| Objective:Osteosarcoma is the most common primary malignancy of bone in children and adolescents.Although the survival rate of patients has been improved thanks to neoadjuvant chemotherapy and advances in surgical techniques,the 5-year survival rate is still low.The most common site of metastasis in patients with osteosarcoma is the lung,and more than 90% of patients’ deaths are related to metastasis.Due to the young age and poor prognosis of patients with osteosarcoma,the strike to a family is devastating,so the study on metastasis of osteosarcoma is of great significance.Metastasis is a complex process in which cells detach from the primary tumor,cross the extracellular matrix and veins,enter the bloodstream,exudate at a distance,and colonize a new site to begin a new process of expansion.At present,further studies on lung metastasis of osteosarcoma need to explore new treatment methods.Although the prognosis of osteosarcoma has improved with the introduction of neoadjuvant chemotherapy,patients with metastatic osteosarcoma are resistant to chemotherapy,with a survival rate of less than 20%.At present,a certain breakthrough has been made in the study of lung metastasis of osteosarcoma.Studies have found that immune cells,including T cells and tumor-associated macrophages,play an important role in the process of tumor invasion and migration.Immunotherapy is expected to become an important means for the treatment of lung metastasis of osteosarcoma.Most monocytes in the body are classical monocytes,also known as inflammatory monocytes,which make up about 80 to 90 percent of monocytes,and when they are recruited to the site of infection,they devour bacteria,viruses,and dead cells.About 10 to 25 percent of nonclassical monocytes,also known as patrolling monocytes,can go against the bloodstream,clearing out pathogens and other harmful cells.PMO can sense tumor cells,move toward them,help coordinate killing them,and remove debris from tumor cells before they can metastasize.The purpose of this study is to discuss the effect of monocytes on osteosarcoma cell metastasis and the potential mechanism,whether monocytes can regulate the metastatic ability of osteosarcoma cells through the expression of MAPK/JNK signaling pathway.Recent hot spots are mainly concentrated in the study of tumor associated macrophage,macrophages and monocytes as precursor cells get attention is limited,however,the recent Hanna et al.,published in Science research results found that the PMO may have the function of the melanoma lung metastasis,mainly by raising the role of NK cells to kill tumor cells,It provides a new idea for the treatment of tumor metastasis.PMO as a immune cell,is involved in lung metastasis of osteosarcoma cells has not been reported.In this study,bioinformatics method was used to screen immune cells that may affect lung metastasis of osteosarcoma,and the influence of different monocytes on lung metastasis of osteosarcoma and the potential mechanism were explored.Combined with in vivo and in vitro experiments,the study was verified,which may provide a new effective strategy for the treatment of osteosarcoma metastasis.Methods: 1.In this study,osteosarcoma was selected as the research object,and the immune cell infiltration affecting the metastasis of osteosarcoma was analyzed by bioinformatics technology.Firstly,the cell quality was screened according to the single cell sequencing data GSE152048 downloaded from GEO Dataset related to metastasis of osteosarcoma,and the relationship between single cells was described and the published literature was combined.Cell populations were annotated.According to the high-throughput gene expression data GSE21257 in the GEO Dataset database,CIBERSORT algorithm was used to analyze the invasion level of 22 kinds of infiltrating immune cells in the osteosarcoma microenvironment,and bioinformatics analysis was conducted to analyze the immune cell infiltration after chemotherapy.To investigate the correlation between monocyte and metastasis and prognosis of osteosarcoma.2.In vivo experiment,chlorophosphate-scavenger was used to remove monocytes in nude mice,and an animal model of monocyte deficiency in nude mice was made.Human IMO and PMO obtained by flow cytometry were injected into nude mice together with osteosarcoma MG-63 cells to detect the effect of two different monocytes on metastasis of osteosarcoma.3.In vitro experiments,bioinformatics techniques were used to screen the pathway in which monocytes play a role.IMO and PMO were obtained by flow separation technology and co-cultured with osteosarcoma cells MG-63 and SAOS-2 in vitro.The effects of different monocyte types on osteosarcoma cells were compared by scratch test,Transwell migration test,invasion test and proliferation test.The differences of monocyte infiltration in different tissue samples were examined by immunofluorescence,and the expression levels of related pathway proteins in IMO and PMO after co-culture with osteosarcoma cells were detected by Western.Result: 1.Monocytes play an important role in pulmonary metastasis of osteosarcoma.The results of single cell sequencing showed that there was abundant infiltration of immune cells mainly medullary cells in osteosarcoma tissues,and there was a great difference in the infiltration of immune cells between metastatic and primary osteosarcoma tissues,and the proportion of medullary cells,mast cells and B cells decreased significantly.CIBERSORT algorithm analysis showed that there were significant differences in the proportion of immune cells in osteosarcoma tissues after chemotherapy,among which monocytes were relatively high in osteosarcoma tissues without metastasis.The overall survival of osteosarcoma patients with high infiltration of monocytes was longer.In addition,patients with high infiltration of monocytes,mast cells and follicular helper T cells had better chemotherapy effect.2.In vivo studies showed that the number of lung metastases was significantly lower in nude mice that were treated with osteosarcoma cells and PMO via tail vein than in nude mice that were treated with osteosarcoma cells and IMO via tail vein.Immunohistofluorescence assay showed that the infiltration of PMO cells in osteosarcoma without metastasis was higher than that in metastatic osteosarcoma.3.PMO can inhibit the migration and invasion of osteosarcoma cells.After co-culture of monocytes and OSTEosarcoma MG-63 cells,scratch,Transwell migration and invasion experiments confirmed that PMO could inhibit the migration and invasion of osteosarcoma cells,while IMO could promote the migration and invasion of osteosarcoma cells.Further analysis by Western Blot showed that patrol monocytes could inhibit the protein expression of the MAPK/JNK signaling pathway.After co-culture of patrol monocytes and osteosarcoma cells,E-cadherin level was up-regulated(p<0.01),D-cadherin level decreased(p<0.01),Vimentin level decreased(p<0.0001).However,classical monocytes can up-regulate the protein expression of MAPK/JNK signaling pathway.After co-culture of classical monocytes and osteosarcoma cells,the level of E-cadherin is down-regulated(p<0.01),D-cadherin level increased(p<0.01),upregulated Vimentin level.Conclusion: 1.Based on the combination of gene chip analysis and bioinformatics methods,this study proved that patients with osteosarcoma with high infiltration of monocytes had a better prognosis and a longer survival time,suggesting that monocytes may inhibit pulmonary metastasis of osteosarcoma cells.2.P PMO is easy to infiltrate in clinical tissues of osteosarcoma without metastasis.In vivo administration of PMO can inhibit lung metastasis in animal models of osteosarcoma,and the mechanism may be related to PMO inhibition of MAPK/JNK pathway and EMT 3.This study preliminarily confirmed that PMO,as a new type of immune cell,can inhibit lung metastasis of osteosarcoma,and the results of this study are expected to provide a new experimental basis for the immunotherapy of osteosarcoma. |
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