Extracellular Matrix-Derived Mechanical Force Governs Breast Cancer Cell Stemness And Quiescence

Background:Extracellular matrix is an important structural component in tissues and organs.It is a complex network composed of extracellular proteins,proteoglycan and glycoproteins,etc.In the tumor microenvironment,extracellular matrix not only provides physical support and protection for the growth of tumor cells,but also provides covalent molecular cross-linking network for biochemical signaling factors,which plays an important role in the occurrence and development of tumors.Previous studies have shown that extracellular matrix molecules interact with cellular receptors to promote the activation of molecular signaling pathways related to proliferation,migration and invasion in tumor cells.In addition,dynamic remodeling of extracellular matrix,including matrix component dissolution,precipitation and crosslinking,plays an important role in local invasion and distant metastasis of tumor cells.In fact,extracellular matrix has not only biochemical characteristics,but also biophysical characteristics,including morphological structure,molecular density,hardness and physical tension,etc.Recent studies have gradually demonstrated that biomechanical force signals play an important role in the development of tumor.However,how extracellular matrix related biomechanical force signals are transmitted and affect the growth and proliferation of tumor cells remains unknown.The objective of this study was to explore the molecular mechanism of dynamic regulation of extracellular matrix related biomechanical forces on tumor stemness and dormancy state of breast cancer cells.Methods:(1)In this study,three kinds of 3D soft gel culture systems(collagen,fibrinogen,Matrigel)were established for 3D culture of breast cancer cell lines(MCF-7,4T1,MDA-MB-231),to explore the effects of biomechanical forces on the biological behavior of breast cancer cells.(2)The effect of biomechanical forces on the tumorigenic potential of breast cancer cells was studied by in vitro colony formation and in vivo subcutaneous tumor formation in mice.(3)In this study,the expression of stem-related genes and the expression of ALDH1,a marker of breast cancer stem cells,were detected by fluorescence quantitative polymerase chain reaction and flow cytometry.(4)In this study,western blotting,immunohistochemistry and immunofluorescence were used to detect the activation of integrin/DDRs signaling pathways in breast cancer cells.(5)In this study,tumor tissue samples from 86 clinical breast cancer patients and prognosis information of 1358 breast cancer patients from public database were collected,and the correlation between extracellular matrix signature and prognosis of patients was analyzed.Results:(1)Breast cancer cells were cultured in three kinds of 3D soft gel.The in vitro colony formation,in vivo tumorigenic potential analysis,and stem-related gene expression were detected.We found that the extracellular matrix derived biomechanical force could promote the tumor stemness and tumorigenic potential of breast cancer cells.(2)We found that excessive biomechanical force can promote breast cancer cells to enter a quiescent state through cell cycle analysis and in vivo tumor growth evaluation;(3)We found that extracellular matrix derived biomechanical forces regulate breast cancer stemness through integrin receptors and cell quiescence through DDRs signaling,by measuring integrin and DDRs signaling molecules expression at mRNA and protein levels;(4)We established a novel extracellular matrix signature,which can effectively predict the prognosis and recurrence risk of breast cancer patients,by clinical samples evalualtaion and prognosis-realted regression analysis;Conclusion:Our findings revealed that ECM-derived mechanical force promoted tumor stemness and cell quiescence in breast cancer cells.A mechanical force of approximately 45 Pa derived from the extracellular substrate activated integrin β1/3 receptors,stimulating stem cell signaling pathways through the cytoskeleton/AIRE axis and promoting tumorigenic potential and stem-like phenotypes.However,excessive mechanical force(450 Pa)could drive stem-like cancer cells into a quiescent state,with the removal of mechanical forces leading to vigorous proliferation in quiescent cancer stem cells.Mechanical force facilitated cell cycle arrest to induce quiescence,dependent on DDR2/STAT1/P27 signaling.Therefore,ECM-derived mechanical force governs breast cancer cell status and proliferative characteristics through stiffness alterations.We further established an ECM signature based on the fibrinogen/fibronectin/vitronectin/elastin axis,which efficiently predicts patient prognosis in breast cancer.Our findings highlight the vital role of ECM-derived mechanical force in governing breast cancer cell stemness/quiescence transition and suggest the novel use of ECM signature in predicting the clinical prognosis of breast cancer.