CD147 Promotes Non-small Cell Lung Cancer Metastasis By Inducing CTC Cluster Formation And Extravasation

Metastasis is the main cause of tumor-associated death.Circulating tumor cells(CTCs)are a critical part of the metastasis process.Circulating tumor cells from the primary site are circulating in the bloodstream and distributed to distant organs for colonization.CTCs are usually formed by two or more homogeneous or heterogeneous cells gathering,which have stronger metastasis and survival potential than a single CTC.Due to the lack of reliable separation equipment and the inability to ensure the integrity of the CTC cluster,the molecular phenotype study of the CTC cluster is severely hindered,and the formation and transfer mechanism of the CTC cluster has not been fully clarified.CD147 is an important multifunctional cell adhesion molecule,widely involved in the adhesion between cells and between cells and stroma,regulating the proliferation,invasion and metastasis of tumor cells and other biological behaviors,and is an important target for tumor therapy.Whether CD147 can induce cell clustering and promote tumor metastasis has not been studied.In view of the above scientific issues,this study aims to identify 1)key genes in the formation of vascular circulating tumor cell clusters;2)To explore the function and molecular mechanism of CD147 driving the formation of vascular circulating tumor cell clusters;3)To preliminarily investigate the role of tumor-related macrophages in regulating vascular infiltration of circulating tumor cell clusters.This study is divided into three parts.Part I: Screening the key genes for the formation of vascular circulating tumor cell clustersWe systematically analyzed clinical case data and pathological section information in the NSCLC cohort in the TCGA database,and found that 185 of 494 patients with lung adenocarcinoma and 40.6% of 475 patients with lung squamous cell carcinoma had vascular thrombus.The later the stage,the higher the proportion of vascular cancer thrombus.The results of survival analysis showed that patients with vascular thrombus detection had worse prognosis and shorter survival.In order to identify the key genes driving the formation of vascularizing tumor cell clusters,we divided patients into groups according to the presence or non-presence of vascularizing thrombus,and separately analyzed the transcriptome changes in lung adenocarcinoma and lung squamous cell carcinoma.A total of 3,071 differential genes were identified in lung adenocarcinoma,among which 1,010 up-regulated genes and 2,061 down-regulated genes.GO/KEGG pathway enrichment analysis of these differential genes showed that the cell adhesion related pathway was significantly activated.A total of 2,718 differential genes were identified in lung squamous cell carcinoma,of which1,689 up-regulated genes and 1,029 down-regulated genes were identified.GO/KEGG pathway enrichment analysis showed that differential genes were significantly enriched into cell adhesion related pathways,and then we further screened key genes with cell adhesion function.We found that CD147 molecule was present in both lung squamous cell carcinoma and lung adenocarcinoma differential genes,and CD147 molecule significantly upregulated m RNA levels in patients with NSCLC vascular cancer suppositories.Using the TCGA database and 46 NSCLC tissue samples,we analyzed the expression of CD147 in cancer and adjacent cancer,and found that the expression of CD147 in tumor was significantly higher than adjacent cancer,and patients with high expression of CD147 had worse prognosis.Part II: To explore the function and molecular mechanism of CD147 driving the formation of vascular circulating tumor cell clusters.We collected 60 cases of advanced non-small cell lung cancer tissue samples for continuous sections,and cancer emboli was found in the blood vessels of a total of 18 cases.Immunohistochemical staining of these tissues revealed that CD147 expression in the CTC cluster was higher than that of a single CTC.Then we sorted out CTCS in peripheral blood of 16 cases of advanced NSCLC.Detection of CD147 expression found that CD147 expression in CTC cluster was significantly higher than that of single CTCs.In order to clarify the function and potential mechanism of CD147 promoting the formation of CTCS at the cellular level.We detected the expression of CD147 in different NSCLC cell lines,and found that A549 cells expressed low CD147,while NCI-H460 cells expressed high CD147.Therefore,we constructed two cell models of H460 knockdown and A549 overexpression of CD147.CD147 overexpressed cells were suspended in culture to simulate the lost-nest environment and observe the adhesion and aggregation of cells.It was found that CD147 overexpressed cells were more likely to form clusters than normal control cells,and more cells aggregated with each other,while the aggregation of cells with CD147 knockdown was significantly weakened.Next,we used a previously constructed PDX model of NSCLC to separate CD147-positive and CD147-negative cells from the PDX model for suspension culture.It was found that CD147-positive cells aggregate and adhere to each other more significantly than CD147-negative cells,further confirming the CD147-mediated cell adhesion and aggregation.At the same time,dynamic cell movement was recorded by living cell fluorescence microscopy,and it was found that CD147 overexpressed cells were more likely to aggregate with each other in the lost-nest environment.We then injected clusters of CD147 overexpressed cells and control cells into mice via caudal vein to observe cell metastasis.The results showed that CD147 overexpressed cells had stronger lung colonization ability than control cells.The cells were injected into BALB/C immunodeficient mice to observe the effects of the two groups of cells on the survival of mice.It was found that the survival of cells in the overexpression group was significantly shorter than that in the control group.HE and immunohistochemical staining were performed on the lung tissues of the two groups of mice,and it was found that the number of lung metastases in the overexpression group increased significantly and the cell proliferation ability was stronger.Next,we further explore the possible molecular mechanism of CD147-mediated cell aggregation.The interaction between CD147 molecules was found by solid phase adhesion experiments.Glutaraldehyde crosslinking experiment confirmed that CD147 can self-polymerize to form dimer or tetramer structure.CD147 extracellular overexpressed plasmids with different labels were transferred into 293 T cells for mixed culture,and co-immunoprecipitation experiments confirmed that CD147 could promote the mutual aggregation of cells through its own interaction.Next,through molecular docking and molecular dynamics simulation,we found four dimer forms of CD147 interaction,namely AD,AC,BC,and DD ’chain.Mutants were constructed based on the results of molecular docking and predicted potential binding sites.Coimmunoprecipitation confirmed that extracellular segment Thr28 and Lys63 may be the key epitope residues for the formation of CD147 dimer structure.Part III: Preliminary study on the role of tumor-related macrophages in regulating vascular infiltration of circulating tumor cell clusters We observed in the tissue sections of the vascular cancer thrombus collected that there were other cellular components besides tumor cells in the cancer thrombus.In order to clarify the possible cellular components,we analyzed the infiltration of immune cells in NSCLC tumor tissues through the TCGA database,and found that the proportion of M2 macrophages in tumor microenvironment was significantly higher than that of other immunosuppressive cells.We purchased pathological tissue sections of 12 cases of NSCLC with vascular cancer embolus for multicolor staining to analyze the proportion and distribution of CD8+T cells,FOXP3+ Treg cells and CD68-positive macrophages in vascular cancer embolus.We found that CD68-positive macrophages were positively correlated with CD147+CTC cluster(p=0.0011),indicating that there was an interaction between tumor-associated macrophages and CD147+ tumor cell cluster.To explore the mutual regulatory relationship between tumor-associated macrophages and CD147-positive tumor cell clusters,we adopted a coculture system in which CD147 overexpressed and knockdown tumor cells were co-cultured with PMA-induced M0 macrophages for 48 hours and macrophages and tumor cells were collected.Detection of the state of macrophages after co-culture showed that the proportion of M2-type macrophages increased after co-culture with CD147 overexpressed cells(p <0.05),while the proportion of M2-type macrophages decreased after CD147 knockdown(p<0.05),indicating that CD147 can induce M2-type polarization of macrophages.Next,we tested the invasion and migration ability of tumor cells after co-culture,and found that the invasion and migration ability of CD147 overexpressed cells was enhanced after co-culture compared with control cells(p < 0.001),and the invasion and migration ability was significantly decreased after CD147 knockdown(p < 0.001).The expression of EMT-related proteins was detected,and the expression of EMT-related proteins in CD147 overexpressed cells decreased after co-culture,while the expression of interstitial related proteins increased.The trans-endothelial migration ability of CD147+ tumor cell clusters was observed in the co-culture system of tumor cells,macrophages,and HUVEC cells,and it was found that CD147 overexpressed cells had stronger trans-endothelial migration ability under the regulation of macrophages(p < 0.001).CD147 overexpressed cells and control cells were injected into the caudal vein after the removal of macrophages from mice by chlorophosphite liposomes.It was found that the metastasis and colonization ability of tumor cells was significantly weakened after the removal of macrophages,indicating that tumor-related macrophages promoted the invasion and metastasis ability of CD147+ tumor cells.