) relative to CD44+ALDH-, CD44-ALDH-, CD44-ALDH+ cells, and the whole-sample control (with 18-44% indenting cells reaching average depths of 4.4-5 µm). The CD44+ALDH+ similarly demonstrates twofold higher migratory capacity in Boyden chambers. The higher invasiveness of CD44+ALDH+ cells reveals their likely role in facilitating disease progression, providing prognostic markers for increased risk of recurrence and metastasis.

Actin as a Target to Reduce Cell Invasiveness in Initial Stages of Metastasis.

We demonstrate the relative roles of the cell cytoskeleton, and specific importance of actin in facilitating mechanical aspects of metastatic invasion. A crucial step in metastasis, the typically lethal spread of cancer to distant body-sites, is cell invasion through dense tissues composed of extracellular matrix and various non-cancerous cells. Cell invasion requires cell-cytoskeleton remodeling to facilitate dynamic morphological changes and force application. We have previously shown invasive cell subsets in heterogeneous samples can rapidly (2 h) and forcefully indent non-degradable, impenetrable, synthetic gels to cell-scale depths. The amounts of indenting cells and their attained depths provide the mechanical invasiveness of the sample, which as we have shown agrees with the in vitro metastatic potential and the in vivo metastatic risk in humans. To identify invasive force-application mechanisms, we evaluated changes in mechanical invasiveness following chemical perturbations targeting the structure and function of cytoskeleton elements and associated proteins. We evaluate effects on short-term (2-hr) indentations of single, well-spaced or closely situated cells as compared to long-time-scale Boyden chamber migration. We show that actomyosin inhibition may be used to reduce (mechanical) invasiveness of single or collectively invading cells, while actin-disruption may induce escape-response of treated single-cells, which may promote metastasis.

Micropatterned topographies reveal measurable differences between cancer and benign cells.

During metastasis, cancer cells migrate away from the primary tumor-site, encountering different microenvironment topographies that may facilitate or inhibit cancer cell adherence and growth; those relate to sites of invasion and seeding. To evaluate topography effects, poly-lactic-poly-glycolic (PLGA) gels are generated as flat substrates, porous, or with rectangular microchannels of varying widths (5-100 µm) and depths (10/20 µm). The topography effect on time-dependent adherence, proliferation, morphology, alignment and long-term structural development of metastatic breast-cancer and benign cells are evaluated; adherence at short time-scales (3 h) is compared to developed morphologies and multicellular structures (>2 days) indicating function. At short time-scales, both cell types exhibit rounded morphologies, however, while the benign cells tend to cluster the cancer cells preferentially adhered as single cells at high-curvature substrate-sites (e.g. convex pore-edges or channel-edges). At long times, the benign cells develop extensive, tissue-like multicellular sheets spanning across several 10 µm deep channels or filling in single-file 20 µm-deep narrow channels (5-15 µm). Contrastingly, cancer cells mainly attach as single cells to high-curvature channel bottoms, in alignment with narrow channels. Thus, cell responses to topography, specifically their localization and growth in narrow microchannels, may provide a way to distinguish cancer from benign cells, by demonstrating their inherent function.

Sodium pyruvate pre-treatment prevents cell death due to localised, damaging mechanical strains in the context of pressure ulcers.

We demonstrate sodium pyruvate (NaPy) pre-treatment as a successful approach for pressure ulcer (PU) prevention by averting their aetiological origin-cell-level damage and death by large, sustained mechanical loads. We evaluated the NaPy pre-treatment effect on permeability changes in the cell's plasma membrane (PM) following application of in vitro damaging-level strains. Fibroblasts or myoblasts, respectively, models for superficial or deep-tissue damage were grown in 0 or 1 mM NaPy, emulating typical physiological or cell culture conditions. Cells were pre-treated for 4 hours with 0 to 5 mM NaPy prior to 3-hour sustained, damaging-level loads (12% strain). PM permeability was quantified by the cell uptake of small (4 kDa), fluorescent dextran compared with unstrained control using fluorescence-activated cell sorting (FACS). Pre-treatment with 1 mM, and especially 5 mM, NaPy significantly reduces damage to PM integrity. Long-term NaPy pre-exposure can improve protective treatment, affecting fibroblasts and myoblasts differently. Pre-treating with NaPy, a natural cell metabolite, allows cells under damaging-level mechanical loads to maintain their PM integrity, that is, to avoid loss of homeostasis and inevitable, eventual cell death, by preventing initial, microscale stages of PU formation. This pre-treatment may be applied prior to planned periods of immobility, for example, planned surgery or transport, to prolong safe time in a position by preventing initial cell damage that can cascade and lead to PU formation.

Non-damaging stretching combined with sodium pyruvate supplement accelerate migration of fibroblasts and myoblasts during gap closure.

BACKGROUND: Sustained, low- and mid-level (3-6%), radial stretching combined with varying concentrations of sodium pyruvate (NaPy) supplement increase the migration rate during microscale gap closure following an in vitro injury; NaPy is a physiological supplement often used in cell-culture media. Recently we showed that low-level tensile strains accelerate in vitro kinematics during en masse cell migration; topically applied mechanical deformations also accelerate in vivo healing in larger wounds. The constituents and nutrients at injury sites change. Thus, we combine a supplement with stretching conditions to effectively accelerate wound healing. METHODS: Monolayers of murine fibroblasts (NIH3T3) or myoblasts (C2C12) were cultured in 1 mM NaPy on stretchable, linear-elastic substrates. Monolayers were subjected to 0, 3, or 6% stretching using a custom three-dimensionally printed stretching apparatus, micro-damage was immediately induced, media was replaced with fresh media containing 0, 1, or 5 mM NaPy, and cell migration kinematics during gap-closure were quantitatively evaluated. FINDINGS: In myoblasts, the smallest evaluated strain (3%, minimal risk of damage) combined with preinjury (1 mM) and post-injury exogenous NaPy supplements accelerated gap closure in a statistically significant manner; response was NaPy concentration dependent. In both fibroblasts and myoblasts, when cells were pre-exposed to NaPy, yet no supplement was provided post-injury, mid-level stretches (6%) compensated for post-injury deficiency in exogenous NaPy and accelerated gap-closure in a statistically significant manner. INTERPRETATION: Small deformations combined with NaPy supplement prior-to and following cell-damage accelerate en masse cell migration and can be applied in wound healing, e.g. to preventatively accelerate closure of microscale gaps.

Cell-Gel Mechanical Interactions as an Approach to Rapidly and Quantitatively Reveal Invasive Subpopulations of Metastatic Cancer Cells.

We present a novel mechanobiology-based invasiveness assay to rapidly and quantitatively evaluate the mechanical invasiveness of metastatic cancer cells and identify invasive subpopulations, without need for chemoattractants and independent of serum content. A commonly accepted assay to determine metastatic potential in vitro is the Boyden chamber assay, where the percentage of serum-starved cells that can long-term transmigrate/invade through subcell size membrane pores is quantified; those experiments typically take 2-3 days and require serum-starvation. To squeeze through the small pores, the invasive cells must be pliable, yet they are also able to force their way through flexible microenvironments. We have previously shown that metastatic breast cancer cells will deform and indent soft, impenetrable, elastic gels within 2 h of seeding, without requiring serum starvation. Specifically, in cell lines with higher metastatic potential, a larger percentage of cells will indent gels and typically also to deeper depths. Thus, we are able to rapidly reveal mechanically invasive subpopulations, which are likely those that lead to high metastatic potential. By comparing the Boyden chamber and gel mechanobiology assays, we show that the gel-indenting cell subpopulations are part of the group that successfully transmigrates through the Boyden chamber membrane (8 µm pores). Thus, we are able to rapidly (within 2 h of seeding and using the standard cell media), provide a quantitative measure of the mechanical invasiveness of cancer cells, which is correlated to the metastatic potential but is an independent parameter; we evaluate numbers of indenting cells and their indentation depth. Moreover, the mechanical invasiveness assay allows focus on specific (invasive or noninvasive) cells within the sample to identify specific surface markers, determine invasive mechanisms, and evaluate effects of applied drugs and treatments on the different subpopulations.

Molecular interference of fibrin's divalent polymerization mechanism enables modulation of multiscale material properties.

Protein based polymers provide an exciting and complex landscape for tunable natural biomaterials through modulation of molecular level interactions. Here we demonstrate the ability to modify protein polymer structural and mechanical properties at multiple length scales by molecular 'interference' of fibrin's native polymerization mechanism. We have previously reported that engagement of fibrin's polymerization 'hole b', also known as 'b-pockets', through PEGylated complementary 'knob B' mimics can increase fibrin network porosity but also, somewhat paradoxically, increase network stiffness. Here, we explore the possible mechanistic underpinning of this phenomenon through characterization of the effects of knob B-fibrin interaction at multiple length scales from molecular to bulk polymer. Despite its weak monovalent binding affinity for fibrin, addition of both knob B and PEGylated knob B at concentrations near the binding coefficient, Kd, increased fibrin network porosity, consistent with the reported role of knob B-hole b interactions in promoting lateral growth of fibrin fibers. Addition of PEGylated knob B decreases the extensibility of single fibrin fibers at concentrations near its Kd but increases extensibility of fibers at concentrations above its Kd. The data suggest this bimodal behavior is due to the individual contributions knob B, which decreases fiber extensibility, and PEG, which increase fiber extensibility. Taken together with laser trap-based microrheological and bulk rheological analyses of fibrin polymers, our data strongly suggests that hole b engagement increases in single fiber stiffness that translates to higher storage moduli of fibrin polymers despite their increased porosity. These data point to possible strategies for tuning fibrin polymer mechanical properties through modulation of single fiber mechanics.

Adenosine A(1) and prostaglandin E receptor 3 receptors mediate global airway contraction after local epithelial injury.

Epithelial injury and airway hyperresponsiveness are prominent features of asthma. We have previously demonstrated that laser ablation of single epithelial cells immediately induces global airway constriction through Ca(2+)-dependent smooth muscle shortening. The response is mediated by soluble mediators released from wounded single epithelial cells; however, the soluble mediators and signaling mechanisms have not been identified. In this study, we investigated the nature of the epithelial-derived soluble mediators and the associated signaling pathways that lead to the L-type voltage-dependent Ca(2+) channel (VGCC)-mediated Ca(2+) influx. We found that inhibition of adenosine A1 receptors (or removal of adenosine with adenosine deaminase), cyclooxygenase (COX)-2 or prostaglandin E receptor 3 (EP3) receptors, epidermal growth factor receptor (EGFR), or platelet-derived growth factor receptor (PDGFR) all significantly blocked Ca(2+) oscillations in smooth muscle cells and airway contraction induced by local epithelial injury. Using selective agonists to activate the receptors in the presence and absence of selective receptor antagonists, we found that adenosine activated the signaling pathway A1R→EGFR/PDGFR→COX-2→EP3→VGCCs→calcium-induced calcium release, leading to intracellular Ca(2+) oscillations in airway smooth muscle cells and airway constriction.

Local small airway epithelial injury induces global smooth muscle contraction and airway constriction.

Small airway epithelial cells form a continuous sheet lining the conducting airways, which serves many functions including a physical barrier to protect the underlying tissue. In asthma, injury to epithelial cells can occur during bronchoconstriction, which may exacerbate airway hyperreactivity. To investigate the role of epithelial cell rupture in airway constriction, laser ablation was used to precisely rupture individual airway epithelial cells of small airways (<300-µm diameter) in rat lung slices (∼250-µm thick). Laser ablation of single epithelial cells using a femtosecond laser reproducibly induced airway contraction to ∼70% of the original cross-sectional area within several seconds, and the contraction lasted for up to 40 s. The airway constriction could be mimicked by mechanical rupture of a single epithelial cell using a sharp glass micropipette but not with a blunt glass pipette. These results suggest that soluble mediators released from the wounded epithelial cell induce global airway contraction. To confirm this hypothesis, the lysate of primary human small airway epithelial cells stimulated a similar airway contraction. Laser ablation of single epithelial cells triggered a single instantaneous Ca(2+) wave in the epithelium, and multiple Ca(2+) waves in smooth muscle cells, which were delayed by several seconds. Removal of extracellular Ca(2+) or decreasing intracellular Ca(2+) both blocked laser-induced airway contraction. We conclude that local epithelial cell rupture induces rapid and global airway constriction through release of soluble mediators and subsequent Ca(2+)-dependent smooth muscle shortening.

Concentration independent modulation of local micromechanics in a fibrin gel.

Methods for tuning extracellular matrix (ECM) mechanics in 3D cell culture that rely on increasing the concentration of either protein or cross-linking molecules fail to control important parameters such as pore size, ligand density, and molecular diffusivity. Alternatively, ECM stiffness can be modulated independently from protein concentration by mechanically loading the ECM. We have developed a novel device for generating stiffness gradients in naturally derived ECMs, where stiffness is tuned by inducing strain, while local mechanical properties are directly determined by laser tweezers based active microrheology (AMR). Hydrogel substrates polymerized within 35 mm diameter Petri dishes are strained non-uniformly by the precise rotation of an embedded cylindrical post, and exhibit a position-dependent stiffness with little to no modulation of local mesh geometry. Here we present the device in the context of fibrin hydrogels. First AMR is used to directly measure local micromechanics in unstrained hydrogels of increasing fibrin concentration. Changes in stiffness are then mapped within our device, where fibrin concentration is held constant. Fluorescence confocal imaging and orbital particle tracking are used to quantify structural changes in fibrin on the micro and nano levels respectively. The micromechanical strain stiffening measured by microrheology is not accompanied by ECM microstructural changes under our applied loads, as measured by confocal microscopy. However, super-resolution orbital tracking reveals nanostructural straightening, lengthening, and reduced movement of fibrin fibers. Furthermore, we show that aortic smooth muscle cells cultured within our device are morphologically sensitive to the induced mechanical gradient. Our results demonstrate a powerful cell culture tool that can be used in the study of mechanical effects on cellular physiology in naturally derived 3D ECM tissues.

Generation of Mathieu-Gauss modes with an axicon-based laser resonator.

We report what is to our best knowledge the first observation of Mathieu-Gauss modes directly generated in an axicon-based stable resonator. By slightly breaking the symmetry of the cavity we were able to generate single lowest and high-order Mathieu-Gauss modes of high quality. The observed transverse modes have an inherent elliptic structure and exhibit remarkable agreement with theoretical predictions.