Compressive Force Induced Remolding Of Chromatin Structure In 3D Cultured Tumor Cells

Cancer has been characterized as a genetic disease,associated with mutations that cause pathological alterations of the cell cycle,prolifer,metabolism,or DNA repair.The malignant progression of tumors is not only on the biological characteristics of tumor cells but also on the tumor microenvironment.Recently,the importance of the anomalous mechanical properties of tumor tissues,which activate tumorigenic biochemical pathways,has become apparent.Solid tumors are crowded with malignant cells due to uncontrolled growth and proliferation,which would mechanically interact with peripheral tissues and cause the emergence of solid stress.Subsquently,the tumor cells are contstantly subjected to intrinsic and extrinsic force that can lead to small and large cellar morphological changes.Meanwhile,tumor cells generate compression force for resisting mechanical force.Many reseachers found that compression force plays an important role in the development of carcinogenesis.Compression stress of cancer cells can hinder their proliferation rate,induce apoptosis and increase their invasion and metastatic potential.However,mechanotransdution often refers to long-term phenotypic changes in the cells,commonly arising from mechanically induced changes in gene expression.Cells can sense mechanical stimulation and changes in their physical environment through force induced conformational changes on the molecular level,but many of the molecular mechanisms are still incompletely understood.Hence,the aim of the thesis was to investigate the biomechanical mechanisms of compression force induced remodeling of structure chromatin and regulated gene expression in tumor cells through combining biomechanics,materials science,and bioinformatics.Herein,we provide a new perspective to descripte the physical changes that promote tumor progression and aggression,which will be used in therapeutic strategies to alleviate the mechanical stresses driving cancer to malignancy.Firstly,to mimic the tumor microenvironment in vitro,we constructed a 3D in vitro model using collagen I hydrogel.We also selected the suitable compression force loading on 3D cell culture model.The tumor cells survived well and responed to compression force immediately in the 3D cell culture system.Meanwhile,compression force also induced the nuclear deformation,including a decreased in nuclear height,volumes,nuclear envelop folding and an increased in nuclear projective area.All results showed that the compression force rebuild the mechanical homeostasis of the nucleus.Moreover,we observed the process of nuclear deformation and diminutive and confine changes of chromatin’s location after loading compression force by time-lapse photography of live cells with fluorescent reporters of chromatin and nuclear membranes.To clarify the reason of regulation gene expression by compression force,we detected chromatin histone modifications and used DNA hairpin tension sensors for mechanical force.We analyzed the expression of H3K27me3 and H3K9 ac in tumor cells by immunofluorescence in the control and load group.We observed the dynamic expression changes of H3K27me3 and HDAC4 after loading compression which tracked by live cell time-lapse photography of real-time fluorescent reporters;We detected the changes of chromatin spatial structure after loading compression stress using DNA hairpin tension sensors.All results were suggested that short duration of compression stress induced nuclear shape causing conformational changes in chroatin structure and organization and directly affecting transcriptional regulation with independent manipulation of histone modifications.As a part of nuclear mechanosensory,chromatin directly was changed by compression stress.In summary,in this thesis,we successfully constructed a 3D tumor cell culture model with compressive stress loading system,which simulate the solid stress microenvironment in solid tumor.We found that compression stress can rapidly transfer to the nucleus,disrupte the nuclear mechanical homeostasis and remodel the chromatin spatial structure;Meanwhile,as a mechanical element in the nucleus,chromatin can be directly stretched or compressed by mechanical force,which changed the threedimensional structure,promoting the chromatin accessibility,regulating gene expression and participating in the regulation of tumor malignant process.