Regulation Of Suspension Mechanical State On The Metastasis Of Breast Tumor Cells And Its Mechanobiological Mechanism

Organ failure caused by tumor metastasis is the leading reason for mortality of cancer patients.Tumor metastasis is a complex process involving in multiple steps,multiple stages and multiple genes.In addition to biochemical factors,the mechanical property of tumor cells and the mechanical microenvironment(such as matrix stiffness,shear force,etc.)are also important aspects of tumor metastasis.Suspension mechanical state is a subsistent biomechanical context during tumor cell detachment from extracellular matrix in the process of metastasis.Studies have shown that suspension state was involved in re-attachment and anoikis resistance of breast tumor cells by regulating cytoskeleton structure and gene expression.However,the current research about the effect of suspension mechanical state on tumor metastasis is not comprehensive enough,and the mechanobiological mechanism is not clear.Therefore,this study intends to systematically explore the effect of suspension state on tumor metastasis and its mechanobiological mechanisms from the perspective of biomechanics.The main research contents and results are as follows:(1)The effect of suspension state on reattachment of breast cancer cells and its mechanobiological mechanismMDA-MB-231 and SK-BR-3 breast cancer cells were cultured under suspension and adherent condition to investigate the effect of suspension state on adhesion and reattachment of tumor cells and its mechanobiological mechanism.It's shown that suspension state changed the actin cytoskeleton polymerization and cell contraction.Suspension state could rapidly and reversibly improve lamin A/C protein expression at post-transcriptional level,which contributed to cells adhesion and spreading,promoted the formation of stress fibers and focal adhesion,reduced cell motility and improved cells stiffness and nuclear roundness in reattached tumor cells.The research on reattachment of suspension tumor cells from malignant pleural effusion in vitro proved that suspension state of tumor cells in vivo might also promote reattachment.Suspension-induced lamin A/C accumulation was associated with disruption of actin cytoskeleton.In addition,suspension state enhanced integrin ?1 expression to contribute to adhesion.(2)Effects of suspension state on survival,migration,invasion and metastasis of breast cancer cellsIn order to explore the effect of suspension state on the ability of metastasis,the cells cycles,apoptosis,migration and invasion of MDA-MB-231 and SK-BR-3 breast tumor cells in suspension state were investigated at first.The proliferation,clonal formation,and survival under hunger stress conditions of reattached breast tumor cells were also detected.Then,the effects of suspension state on the tumorigenic ability and metastasis ability of tumor cells were investigated.Our results showed that suspension state leaded cycles arrest and a certain degree of apoptosis of breast cancer cells.Although suspension state could affect clonal formation ability of tumor cells and caused growth delay in re-adherent cells,suspension state increased survival of breast tumor cells under hunger stress conditions after reattaching,and improved the ability of migration and invasion.Animal experiments showed that suspension state increased tumor cell dormancy,inhibited tumor growth,but significantly promoted lung metastasis.(3)Regulation of suspension state on gene expression in breast cancer cellsIn order to investigate the changes of gene expression profile of tumor cells by suspension state and to explore the mechanism of suspension-promoted tumor metastasis,total RNA of MDA-MB-231 cells cultured in suspension or normal adherent condition were extracted for RNA sequencing by Illumina HiSeq 2000 sequencing platform.RT-PCR and western blotting were used to validate the expression of tumor metastasis-related genes.Compared with adherent culture cells,254 genes were up-regulated and 190 genes were down-regulated by suspension state(|log2 Fold change| > 2,FDR < 0.05).Differently expressed genes were mainly enriched in immune response,cell cycle,cell adhesion,extracellular matrix part,cell migration,apoptosis regulation and other biological processes.The expression of pro-metastasis genes PTGS2,FN,LOX1,ABCC3,ICAM-1,MUC1,SELPLG,TGFBR2 and TGF-?1 were up-regulated in a time-dependent manner by suspension state.The expression of these genes was decreased gradually to the level of adherent culture cells after reattaching.(4)The mechanobiological mechanism of suspension-induced cyclooxygenase-2(COX-2)up-regulation and its role in metastasis of breast cancer cellsIn order to investigate the mechanism of COX-2 up-regulation,effects of Ca2+/calcineurin/nuclear factor of activated T cells(NFAT)/COX-2 signaling pathway on biological behaviors of breast tumor cells were detected.Breast cancer cells were cultured in suspension and adherent condition with cytoskeletal inhibitors and cultured on polyacrylamide gel with different stiffness to examine the effect of cytoskeletal state on the expression of COX-2.Then,at the animal level,the effect of COX-2 knockdown on the lung metastatic capacity of suspension tumor cells was examined.Cell level studies showed that suspension state increased the intracellular Ca2+ level and enhanced nuclear transport of NFAT2 to promote the expression of COX-2,which promoted the survival,migration and invasion of tumor cells.Both low matrix stiffness and cytochalasin D treatment were beneficial to COX-2 expression,but the inhibitor of cell contraction Y27632 had no effect on COX-2 expression.Animal experiments showed that suspension-induced COX-2 promoted tumor metastasis by increasing the retention and survival of tumor cells in lungs.This paper systematically studied the regulation of suspension state on metastasis of breast cancer cells.It was proved that suspension state could reversibly regulate the expression of related genes both at transcriptional level and post-transcriptional level.Suspension state promoted metastasis of breast cancer cells by enhancing the re-adhesion,migration,invasion and survival of breast cancer cells.This study initially confirmed the mechanobiological role of suspension-induced cytoskeleton changes.The results of this study were helpful to understand the importance of suspension mechanical state in the process of breast cancer metastasis and provied theoretical support for anti-metastasis therapy strategies targeting lamin A/C and COX-2.