The Role And Mechanism Of Netrin-1 In Promoting The Stemness Of Colorectal Cancer Cells

Cancer stem cells（CSCs）are a group of tumor cells with the potential of self-renewal and differentiation,which can contribute to tumor initiation,recurrence,and metastasis of colorectal cancer（CRC）.Myeloid-derived suppressor cells（MDSCs）are the most important immunosuppressive cells in tumor microenvironment.On the one hand,they can directly inhibit the anti-tumor immune response;on the other hand,MDSCs also can promote tumor growth and angiogenesis.At present,it is known that axon guidance factor netrin-1 can participate in tumor progression,but the regulation of netrin-1 on CRC stem cells and tumor immune microenvironment is not clear.Objective:The purpose of this study was to explore the roles of axon guidance factor netrin-1 in the progression of CRC.To investigate the direct regulation of tumor cell-derived netrin-1 on the stemness of CRC cells and the effect of netrin-1-treated MDSCs on the stemness of CRC cells.Methods:1.Analysis of the effect of netrin-1 on the stemness of CRC cellsCancer and adjacent tissues of CRC patients were collected,and the expression and localization of netrin-1 in CRC and adjacent tissues were detected by Western blot and IHC.The expression of netrin-1 and the proportion of CD133⁺CSCs in cancer and adjacent tissues of CRC patients were analyzed by immunofluorescence.q RT-PCR and Western blot were used to detect the expression of netrin-1 in two murine CRC cell lines CT26 and MC38.ELISA was used to detect the content of netrin-1 in the supernatant of the two kinds of cells.Immunofluorescence was used to analyze the localization and fluorescence intensity of netrin-1 in the two kinds of cells.q RT-PCR and Western blot were used to detect the expression of netrin-1 receptors（UNC5A,UNC5B,UNC5C,UNC5D,DCC,neogenin,DSCAM and A2BR）in CT26cells and MC38 cells.The sh RNA-NTN1 Lentivirus vector was constructed and transfected into MC38cells to obtain MC38^{sh RNA-NTN1}cells.q RT-PCR and Western blot were used to verify the knockdown efficiency of netrin-1.and the apoptosis ratio was detected by FCM.The expressions of SOX2,Nanog,OCT-4 andβ-catenin in MC38^{sh RNA-NTN1} cells were detected by q RT-PCR and Western blot.The proportion of CD133⁺and ALDH1⁺stem-like MC38^{sh RNA-NTN1} cells was analyzed by FCM.The sphere forming ability of MC38^{sh RNA-NTN1} cells was detected by tumor sphere formation assay.The migration ability of MC38^{sh RNA-NTN1} cells was detected by Transwell assay.MC38 cells were treated with exogenous recombinant netrin-1 protein to further verify the effect of netrin-1 on the stemness of MC38 cells as described above.The subcutaneous MC38^{sh RNA-NTN1}transplanted tumor model was established,and the tumor growth of MC38^{sh RNA-NTN1}-bearing mice was dynamically monitored.The proportion of CD133⁺CSCs and the expression of Ki67 in tumor tissue were detected by FCM.2.To explore the main mechanism of netrin-1 in stemness regulation of CRC cellsThe expression of UNC5B and neogenin in MC38 cells was knocked down by si RNA,and the stemness of MC38 cells were detected according to the above method.After recombinant netrin-1 protein treatment,or UNC5B and neogenin knockdown,the phosphorylation level of NF-κB p65 and ERK1/2 in MC38 cells were detected by Western blot.MC38 cells were treated with NF-κB inhibitor BAY 11-7085 or ERK1/2inhibitor PD98059,respectively,and the effects of BAY 11-7085 or PD98059 on the stemness of MC38 cells were detected.3.Effect of Netrin-1 on the pro-stemness ability of MDSCsThe MC38 subcutaneous tumor model was constructed,and the splenic MDSCs were isolated by immunomagnetic beads.The expression of netrin-1 and its receptors was detected by q RT-PCR and western blot.MDSCs were pretreated with exogenous recombinant netrin-1 protein for 24 hours（referred to as NTN1-MDSCs）,NTN1-MDSCs were collected,washed fully and co-cultured with MC38 cells for another 24hours.The expressions of SOX2,Nanog andβ-catenin in MC38 cells were detected by q RT-PCR and Western blot.The proportion of CD133⁺stem cell-like MC38 cells was analyzed by FCM.MDSCs were pretreated with A2BR antagonist PSB1115 for30 minutes,and then treated with recombinant netrin-1 protein for 24 hours（referred to as PSB+NTN1-MDSCs）.PSB+NTN1-MDSCs were collected,washed fully and co-cultured with MC38 cells to detect stemness in MC38 cells as mentioned above.The proportion of CD133⁺and ALDH1⁺stem-like MC38 cells was analyzed by FCM.The sphere forming ability of MC38 cells was detected by tumor sphere formation assay.The proliferation ability of MC38 cells was detected by colony-forming assay and the migration ability of MC38 cells was detected by Transwell assay.Construct MC38 subcutaneous tumor models,each mouse was intraperitoneally administrated 100μg anti-DR-5 monoclonal antibody twice for depleting endogenous MDSCs during tumor development,and adoptive transferring 1×10⁶NTN1-MDSCs or PSB+NTN1-MDSCs to tumor tissues on the 16th and 22nd day of tumor injection,and tumor growth was monitored dynamically.The proportion of CD133⁺CSCs in tumor tissues was determined by FCM.4.Clarify the pro-stemness mechanism of NTN1-MDSCsTranscriptome sequencing was conducted on NTN1-MDSCs and GO enrichment and KEGG pathway enrichment were used to analyze the signaling pathways with significant differentially expressed genes in the results.The oxidative phosphorylation（OXPHOS）pathway was screened for further study.Mito-Tracker Green fluorescent probe and JC-1 probe were used to label mitochondria,and the effects of netrin-1 on mitochondrial quality and membrane potential in MDSCs were detected by immunofluorescence.The extracellular oxygen consumption rate of MDSCs was detected by oxygen probe method.Western blot analysis of AMPK-PGC-1αsignaling pathway in NTN1-MDSCs.ATP content in MDSCs was detected by ATP detection kit.MDSCs were treated with netrin-1 or A2BR antagonist PSB1115,MDSCs were treated with cultured medium of MC38^{sh RNA-scramble} cells or MC38^{sh RNA-NTN1} cells,respectively;or MDSCs in tumor tissue and spleen were isolated from MC38^{sh RNA-scramble} and MC38^{sh RNA-NTN1}-bearing mice to detect ATP content in MDSCs.MDSCs were treated with four OXPHOS inhibitors（mitochondrial respiratory inhibitors Rotenone and Antimycin A,phosphorylation inhibitor oligomycin and uncoupling agent FCCP）for 24 hours.The content of ATP in MDSCs was detected.MDSCs were collected,washed fully and co-cultured with MC38 cells.The regulatory roles of MDSCs on the stemness of MC38 cells was detected as indicated above.In order to determine the effect of ATP on the stemness of MC38 cells,exogenous ATP was added to treat MC38 cells,and the stemness of MC38 cells was detected by the above method.In order to clarify the main energy metabolic pathway involved in stemness regulation of MC38 cells,MC38 cells were treated with glycolysis inhibitor 2-DG and oxamate or OXPHOS inhibitor oligomycin,respectively,to detect the stemness of MC38 cells.EDTA anticoagulant blood of CRC patients and healthy controls was collected,PBMCs were extracted from Ficoll isolation solution,and the proportions of CD33⁺CD11b⁺HLA-DR^-/low MDSCs in PBMC were detected by FCM.Plasma netrin-1 levels in CRC patients and healthy controls were determined by ELISA.The correlation between the proportion of MDSCs and the plasma netrin-1 levels were analyzed.GEPIA database was used to analyze the correlation between the expression level of netrin-1 and the expression levels of MDSCs markers（CD33,ITGAM）and pluripotency related transcription factor（CNTTB1）in CRC tissues.Results:1.Netrin-1 enhances the stemness of MC38 cells through UNC5B/neogenin receptorThe expression of netrin-1 in cancer tissues of CRC patients was significantly higher than that in adjacent tissues,and the expression of netrin-1 was colocalized with CD133.The expression of netrin-1 in two kinds of mouse CRC cell lines showed that the expression of netrin-1 m RNA and protein were higher in MC38 cells and lower in CT26 cells.At the same time,MC38 cells secreted high levels of netrin-1（4.04±0.16 ng/m L）,while CT26 cells had lower expression levels（1.52±0.25ng/m L）.Compared with MC38^{sh RNA-scramble} cells,the m RNA and protein levels of netrin-1were significantly reduced in MC38^{sh RNA-NTN1} cells.SOX2 and Nanog m RNA and protein levels were significantly decreased in MC38^{sh RNA-NTN1} cells.The sphere formation number of MC38^{sh RNA-scramble} cells was 118±7.07,the sphere formation number of MC38^{sh RNA-NTN1} cells was 50±2.83 and migration numbers of MC38^{sh RNA-scramble} cells were reduced by 50%than MC38^{sh RNA-scramble} cells.Netrin-1 recombinant protein treatment increased the expression of SOX2 and Nanog m RNA and protein in MC38 cells（P<0.01）.The proportions of CD133⁺/ALDH1⁺stem-like MC38 cells were increased（P<0.01）.Netrin-1 can also enhance sphere formation,colony-forming and migration ability of MC38 cells.In vivo,the tumor growth in MC38^{sh RNA-NTN1}-bearing mice was significantly delayed;the proportion of CD133⁺CSCs was significantly lower than that of the MC38^{sh RNA-scramble} group（P<0.01）.These results suggest that MC38 cells can produce high levels of netrin-1 to promote MC38 cells stemness.MC38 cells mainly express two kinds of netrin-1 receptors,UNC5B and neogenin.Knockdown UNC5B and neogenin in MC38 cells decreased the expression of SOX2 and Nanog m RNA and protein（P<0.01）.Knockdown of UNC5B and neogenin expression could also reduce the ball formation,cell migration and clone formation of MC38 cells（P<0.05）.When MC38 cells were treated with exogenous netrin-1 recombinant protein,the levels of intracellular NF-κB p65 and ERK1/2 phosphorylation were significantly increased,and after knocking down the expression of UNC5B and neogenin in MC38cells,exogenous netrin-1 recombinant protein was added,the phosphorylation levels of NF-κB p65 and ERK1/2 in knockdown group were significantly lower than those in si RNA control group,suggesting that netrin-1 can activate intracellular NF-κB p65and ERK1/2 signaling pathways through UNC5B or neogenin.Compared with DMSO control group,BAY 11-7085 or PD98059 treatment decreased SOX2 and Nanog m RNA expression in MC38 cells（P<0.05）,the proportion of CD133⁺/ALDH1⁺stem cell-like MC38 cells（P<0.05）,and the numbers of tumor sphere formation,cell migration and colony formation of MC38cells（P<0.01）.These results suggest that netrin-1 can activate NF-κB and ERK1/2 signaling pathways through UNC5B and neogenin to promote the stemness of MC38 cells.2.Netrin-1 enhances the OXPHOS of MDSCs,thereby promoting the stemness of MC38 cellsCompared with MC38 cells,MDSCs showed lower expression of netrin-1.A2BR is the main netrin-1 receptor expressed by MDSCs.MDSC could promote the stemness of MC38 cells,and when MDSCs were pretreated with recombinant netrin-1 protein（NTN1-MDSCs）,NTN1-MDSCs increased the stemness-related markers expression and up-regulated the proportion of stem cell-like MC38 cells（P<0.05）.Treatment of MDSCs with A2BR specific antagonist PSB1115 could partially block the promoting effect of NTN1-MDSCs on the stemness of tumor cells.Adoptive transfer of MDSCs into tumor-bearing mice can promote tumor progression,NTN1-MDSCs increased the proportion of CD133⁺CSCs in tumor tissues（P<0.05）,while PSB+NTN1-MDSCs decreased the proportion of CD133⁺CSCs in tumor tissues（P<0.05）.These results confirm that netrin-1 acts on MDSCs through A2BR and enhances the pro-stemness effects of MDSCs.Analyzed transcriptional sequencing results and compared with MDSCs group,there are 206 genes were up-regulated and 17 genes were down-regulated in NTN1-MDSCs group.GO enrichment analysis and KEGG pathway analysis of differentially expressed genes showed that up-regulated differentially expressed genes were mainly involved in ATP synthesis,mitochondrial respiratory chain assembly and OXPHOS process.The OXPHOS process was screened for further study.Exogenous recombinant netrin-1 protein treatment could increase the mitochondrial mass and mitochondrial membrane potential of MDSCs.Netrin-1 treatment enhanced the phosphorylation of AMPK and the expression of PGC-1αprotein in MDSCs.The content of ATP in NTN1-MDSCs was 4.6 times higher than that in the control group.Compared with MDSCs treated with MC38^{sh RNA-scramble} CM,the content of ATP in MDSCs treated with MC38^{sh RNA-NTN1} CM was decreased by 1.5times.These results indicate that netrin-1 is involved in the synthesis of ATP in MDSCs.Treated MDSCs with OXPHOS inhibitors,the ATP content in MDSCs was significantly decreased.When OXPHOS inhibitors pretreated MDSCs were co-cultured with MC38 cells,OXPHOS inhibitors pretreated MDSCs could reduce the stemness-related markers expression of MC38 cells and the proportion of CD133⁺/ALDH1⁺stem-like MC38 cells（P<0.05）.Exogenous ATP treatment increased the stemness-related markers expression in MC38 cells（P<0.05）.These results suggest that netrin-1 enhances OXPHOS and ATP production in MDSCs,thereby regulating MDSCs to promote MC38 cell stemness.Glycolytic inhibitors 2-DG and oxamate significantly inhibited the expression of stemness-related markers in MC38 cells（P<0.001）.In contrast,use of the OXPHOS inhibitors FCCP,oligomycin,and rotenone resulted in the increased stemness-related marker levels of MC38 cells（P<0.001）.2-DG and oxamate significantly inhibited the tumor sphere formation and colony-forming ability of MC38 cells.This shows that MC38 cells mainly rely on glycolysis pathway to maintain stemness.In addition,the proportion of MDSCs in PBMC of CRC patients and healthy controls was 17.55±10.64%and 6.23±4.44%,respectively.The proportion of MDSCs was significantly increased in PBMC of CRC patients（P<0.001）.The plasma levels of netrin-1 in CRC patients and healthy controls were 3.91±2.58 ng/m L and1.64±1.61 ng/m L,respectively.The plasma netrin-1 levels in CRC patients were also significantly higher than those in healthy controls（P<0.05）.The netrin-1 levels in peripheral blood of CRC patients are positively correlated with the proportion of MDSCs（R²=0.5895,P<0.001）.GEPIA database analysis found that there are strong correlations among the expression of netrin-1,pluripotency related transcription factor CNTTB1（the gene encodingβ-catenin）and the two key surface markers of MDSCs:CD33 and ITGAM（the gene encoding CD11b）（P<0.001）.These results suggest that there is a correlation among netrin-1,MDSCs and stemness-related molecule in CRC patients.Conclusion:Netrin-1 can enhance the stemness of CRC cells by activating NF-κB and ERK1/2 signaling pathways.Netrin-1 can also act on MDSCs through A2BR to up-regulate OXPHOS and increase ATP synthesis in MDSCs,further enhancing the stemness of CRC cells and promoting tumor growth.