The Role And Mechanisms Of Tumor Cells-derived Exosomal Twist1 In Breast Cancer-induced Depression

Objective: Twist1 is a basic transcription factor,a member of the basic helix-loop-helix protein family,and is involved in the occurrence and metastasis of a variety of cancers(including breast cancer).Studies have found that a wide variety of cancers induce depression in patients,which is called cancer-induced depression(CID).However,the mechanism of CID is still unclear.Exosomes are vesicles secreted by different types of cells and package various biologically active molecules,such as protein,RNA,DNA and lipids.They can act as intercellular communication materials transmitted to other target cells and are involved in occurrence and prognosis of systemic degenerative diseases and cancers.Previous work has proved that Twist1 is significantly increased in the medial prefrontal cortex(mPFC)of chronic stress induced depressive mice and increases susceptibility to stress.This study mainly explored whether breast cancer 4T1 cells could secrete exosomes Twist1 to promote the depressive-like behaviors in mice.Methods: A 4T1 cell line with stable and low expression of Twist1(referred to as stable expressing sh Twist1 4T1 cells)were obtained via lentivirus and puromycin.4T1 cells and stable expressing sh Twist1 4T1 cells were subcutaneously inoculated into mice to construct a tumorbearing mice model.Tail suspension test(TST),forced swimming test(FST)and sucrose preference test(SPT)were used to detect depressive-like behaviors in mice.Ultra-high-speed centrifugation were used to extract exosomes in cell medium or serum.Quantitative polymerase chain reaction(q PCR)and western blot(WB)were used to detect m RNA and protein expression levels of Twist1 in the cells,exosomes and mPFC.The role of Twist1 in depressive-like behavior in mice was studied by injecting lentivirus into the mPFC to knock down Twist1.The exosomes were administered to mice through the intranasal administration or intravenous injection,or mice were treated with exosomal inhibitors to study the role of exosomal Twist1 in depressive-like behaviors in mice.Confocal microscope was used to study the distribution of exosomes in the brain regions and the morphological changes of pyramidal neurons in the mPFC.Results:(1)Mice inoculated with 4T1 cell suspension showed significant depressive-like behaviors,including increased immobility time in TST and FST and decreased sucrose preference in SPT.(2)WB and transmission electron microscope(TEM)were used to prove the purity of exosomes.(3)The mice showed significant depressive-like behavior after absorbing the exosomes extracting from the 4T1 cell culture supernatant through the intranasal administration or intravenous injection,including increased immobility time in TST and FST and decreased sucrose preference in SPT.(4)After the mice absorbed the exosomes labeled with PKH67 through the intranasal administration or intravenous injection,the exosomes were detected by the confocal microscope and showed extensive expression in the brain.(5)The m RNA and protein expression levels of Twist1 in mPFC of tumor-bearing mice were increased.(6)Knockdown of Twist1 via lentivirus in the mPFC reversed depressive-like behavior in tumor-bearing mice,including reduced immobility time in TST and FST and increased sucrose preference in SPT.(7)Stable expressing sh Twist1 4T1 cells were obtained via lentivirus and puromycin,the m RNA and protein expression levels of Twist1 in stable expressing sh Twist1 4T1 cells were decreased significantly.The exosomes from this cell line were extracted and the m RNA expression level of Twist1 in the exosomes was decreased significantly.(8)Tumor-bearing mice inoculated with stable expressing sh Twist1 4T1 cells reversed depressive-like behaviors,including decreased immobility time in TST and FST and increased sucrose preference in SPT.The m RNA and protein expression levels of Twist1 in the mPFC of tumor-bearing mice inoculated with stable expressing sh Twist1 4T1 cells were decreased significantly compared with tumor-bearing mice inoculated with 4T1 cells.(9)GW4869 inhibited the secretion and release of exosomes significantly,and reversed the depressive-like behavior of tumor-bearing mice,including decreased immobility time in TST and increased sucrose preference in SPT.(10)Mice were respectively inoculated with normal 4T1 cells or stable expressing sh Twist1 4T1 cells.The exosomes(serum-derived exosomes,SDE)in the serum of tumor-bearing mice were extracted,and were given to new normal mice via intranasal administration or intravenous injection.SDE derived from tumor-bearing mice inoculated with 4T1 cells induced significant depressive-like behaviors,including increased immobility time in TST and FST and decreased sucrose preference in SPT,while SDE derived from tumor-bearing mice inoculated with stable expressing sh Twist1 4T1 cells had no effects.SDE derived from tumor-bearing mice inoculated with 4T1 cells increased the m RNA and protein expression level of Twist1 in the mPFC of normal mice,while the SDE derived from tumorbearing mice inoculated with stable expressing sh Twist1 4T1 cells had no effect.(11)SDE were extracted from mice inoculated with normal 4T1 cells or stable expressing sh Twist1 4T1 cells,and were given to new normal mice via intravenous injection.SDE derived from tumor-bearing mice inoculated with 4T1 cells reduced total dendrite length and branching and dendritic complexity of pyramidal neurons in the mPFC,while SDE derived from tumor-bearing mice inoculated with stable expressing sh Twist1 4T1 cells had no effect.Conclusion: Tumor-bearing mice inoculated with breast cancer 4T1 cells showed depressive-like behavior,and tumor cell-derived Twist1 was involved in this process.4T1 cells secreted exosomal Twist1 into the peripheral circulatory system and targeted mPFC of mice,reducing the dendritic complexity,the number of branch and length of dendrites to induce depressive-like behavior.