| Background:Colorectal Cancer(CRC)is the third most common malignant tumor in the world.Many patients with CRC have distant metastasis at the first diagnosis,or even after radical surgical resection,distant metastasis can also develop in the follow-up process.The therapeutic effect is not satisfactory.Distant metastasis of tumors is a major cause of CRC-related death,and there is relatively limited research on lung metastasis of CRC.The tumor microenvironment(TME)plays a key role in tumor genesis,development,and distant metastasis.Many studies found that tumorassociated macrophages(TAMs)are the most important immune cells in the TME,which can enter the TME under the mediation of chemokines and be closely related to the occurrence and development of tumors.However,the regulatory networks between them have not been thoroughly studied.The purpose of this study is to explore the relationship between chemokines and TAMs and the mutual regulation of tumor cells and to seek the potential mechanism of CRC lung metastasis,finally,to provide a theoretical basis for the precise treatment of CRC lung metastasis in clinic.Methods:1.Surgical specimens of CRC patients with lung metastasis were collected,and then analyzed the primary tumors and the lung metastases by the next-generation sequencing.The different genes were screened out.Through bioinformatics analysis,core genes were identified and the main pathways involved in lung metastasis were searched by enrichment analysis.2.Through public databases,the related gene chip data of CRC primary tumors and their adjacent tissues,as well as the lung metastatic tumors and their adjacent tissues were analyzed and verified.3.Immunohistochemical staining was used to detect the expression of related proteins in clinical specimens of CRC patients with lung metastasis.4.CRC cell lines with overexpression or knockout of related genes were constructed and verified by Western Blotting.5.The ability of proliferation and migration of CRC cell lines with overexpressing related genes were detected by plate clone formation assay and Transwell assay.6.qRT-PCR was used to detect whether overexpression of related genes in CRC cells affects the changes of other related genes.7.The influence of coculture with TAMs on cell proliferation and migration was tested again,by performing plate clone formation assay and Transwell assay on CRC cells transfected with the overexpression of related genes.8.BALB/c nude mice and NOD-SCID immunodeficient mice were selected to construct subcutaneous tumor-forming and caudal vein lung metastasis models,and the effects of the change of endogenous protein on the proliferation and metastasis of CRC cells in vitro were observed by immunohistochemistry and immunofluorescence co-staining.Results:1.A total of 30 differential genes were screened out by transcriptome sequencing of CRC primary tumors and lung metastases,among which several genes(such as Vimentin,EPCAM,MMP3,MMP10,CDH1,and SNAI1)were involved in the process of Epithelial-Mesenchymal Transition(EMT)and played a key role.In addition,chemokine ligands(such as CXCL3,CXCL2,CXCL8,CXCL1,and CCL20)and other immune-related indicators also showed significant differences in expression.By using enrichment analysis,it was also found that lung metastases were not only enriched in EMT proliferation-related pathways,but also in the immune microenvironment and chemokine-related pathways significantly.2.Transcriptome sequencing(RNAseq)of CRC primary tumors and lung metastases were analyzed by the WGCNA method,and it was found that CXCL2 was an important core gene of lung metastasis.3.By analyzing transcriptome data from the public databases,we found that CXCL2 expression was increased in lung metastases compared with primary CRC tumors,while CXCL2 expression was increased in primary CRC tumors compared with paracancer tissues.4.CIBERSORT deconvolution algorithm was used to analyze the transcriptome sequencing data of CRC primary tumor and the lung metastases,and the transcriptome data of the public database.It was found that the proportion of M2-type macrophages was significantly increased in lung metastases compared with CRC primary tumors.5.Immunohistochemical staining was used to detect the expression of EMT markers(E-cadherin and Vimentin),M2-type macrophage surface markers(CD 163),Snail,and CXCL2 in the specimens of CRC patients with lung metastasis.Vimentin,CXCL2,CD 163,and Snail were highly expressed in the lung metastases,while Ecadherin was in a lower expression.In addition,we found that Snail was highly expressed in the primary specimens of CRC patients with lung metastases compared with the primary specimens without distant metastasis.6.In vitro cell experiments,it was found that overexpression of Snail could increase the ability of proliferation and migration of DLD-1 and RKO CRC cell lines,but significantly lower than that after co-culture with TAMs.7.In vitro cell experiments,it was found that Snail can promote the expression of Vimentin and CXCL2 in DLD-1 and RKO CRC cell lines,and reduce the expression of E-cadherin,while the co-culture of Snail-overexpressing DLD-1 and RKO CRC cell lines with TAMs did not increase the expression of CXCL2.8.In vivo subcutaneous tumorigenic experiments in nude mice,it was found that overexpression of Snail promotes the ability of subcutaneous tumorigenicity of CRC cells,and increase the expressions of Vimentin,CXCL2,CD 163,and Ki67,while decreasing the expression of E-cadherin.9.Constructed CRC cell lines with Snail knockdown or overexpression,and CXCL2 knockdown or overexpression.Further experiments found that CXCL2 directly promoted the ability of migration and proliferation of CRC cells compared with Snail,in vitro cell as well as in vivo animal experiment(construction of subcutaneous tumorigenesis and caudal vein lung metastasis model).It promotes the expression of Vimentin,CD 163,and Ki67,but decreases the expression of Ecadherin,which is the main factor promoting tumor growth and lung metastasis.Conclusion:CXCL2 may plays a regulatory role in the networks of mediating the interaction between TAMs and CRC cancer cells.In this process,it may be after EMT,the CRC cells acquire the mesenchymal phenotype which secretes CXCL2 to mediate TAMs infiltration,and promotes CRC lung metastasis.This discovery may provide a new therapeutic target for CRC with lung metastasis. |
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