Isolation Of Exosomes Derived From Tumor-associated Macrophages And Their Effects On The Stemness Characteristics Of Breast Cancer Cell Line 4T1

Objective:Exosomes are 30-150nm membrane vesicles of endocytic origin released by most cells.In recent years,macrophage has emerged as a key regulator in tumor microenvironment.And bone marrow-derived macrophages(BMDMs)is increasingly used to study tumor associated macrophages(TAMs)in vitro.But there is no standardized exosome isolation protocol for bone marrow-derived macrophages from Balb/c mice.Thus,this work was aimed to explore an optimized protocol to obtain purer exosome with higher yields from BMDMs.Methods:We have evaluated three methods of isolating exosomes from cell culture supernatants including ultracentrifugation,polyethylene glycol(PEG)precipitation and immunoaffinity methods.Transmission electron microscope(TEM),nanoparticle tracking analysis(NTA)and immunoblot were performed to characterize the isolated vesicles.Results:1.All methods were useful to isolate exosomes.Each method has advantages and disadvantages.2.According to the results of TEM and NTA,a higher purity but lower productivity of exosomes could be achieved by ultracentrifugation.Compared to ultracentrifugation,PEG precipitation and immunoaffinity methods generated a relatively high yield of exosomes.Nevertheless,there was more contaminated proteins in the isolated vesicles.3.Western blot results showed that except immunoaffinity methods,the vesicles isolated by PEG precipitation and ultracentrifugation were all positive for exosome markers(TSG101,Alix),while GAPDH was negative.4.Moreover,we refined the ultracentrifugation protocol and discovered that with the prolonged centrifugation time,at 4-hour,ultracentrifugation could separate highest yield of exosomes.And this optimized protocol could be successfully used in isolating exosomes of tumor associated macrophages.Conclusion:By comparing the purity and yield of BMDMs-derived exosomes isolated by three methods,we found that ultracentrigation was the best method.After further optimization,exosomes with high yield and purity could be isolated from the supernatants of BMDMs and TAMs by ultracentrifugation for 4h.Objective Tumor associated macrophages(TAMs)are widely found in most malignant tumors and have the function of promoting tumor fuigiogenesis,helping cancer cells escape into circulation and inhibiting anti-tumor immune.Breast cancer is a common cancer in women,on the other hand,it also is the leading cause of cancer-related deaths among women worldwide due to metastasis and resistance to chemotherapy.At present,studies have shown that tumor metastasis is elosely related to the stemness of tumor cells.Exosomes are around 100nm in diameter,extracellular vesicles secreted by most cells,which contain biological molecules such as proteins,RNA and lipids,and play an important role in cell coumunication.In most human and mouse tumor tissues,the phenotype and function of tumor associated-macrophages are more like M2 macrophages.Recent studies have found that TAMs derived exosomes were widely involved in the process of TAMs promoting tumor.It has been reported that TAMs can release exosomes in epithelial ovarian cancer to disturb the balance of Treg/Th17 cells[36],and TAMs-derived exosomes in gastric cancer can enhance the chemotherapy resistance of gastric cancer cells.Our research focused on the effect of tumor associated-macrophage-derived exosomes on the stemness of 4T1 cells,providing new perspective for the mechanism of TAMs promoting tumor development.Method In vitro induction of M2 macrophages were used to simulate tumor-associated macrophages.M2 macrophage-derived exosomes were isolated by ultracentrifugation.And transmission electron microscopy,nanoparticle tracking technology and Western blot were performed to identify the exosomes.Then,the M2 macrophage-derived exosomes were labeled with Dil which makes it available for us to observe their transfering to breast cancer cell line 4T1 by immunofluorescence.After that,flow cytometry,co-culture system and transwell chamber were used to investigate the tumor phenotype and stemness of 4T1 under the influence of M2 macrophage-derived exosomes.Results1.BMDMs were successfully induced into M2 macrophages by IL-4/13 in vitro,and exosomes derived from M2 macrophages were identified by TEM,NTA and western blot.2.M2 macrophage-derived exosomes labeled with Dil could be transferred into 4T1 cells.3.After co-incubation with M2 macrophage-derived exosomes,the migration and sphere formation abilities of 4T1 cells were significantly enhanced.4.Exosome inhibitor GW4869 reduced the number and volume of spheres formed by 4T1 stem cells.5.The proportion of CD44highCD24low cells in 4T1 cells was significantly increased after incubating with M2 macrophage-derived exosomes.Conclusions TAMs-derived exosomes can increase the migration and sphere formation ability of 4T1 tumor cells,so is the proportion of tumor stem cells in 4T1 cells.