Shear Stress Regulates Tumor Cell Mechanics and Actomyosin-Dependent Survival and Chemoresistance

Metastasis is the main cause of cancer death,and tumor cells mainly disseminate to the distal organs through the blood circulation,in which they experience considerable levels of fluid shear stress.CTCs are heterogeneous with diverse subpopulations of distinct genotypes and phenotypes and the frequency of CTCs is correlated with poor prognosis and overall survival in cancer patients.Less than 0.01%of them may eventually generate metastatic tumors in secondary sites,indicating the inefficiency of metastasis.Nevertheless,metastasis accounts for over 90%of cancer-related deaths,suggesting that a subpopulation of CTCs are able to survive the metastatic process and form metastases.To target metastasis,it is thus essential to understand the roles of various factors during dissemination in the survival and functions of CTCs.However,the effects of hemodynamic shear stress on biophysical properties and functions of CTCs in suspension are not fully understood.This study was to investigate the effect of hemodynamic shear stress on the survival and anti-chemotherapy ability of suspended circulating tumor cells during metastasis,and the effect of actomyosin activity on this regulation.In this study,we developed a circulatory system to generate physiologic levels of hemodynamic shear stress,which mimicked certain important aspects of the CTC microenvironment in blood circulation.The survival of tumor cells in suspension,as a model for real CTCs,under different shear stress and circulation duration was examined.We found that the majority of breast tumor cells s in suspension can be eliminated by hemodynamic shear stress.The surviving cells exhibit unique biophysical properties,including significantly retarded cell adhesion,mesenchymal-like cell morphology,and reduced F-actin expression and cellular stiffness.Cancer stem cells which has been reported in other papers have lower stiffness compared with conventional tumor cells showed significantly higher survival in blood flow.Importantly,low actomyosin activity promotes the survival of CTCs in blood shear flow while high actomyosin activity inhibits tumor cells surviving shear stress treatment.These findings might be explained by the up-and down-regulation of the anti-apoptosis genes.Soft surviving tumor cells held survival advantages in shear flow and higher resistance to chemotherapy.Metastasis is closely linked with chemoresistance.However,the underlying mechanisms have not been fully understood,in particular,the roles of hemodynamic shear stress and actomyosin-dependent cell mechanics in drug resistance of CTCs remain unclear.Inhibiting actomyosin activity in suspended tumor cells enhanced chemoresistance,while activating actomyosin suppressed this ability.These findings might be associated with the corresponding changes in multidrug resistance related genes.Our study unveils the regulatory roles of actomyosin in the survival and drug resistance of circulating tumor cells in hemodynamic shear flow,which imply the importance of fluid shear stress and actomyosin activity in tumor metastasis.Our findings reveal a new mechanism by which circulating tumor cells are able to survive hemodynamic shear stress and chemotherapy and may offer a new potential strategy to target circulating tumor cells in shear flow and combat chemoresistance through actomyosin.

Hemodynamic Shear Stress Regulates the Survival of Circulating Tumour Cells Through Piezo1-Actomyosin-Mediated YAP/TAZ Nuclear Translocation

It is critical to combat tumor metastasis by eradicating disseminated tumor cells in any step during the metastasis process.After entering the blood circulation system,tumor cells are in suspension and experience considerable levels of fluid shear stress.However,the influence of hemodynamic shear stress on the survival of CTCs and the underlying mechanotransduction mechanisms remain unclear.This study shows that fluid shear stress can eliminate the majority of CTCs and the viability of suspended tumor cells depends on the stress magnitude,indicating that tumor cells can sense and respond to fluid shear stress.Mechanistically,the expression of Piezo1 but not Piezo2 is greatly upregulated in suspended tumor cells after shear stress treatment.Inhibiting/activating Piezo1 increases/decreases the viability of suspended tumor cells in shear flow,which depends on Piezo1-mediated calcium entry.These findings suggest that Piezo1 may be the major mechanosensor by which suspended tumor cells sense fluid shear stress.As the downstream effector of Piezo1,actomyosin in tumor cells is significantly activated under increasing shear stress.Its activity influences the survival of CTCs in shear flow and rescues the effects of Piezo1 on tumor cell survival,suggesting that hemodynamic shear stress regulates the survival of CTCs through Piezo1 mediated actomyosin activity.Importantly,fluid shear stress considerably up-regulates YAP/TAZ activity of suspended tumor cells and promotes their nuclear translocation in a magnitude-dependent manner.Inhibiting YAP/TAZ enhances the viability of suspended tumor cells in shear stress,while activating their activity decreases tumor cell survival,suggesting that YAP/TAZ activation promotes the apoptosis of suspended tumor cells,which is different from the findings that YAP/TAZ facilitates the survival of adherent cells to shear flow.Further,blocking the nuclear import of YAP/TAZ inactivates the sensation of suspended tumor cells to fluid shear flow and attenuates the dependence of tumor cell survival on different magnitudes of hemodynamic shear stress.The influence of Piezo1-actomyosin pathway on suspended tumor cells can be rescued by YAP/TAZ activity,suggesting that Piezo1-mediated signaling induces tumor cell apoptosis via nuclear translocation of YAP/TAZ.In addition,fluid shear stress can also activate the expressions of LATS1/2 and MST1/2 in Hippo pathway through Piezo1,which is known to inhibit YAP/TAZ activity.Silencing/activating LATS1/2 or MST1/2 inhibits/enhances the viability of CTCs under shear stress,the effects of which can be further rescued by YAP/TAZ.These findings suggest that the responses of suspended tumor cells to hemodynamic shear stress are partially mediated by Hippo signaling.After nuclear translocation,YAP/TAZ directly bind p73/PUMA,which further promotes the transcription of pro-apoptotic genes and induces the apoptosis of suspended tumor cells.In summary,these findings show that hemodynamic shear stress considerably influences the survival of CTCs in blood circulation.We have identified the calcium channel Piezo1 as a novel mechanosensor for the response of CTCs to fluid shear stress.Hemodynamic shear stress induces the apoptosis of suspended tumor cells through Piezo1-actomyosin-YAP/TAZ-p73/PUMA signaling,which is different from the mechanotranduction mechanisms for tumor cells in adherent.Therefore,this study has unveiled the novel mechanosensor of suspended CTCs in response to fluid shear stress and the subsequent mechanisms and identified Piezo1 and YAP/TAZ as the potential therapeutic targets,through which CTCs may be effectively eradicated in the vasculature to prohibit tumor metastasis.

Self-renewal memory of tumor-repopulating cells

Tumor-initiating cells(TICs)are a highly tumorigenic subpopulation of solid tumor cells that play a critical role in the initiation of cancer~[1].These tumorigenic cells resist conventional chemotherapeutic drug treatment and are assumed to be playing major roles in cancer relapses after chemotherapy~[2].However,the notion of TICs has been rather controversial.A report shows that a high percentage(】25%)of human melanoma cells can generate a tumor in a NOD-SCID interleukin-2 receptor gamma chain null mouse~[3],suggesting that there is no clonal development of solid tumors,refuting the idea of TICs.We recently developed a method of isolating TICs from cancer cell lines by culturing single individual cells of B16-F1(a melanoma cell line)into 3D soft fibrin gels~[4].In addition to being able to generate local tumors in a

Mechanics and mechanotransduction of tumorigenic cells

Despite significant progress in cancer research during the past decades,yet there are no major breakthroughs that can be translated into major benefits for the general public in terms of treatment or therapy for the complex neoplastic diseases,especially for the malignant solid tumors.This depressing but indisputable fact leads to a call for new ideas to target tumor metastasis by editors of Nature Medicine[1].The real problems are that the fundamental issues of transformation and malignancy in vivo are poorly understood.In a recent review on cancer,

Cellular tension promotes germ layer organization

Understanding the mechanism of gastrulation-the early phase in embryonic development where the blastula loses its symmetry and forms organized germ layers（i.e.endoderm,mesoderm,and ectoderm）-has long been a major challenge to the field of developmental biology.A long standing objective in developmental biology is not only to direct the differentiation of ESCs into specific developmental lineages,but also to organize these differentiated lineages into spatially distinct arrangements resembling the physiological gastrulation.In vivo,research on embryo morphogenesis in lower animals has demonstrated the importance of mechanical forces[1-3].In vitro,experiments of self-sorting utilize pairwise sorting assays where two types of differentiated germ cells are homogeneously mixed[4].It has not been possible to study the or-

Cell Mechanics Regulates Cellular Uptake of Graphene Quantum Dot for Specifically Targeting Cancer Stem Cells

Cancer stem cells(CSCs)are the driving force for sustainable tumor growth and metastasis and responsible for drug resistance and cancer relapse.Nanoparticle-based drug delivery has been demonstrated to be effective in combating tumor growth.However,it has been challenging to selectively eliminate CSCs due to the lack of a general signature for a spectrum of cancers.It is known that CSCs from various types of cancer show lower stiffness compared to non-CSCs.It remains unclear whether low stiffness in CSCs influences cellular uptake in nanoparticle-based drug delivery and thus the chemotherapy efficacy.Graphene quantum dot(GQD)is emerging as a promising carrier material in delivering anti-cancer drugs.We found that breast CSCs were softer than conventional cancer cells,which were further softer compared to healthy breast tissue cells.Importantly,soft CSCs uptook more GQD than conventional cancer cells,while stiff breast cancer cells with relatively low stiffness uptook more GQD than healthy breast cells.Softening cells by pharmacologically inhibiting actomyosin activity using either siRNA or actomyosin inhibitors significantly enhanced the cellular uptake of GQD in breast cancer cells but not CSCs,while stiffening cells by activating actomyosin using CA-MLCK/ROCK or actomyosin activators considerably suppressed the nanoparticle uptake in both cancer cells and CSCs.GQD could specifically target CSC because of low cell stiffness of CSC in breast cancer cell line MCF-7 and MDA-MB-231.Further regulating cell stiffness reflected that decreasing breast cancer cell stiffness by inhibiting actomyosin activity using blebbistatin could promote GQD uptake.Vice versa,stiffening cancer cell by activating actomyosin decreased GQD uptake.The attachment of anti-cancer drug doxorubicin did not alter the trend of GQD uptake in neither soften nor stiffen cancer cells.Actomyosin activity regulates cellular uptake ofGQD might through clathrin and caveolin-mediated endocytosis.Cancer cells are softer than normal cells from the same organ,CSC are softer than non-CSC.Thus we further confirmed that the GQD uptake of normal breast cell line MCF-10 is less than breast cancer cell line MCF-7 and MDA-MB-231.Suggesting the clinical significance that using GQD as drug carrier targeting softer cells could reduce side effects to normal tissue cells.Since CSC are softer than non-CSC,GQD could decreased the percentage of CSC in whole cancer cell population by targeting softer cells.These results suggesting that it would be possible to target cancer cells and CSC by targeting from a perspective of cell mechanical difference.High uptake of nanoparticles in soft cancer cells could not be explained by their differential membrane potentials.Mechanistically,low cell mechanics or inhibiting actomyosin activity activated both clathrin and caveolin-mediated endocytosis signaling pathways,while high cell mechanics or activating actomyosin suppressed these signalings.Pharmacologically inhibiting clathrin or caveolin-mediated endocytosis signaling significantly decreased GQD uptake in CSCs and in conventional breast cancer cells when actomyosin was suppressed.Further,GQD conjugated with doxorubicin could be specifically delivered into CSCs with low stiffness and eliminated more CSCs in the presence of both CSCs and non-CSCs.Taken together,these data reveal the regulatory role of cell mechanics in cellular uptake of nanoparticles and demonstrate that GQD can be utilized to specifically eliminate CSCs,which have important implications in nanoparticle-based drug delivery for cancer therapy.

The Mechanics of Tumor Cells Dictate Malignancy via Cytoskeleton-Mediated APC/Wnt/β-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)andβ-catenin,which facilitatesβ-catenin nuclear/cytoplasmic localization.Nuclearβ-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/β-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.

Inhibition of in-stent restenosis after graphene oxide double-layer drug coating with good biocompatibility

In this study,we designed a double layer-coated vascular stent of 316L stainless steel using an ultrasonic spray system to achieve both antiproliferation and antithrombosis.The coating included an inner layer of graphene oxide(GO)loaded with docetaxel(DTX)and an outer layer of carboxymethyl chitosan(CMC)loaded with heparin(Hep).The coated surface was uniform without aggregation and shedding phenomena before and after stent expanded.The coating treatment was able to inhibit the adhesion and activation of platelets and the proliferation and migration of smooth muscle cells,indicating the excellent biocompatibility and antiproliferation ability.The toxicity tests showed that the GO/DTX and CMC/Hep coating did not cause deformity and organ abnormalities in zebrafish under stereomicroscope.The stents with GO double-layer coating were safe and could effectively prevent thrombosis and in-stent restenosis after the implantation into rabbit carotid arteries for 4–12 weeks.

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.