Circulating tumor cells with metastasis-initiating competence survive fluid shear stress during hematogenous dissemination through CXCR4-PI3K/AKT signaling.

To seed lethal secondary lesions, circulating tumor cells (CTCs) must survive all rate-limiting factors during hematogenous dissemination, including fluid shear stress (FSS) that poses a grand challenge to their survival. We thus hypothesized that CTCs with the ability to survive FSS in vasculature might hold metastasis-initiating competence. This study reported that FSS of physiologic magnitude selected a small subpopulation of suspended tumor cells in vitro with the traits of metastasis-initiating cells, including stemness, migration/invasion potential, cellular plasticity, and biophysical properties. These shear-selected cells generated local and metastatic tumors at the primary and distal sites efficiently, implicating their metastasis competence. Mechanistically, FSS activated the mechanosensitive protein CXCR4 and the downstream PI3K/AKT signaling, which were essential in shear-mediated selection of metastasis-competent CTCs. In summary, these findings conclude that CTCs with metastasis-initiating competence survive FSS during hematogenous dissemination through CXCR4-PI3K/AKT signaling, which may provide new therapeutic targets for the early prevention of tumor metastasis.

Neoteric Semiembedded ß-Tricalcium Phosphate Promotes Osteogenic Differentiation of Mesenchymal Stem Cells under Cyclic Stretch.

ß-Tricalcium phosphate (ß-TCP) is a bioactive material for bone regeneration, but its brittleness limits its use as a standalone scaffold. Therefore, continuous efforts are necessary to effectively integrate ß-TCP into polymers, facilitating a sturdy ion exchange for cell regulation. Herein, a novel semiembedded technique was utilized to anchor ß-TCP nanoparticles onto the surface of the elastic polymer, followed by hydrophilic modification with the polymerization of dopamine. Cell adhesion and osteogenic differentiation of mesenchymal stem cells (MSCs) under static and dynamic uniaxial cyclic stretching conditions were investigated. The results showed that the new strategy was effective in promoting cell adhesion, proliferation, and osteogenic induction by the sustained release of Ca2+ in the vicinity and creating a reasonable roughness. Specifically, released Ca2+ from ß-TCP could activate the calcium signaling pathway, which further upregulated calmodulin and calcium/calmodulin-dependent protein kinase II genes in MSCs. Meanwhile, the roughness of the membrane and the uniaxial cyclic stretching activated the PIEZO1 signaling pathway. Chemical and mechanical stimulation promotes osteogenic differentiation and increases the expression of related genes 2-8-fold. These findings demonstrated that the neoteric semiembedded structure was a promising strategy in controlling both chemical and mechanical factors of biomaterials for cell regulation.

Cell mechanics regulate the migration and invasion of hepatocellular carcinoma cells via JNK signaling.

Hepatocellular carcinoma (HCC) cells, especially those with metastatic competence, show reduced stiffness compared to the non-malignant counterparts. However, it is still unclear whether and how the mechanics of HCC cells influence their migration and invasion. This study reports that HCC cells with enhanced motility show reduced mechanical stiffness and cytoskeleton, suggesting the inverse correlation between cellular stiffness and motility. Through pharmacologic and genetic approaches, inhibiting actomyosin activity reduces HCC cellular stiffness but promotes their migration and invasion, while activating it increases cell stiffness but impairs cell motility. Actomyosin regulates cell motility through the influence on cellular stiffness. Mechanistically, weakening/strengthening cells inhibits/promotes c-Jun N terminal kinase (JNK) phosphorylation, activation/inhibition of which rescues the effects of cell mechanics on their migration and invasion. Further, HCC cancer stem cells (CSCs) exhibit higher motility but lower stiffness than control cells. Increasing CSC stiffness weakens migration and invasion through the activation of JNK signaling. In conclusion, our findings unveil a new regulatory role of actomyosin-mediated cellular mechanics in tumor cell motility and present new evidence to support that tumor cell softening may be one driving force for HCC metastasis. STATEMENT OF SIGNIFICANCE: Tumor cells progressively become softened during metastasis and low cell stiffness is associated with high metastatic potential. However, it remains unclear whether tumor cell softening is a by-product of or a driving force for tumor progression. This work reports that the stiffness of hepatocellular carcinoma cells is linked to their migration and invasion. Importantly, tumor cell softening promotes migration and invasion, while cell stiffening impairs the mobility. Weakening/strengthening cells inhibits/promotes JNK phosphorylation, activation/inhibition of which rescues the effects of cell mechanics on their migration and invasion ability. Further, stiffening liver cancer stem cells attenuates their motility through activating JNK signaling. In summary, our study uncovers a previously unappreciated role of tumor cell mechanics in migration and invasion and implicates the therapeutic potential of cell mechanics in the mechanotargeting of metastasis.

Biophysics in tumor growth and progression: from single mechano-sensitive molecules to mechanomedicine.

Evidence from physical sciences in oncology increasingly suggests that the interplay between the biophysical tumor microenvironment and genetic regulation has significant impact on tumor progression. Especially, tumor cells and the associated stromal cells not only alter their own cytoskeleton and physical properties but also remodel the microenvironment with anomalous physical properties. Together, these altered mechano-omics of tumor tissues and their constituents fundamentally shift the mechanotransduction paradigms in tumorous and stromal cells and activate oncogenic signaling within the neoplastic niche to facilitate tumor progression. However, current findings on tumor biophysics are limited, scattered, and often contradictory in multiple contexts. Systematic understanding of how biophysical cues influence tumor pathophysiology is still lacking. This review discusses recent different schools of findings in tumor biophysics that have arisen from multi-scale mechanobiology and the cutting-edge technologies. These findings range from the molecular and cellular to the whole tissue level and feature functional crosstalk between mechanotransduction and oncogenic signaling. We highlight the potential of these anomalous physical alterations as new therapeutic targets for cancer mechanomedicine. This framework reconciles opposing opinions in the field, proposes new directions for future cancer research, and conceptualizes novel mechanomedicine landscape to overcome the inherent shortcomings of conventional cancer diagnosis and therapies.

The Mechanics of Tumor Cells Dictate Malignancy via Cytoskeleton-Mediated APC/Wnt/ß-Catenin Signaling.

Tumor cells progressively remodel cytoskeletal structures and reduce cellular stiffness during tumor progression, implicating the correlation between cell mechanics and malignancy. However, the roles of tumor cell cytoskeleton and the mechanics in tumor progression remain incompletely understood. We report that softening/stiffening tumor cells by targeting actomyosin promotes/suppresses self-renewal in vitro and tumorigenic potential in vivo. Weakening/strengthening actin cytoskeleton impairs/reinforces the interaction between adenomatous polyposis coli (APC) and ß-catenin, which facilitates ß-catenin nuclear/cytoplasmic localization. Nuclear ß-catenin binds to the promoter of Oct4, which enhances its transcription that is crucial in sustaining self-renewal and malignancy. These results demonstrate that the mechanics of tumor cells dictate self-renewal through cytoskeleton-APC-Wnt/ß-catenin-Oct4 signaling, which are correlated with tumor differentiation and patient survival. This study unveils an uncovered regulatory role of cell mechanics in self-renewal and malignancy, and identifies tumor cell mechanics as a hallmark not only for cancer diagnosis but also for mechanotargeting.

Fluid shear stress enhances natural killer cell's cytotoxicity toward circulating tumor cells through NKG2D-mediated mechanosensing.

Tumor cells metastasize to distant organs mainly via hematogenous dissemination, in which circulating tumor cells (CTCs) are relatively vulnerable, and eliminating these cells has great potential to prevent metastasis. In vasculature, natural killer (NK) cells are the major effector lymphocytes for efficient killing of CTCs under fluid shear stress (FSS), which is an important mechanical cue in tumor metastasis. However, the influence of FSS on the cytotoxicity of NK cells against CTCs remains elusive. We report that the death rate of CTCs under both NK cells and FSS is much higher than the combined death induced by either NK cells or FSS, suggesting that FSS may enhance NK cell's cytotoxicity. This death increment is elicited by shear-induced NK activation and granzyme B entry into target cells rather than the death ligand TRAIL or secreted cytokines TNF-α and IFN-γ. When NK cells form conjugates with CTCs or adhere to MICA-coated substrates, NK cell activating receptor NKG2D can directly sense FSS to induce NK activation and degranulation. These findings reveal the promotive effect of FSS on NK cell's cytotoxicity toward CTCs, thus providing new insight into immune surveillance of CTCs within circulation.

Mechanical manipulation of cancer cell tumorigenicity via heat shock protein signaling.

Biophysical cues of rigid tumor matrix play a critical role in cancer cell malignancy. We report that stiffly confined cancer cells exhibit robust growth of spheroids in the stiff hydrogel that exerts substantial confining stress on the cells. The stressed condition activated Hsp (heat shock protein)-signal transducer and activator of transcription 3 signaling via the transient receptor potential vanilloid 4-phosphatidylinositol 3-kinase/Akt axis, thereby up-regulating the expression of the stemness-related markers in cancer cells, whereas these signaling activities were suppressed in cancer cells cultured in softer hydrogels or stiff hydrogels with stress relief or Hsp70 knockdown/inhibition. This mechanopriming based on three-dimensional culture enhanced cancer cell tumorigenicity and metastasis in animal models upon transplantation, and pharmaceutically inhibiting Hsp70 improved the anticancer efficacy of chemotherapy. Mechanistically, our study reveals the crucial role of Hsp70 in regulating cancer cell malignancy under mechanically stressed conditions and its impacts on cancer prognosis-related molecular pathways for cancer treatments.

Adhesion to the Brain Endothelium Selects Breast Cancer Cells with Brain Metastasis Potential.

Tumor cells metastasize from a primary lesion to distant organs mainly through hematogenous dissemination, in which tumor cell re-adhesion to the endothelium is essential before extravasating into the target site. We thus hypothesize that tumor cells with the ability to adhere to the endothelium of a specific organ exhibit enhanced metastatic tropism to this target organ. This study tested this hypothesis and developed an in vitro model to mimic the adhesion between tumor cells and brain endothelium under fluid shear stress, which selected a subpopulation of tumor cells with enhanced adhesion strength. The selected cells up-regulated the genes related to brain metastasis and exhibited an enhanced ability to transmigrate through the blood-brain barrier. In the soft microenvironments that mimicked brain tissue, these cells had elevated adhesion and survival ability. Further, tumor cells selected by brain endothelium adhesion expressed higher levels of MUC1, VCAM1, and VLA-4, which were relevant to breast cancer brain metastasis. In summary, this study provides the first piece of evidence to support that the adhesion of circulating tumor cells to the brain endothelium selects the cells with enhanced brain metastasis potential.

Unraveling the mechanobiology of cornea: From bench side to the clinic.

The cornea is a transparent, dome-shaped structure on the front part of the eye that serves as a major optic element and a protector from the external environment. Recent evidence shows aberrant alterations of the corneal mechano-environment in development and progression of various corneal diseases. It is, thus, critical to understand how corneal cells sense and respond to mechanical signals in physiological and pathological conditions. In this review, we summarize the corneal mechano-environment and discuss the impact of these mechanical cues on cellular functions from the bench side (in a laboratory research setting). From a clinical perspective, we comprehensively review the mechanical changes of corneal tissue in several cornea-related diseases, including keratoconus, myopia, and keratectasia, following refractive surgery. The findings from the bench side and clinic underscore the involvement of mechanical cues in corneal disorders, which may open a new avenue for development of novel therapeutic strategies by targeting corneal mechanics.

Biomechanics of cancer stem cells.

Cancer stem cells (CSCs) have been believed to be one driving force for tumor progression and drug resistance. Despite the significance of biochemical signaling in malignancy, highly malignant tumor cells or CSCs exhibit lower cellular stiffness than weakly malignant cells or non-CSCs, which are softer than their healthy counterparts, suggesting the inverse correlation between cell stiffness and malignancy. Recent years have witnessed the rapid accumulation of evidence illustrating the reciprocity between cell cytoskeleton/mechanics and CSC functions and the potential of cellular stiffness for specific targeting of CSCs. However, a systematic understanding of tumor cell mechanics and their role in CSCs and tumor progression is still lacking. The present review summarizes the recent progress in the alterations of tumor cell cytoskeleton and stiffness at different stages of tumor progression and recapitulates the relationship between cellular stiffness and CSC functions. The altered cell mechanics may mediate the mechanoadaptive responses that possibly empower CSCs to survive and thrive during metastasis. Furthermore, we highlight the possible impact of tumor cell mechanics on CSC malignancy, which may potentiate low cell stiffness as a mechanical marker for CSC targeting.

Tumorigenic cell selection and substrate rigidity manipulation using 2D and 3D extracellular matrices.

Soft fibrin gels are used to select tumorigenic cells and regulate the stemness and metastasis of colorectal cancer cells via mechanotransduction. This protocol details steps to produce two-dimensional (2D) and three-dimensional (3D) extracellular matrices for substrate rigidity manipulation and tumorigenic cell selection. We also describe how it can be applied to tumor mechanotransductive research by colony growth monitoring and cell isolation. For complete details on the use and execution of this protocol, please refer to Chang et al. (2022).

Fluid shear stress regulates the survival of circulating tumor cells via nuclear expansion.

Distant metastasis mainly occurs through hematogenous dissemination, where suspended circulating tumor cells (CTCs) experience a considerable level of fluid shear stress. We recently reported that shear flow induced substantial apoptosis of CTCs, although a small subpopulation could still persist. However, how suspended tumor cells survive in shear flow remains poorly understood. This study finds that fluid shear stress eliminates the majority of suspended CTCs and increases nuclear size, whereas it has no effect on the viability of adherent tumor cells and decreases their nuclear size. Shear flow promotes histone acetylation in suspended tumor cells, the inhibition of which using one drug suppresses shear-induced nuclear expansion, suggesting that shear stress might increase nuclear size through histone acetylation. Suppressing histone acetylation-mediated nuclear expansion enhances shear-induced apoptosis of CTCs. These findings suggest that suspended tumor cells respond to shear stress through histone acetylation-mediated nuclear expansion, which protects CTCs from shear-induced destruction. Our study elucidates a unique mechanism underlying the mechanotransduction of suspended CTCs to shear flow, which might hold therapeutic promise for CTC eradication.

Wavelength-regulated switchable photoelectrochemical system for concurrent detection of dual antibiotics.

Concurrent detection of antibiotics with high sensitivity and reliability is always of high importance for food safety and environmental monitoring. Herein, a wavelength-dependent photoelectrochemical (PEC) aptasensor based on TiO2-Ag/nitrogen doped graphene composites (TiO2-Ag/NDG) was developed for concurrent detection of two antibiotics with signal amplification function. The as-fabricated ternary nanocomposites could concurrently detect two antibiotics using two different aptamer molecules as recognition elements, amplify the photocurrent outputs and perform a photocurrent-switchable operation, where photocurrents could be switched between anodic direction and cathodic direction via simply regulating the irradiation wavelength. Such proposed wavelength-dependent PEC sensing strategy was capable of concurrently detecting chloramphenicol (CAP) with a wide linear detection range from 50 pM-10 nM under irradiation of 380 nm and tetracycline (TET) with a linear detection range from 100 pM to 100 nM under irradiation of 600 nm, respectively. The limits of detection (LOD) for CAP and TET were 16.7 pM and 30 pM, respectively. Moreover, this PEC sensor also displayed good specificity and outstanding reliability in real sample applications. This wavelength-dependent PEC strategy could be conveniently adapted to other applications in food safety biosensing and environmental monitoring.

Two-stage degradation and novel functional endothelium characteristics of a 3-D printed bioresorbable scaffold.

Bioresorbable scaffolds have emerged as a new generation of vascular implants for the treatment of atherosclerosis, and designed to provide a temporary scaffold that is subsequently absorbed by blood vessels over time. Presently, there is insufficient data on the biological and mechanical responses of blood vessels accompanied by bioresorbable scaffolds (BRS) degradation. Therefore, it is necessary to investigate the inflexion point of degradation, the response of blood vessels, and the pathophysiological process of vascular, as results of such studies will be of great value for the design of next generation of BRS. In this study, abdominal aortas of SD rats were received 3-D printed poly-l-actide vascular scaffolds (PLS) for various durations up to 12 months. The response of PLS implanted aorta went through two distinct processes: (1) the neointima with desirable barrier function was obtained in 1 month, accompanied with slow degradation, inflammation, and intimal hyperplasia; (2) significant degradation occurred from 6 months, accompanied with decreasing inflammation and intimal hyperplasia, while the extracellular matrix recovered to normal vessels which indicate the positive remodeling. These in vivo results indicate that 6 months is a key turning point. This "two-stage degradation and vascular characteristics" is proposed to elucidate the long-term effects of PLS on vascular repair and demonstrated the potential of PLS in promoting endothelium function and positive remodeling, which highlights the benefits of PLS and shed some light in the future researches, such as drug combination coatings design.

Hybrid theranostic microbubbles for ultrasound/photoacoustic imaging guided starvation/low-temperature photothermal/hypoxia-activated synergistic cancer therapy.

Constructing a theranostic agent for high-contrast multimodality imaging-guided synergistic therapy with long-term tumor retention and minimum systemic side effects still remains a major challenge. Herein, a hybrid microbubble-based theranostic platform was developed for dual-modality ultrasound (US) and enhanced photoacoustic (PA) imaging-guided synergistic tumor therapy by combining starvation therapy, low-temperature photothermal therapy (PTT), and hypoxia-activated therapy, based on polydopamine (PDA) doped poly(vinyl alcohol) microbubbles loaded with glucose oxidase (GOx) (PDA-PVAMBs@GOx) and hypoxia-activated prodrug (HAP) tirapazamine (TPZ). For dual-modality US/enhanced PA imaging, PDA-PVAMBs provided 6.5-fold amplified PA signals relative to freely dispersed PDA nanoparticles (PDA NPs). For synergistic cancer therapy, oxygen (O2) carried by PDA-PVAMBs@GOx was first released to promote starvation therapy by loaded GOx. Then, moderate near-infrared (NIR) laser irradiation triggered PTT and improved enzymatic activity of GOx with its optimal activity around 47 °C. Subsequently, GOx-mediated tumor starvation depleted O2 and exacerbated the hypoxia environment, thereby activating the toxicity of TPZ in the tumor site. Through dual-modality US/PA imaging monitoring, PDA-PVAMBs@GOx with long-term retention (∼7 days) combined with PTT and TPZ significantly inhibited the growth of solid tumors with minimum systemic side effects, which might be a powerful tool for effective tumor treatment.

Nanoparticle-mediated specific elimination of soft cancer stem cells by targeting low cell stiffness.

As the driving force of tumor progression, cancer stem cells (CSCs) hold much lower cellular stiffness than bulk tumor cells across many cancer types. However, it remains unclear whether low cell stiffness can be harnessed in nanoparticle-based therapeutics for CSC targeting. We report that breast CSCs exhibit much lower stiffness but considerably higher uptake of nitrogen-doped graphene quantum dots (N-GQDs) than bulk tumor cells. Softening/stiffening cells enhances/suppresses nanoparticle uptake through activating/inhibiting clathrin- and caveolae-mediated endocytosis, suggesting that low cell stiffness mediates the elevated uptake in soft CSCs that may lead to the specific elimination. Further, soft CSCs enhance drug release, cellular retention, and nuclear accumulation of drug-loaded N-GQDs by reducing intracellular pH and exocytosis. Remarkably, drug-loaded N-GQDs specifically eliminate soft CSCs both in vitro and in vivo, inhibit tumor but not animal growth, and reduce the tumorigenicity of xenograft cells. Our findings unveil a new mechanism by which low cellular stiffness can be harnessed in nanoparticle-based strategies for specific CSC elimination, opening a new paradigm of cancer mechanomedicine. STATEMENT OF SIGNIFICANCE: Low cell stiffness is associated with high malignancy of tumor cells and thus serves as a mechanical hallmark of CSCs. However, it remains unclear whether cellular stiffness can be exploited for specific targeting of soft CSCs. This work reports that soft CSCs exhibit high N-GQD uptake compared to stiff tumor cells, which is regulated by cellular stiffness. Further, soft CSCs have enhanced drug release, cellular retention, and nuclear accumulation of drug-loaded N-GQDs, which enable the specific elimination of malignant CSCs both in vitro and in vivo with minimal side effect. In summary, our study demonstrates that CSC's low stiffness can be harnessed as a mechanical target for specific eradication, which provides a new paradigm of cancer mechanomedicine.

Effects of Mechanical Compression on Cell Morphology and Function in Human Corneal Fibroblasts.

Purpose: To explore the effect of mechanical compression (similar to that induced by eye rubbing) on cell morphology, proliferation, apoptosis, and extracellular matrix synthesis and degradation in human corneal fibroblasts.Materials and methods: Human corneal fibroblasts were isolated from corneal lenticule tissue of 10 patients after small incision lenticule extraction surgery. A system was established to mechanically compress corneal fibroblasts with pressure ranging from 0 to 524 Pa. Morphological changes, cell proliferation, apoptosis, and corneal matrix synthesis and degradation were examined using microscopy imaging, bromodeoxyuridine staining, flow cytometry, and qPCR analysis in human corneal fibroblasts after mechanical compression.Results: Human corneal fibroblasts showed short and thick cytoplasmic extensions, as well as a relatively low aspect ratio, suggesting significant morphological alterations caused by high levels of compressive stress. Mechanical compression inhibited cell proliferation and promoted cell apoptosis. Furthermore, compressive stress led to significant elevation in the expression of genes related to extracellular matrix degradation (matrix metalloproteinases MMP1 and MMP9) as early as 6 h after compression and moderate changes in the expression of tissue inhibitors of metalloproteinases. In addition, the mRNA expression levels of COL1A1, lumican, and vimentin were reduced 24 and 48 h after mechanical compression.Conclusions: Mechanical compression alters cell morphology, inhibits proliferation, induces apoptosis, upregulates genes related to extracellular matrix degradation, and downregulates corneal structural genes in human corneal fibroblasts. This study provides evidence that compressive stress significantly influences corneal keratocytes. Our findings suggest that this mechanical effect may be related to keratoconus associated with chronic eye rubbing.

Biophysical properties of corneal cells reflect high myopia progression.

Myopia is a common ocular disorder with significant alterations in the anterior ocular structure, including the cornea. The cell biophysical phenotype has been proposed to reflect the state of various diseases. However, the biophysical properties of corneal cells have not been characterized during myopia progression and their relationship with myopia remains unknown. This study characterizes the biophysical properties of corneal cells in normal, myopic, and recovered conditions, using two classical myopia models. Surprisingly, myopic corneal cells considerably reduce F-actin and microtubule content and cellular stiffness and generate elevated traction force compared with control cells. When myopia is restored to the healthy state, these biophysical properties are partially or fully restored to the levels of control cells. Furthermore, the level of chromatin condensation is significantly increased in the nucleus of myopic corneal cells and reduced to a level similar to healthy cells after recovery. These findings demonstrate that the reversible biophysical alterations of corneal cells reflect myopia progression, facilitating the study of the role of corneal cell biophysics in myopia.