Effects Of Microenvironmental S100A6 On Angiogenesis And CRC Cell Behavior Through Macrophages And Its Mechanism

Backgrounds and objectivesBeing the third most common malignant tumor worldwide,colorectal cancer(CRC)has a high incidence and a high mortality,with one million new cases and nearly 900,000 deaths annually.With living condition improving and lifestyle changing,the incidence of CRC in developing countries has been increasing year by year.Meanwhile the five-year survival rate of patients with advanced stages is not ideal.Therefore,we need to do further researches and provide more experimental supports for clarifying the mechanism of its occurrence and development and for developing new diagnosis approaches.Tumor microenvironment(TME)is an important determinant of tumor behavior,which significantly affects tumor progression,treatment effeciency and prognosis.Macrophages are the most important immune cells infiltrated in the tumor microenvironment reaching nearly 50%of tumor mass.Macrophages are highly plastic and are usually polarized into an immunosuppressive phenotype(type M2)under the stimulation of TME components.These macrophages then accelerate tumor progression by stimulating neovascularization and promoting tumor growth and metastasis.High infiltration of macrophages exists in CRC and are associated with the stages and metastasis of CRC.Angiogenesis which is a crucial factor influencing tumor progression is a novel target for tumor researches and clinical tumor treatment.In TME,there are many factors involved in promoting angiogenesis,including various growth factors and cytokines such as vascular endothelial growth factor(VEGF)and angiogenin.Macrophages also have the abilities to induce angiogenesis and increase blood vessel density.However,how do the various protein molecules in TME interact with tumor cells and macrophages,and the effects of their interactions on angiogenesis,tumor growth and metastasis have not been fully elucidated.The secreted protein S100A6,a member of the S100 family,is highly expressed in CRC and is associated with its stage and lymphatic metastasis.Our previous research has found that S100A6 can not only promote tumor cell proliferation and migration by directly interacting with tumor cells,but also through macrophages indirectly.Therefore,we raised two hypotheses:1.S100A6 participates in regulating the recruitment and activation of macrophages;2.S100A6promotes angiogenesis and CRC cell proliferation,migration and invasion through recruitment and activation of macrophages.In this study,the human mononuclear cell line THP-1,human umbilical vein endothelial cell line HUVEC and human colorectal cancer cell lines(LoVo and SW480)were used to explore the role of S100A6 on recruiting and activating macrophages and their mechanisms,and to further explore the role of S100A6-acivated macrophages on angiogenesis and the proliferation,migration and invasion of CRC cells.Methods1.Preparation and identification of recombinant proteins GST-hS100A6 and GST:prokaryotic expression and identification by SDS-PAGE and Western blot.2.Induction of human mononuclear cell line THP-1 into macrophages:THP-1 cells were treated with PMA(Phorbol 12-myristate 13-acetate,final concentration at 100ng/ml)for 24 hours.3.The role of S100A6 in recruiting and activating macrophages and the underlying mechanisms3.1 The role of S100A6 in recruiting macrophages and its mechanism3.1.1 Transwell assay was used to detect whether GST-hS100A6(final concentration 30?g/ml)recruits macrophages.3.1.2 Western blot was used to detect the protein levels of RGAE,JAK2,STAT3,p-JAK2(Tyr1007/1008),and p-STAT3(Tyr705)of macrophages treated with GST-hS100A6.3.1.3 The macrophages were pretreated with the RAGE blocker FPS-ZM1 and the JAK2 inhibitor XL019 for 30 min respectively,and then treated with GST-hS100A6.The migration abilities of macrophages were detected by transwell assay.Western blot was used to verify the inhibitory effects of FPS-ZM1 and XL019 on RGAE or JAK2/STAT3 signaling pathway.3.2 The role of S100A6 in activating macrophages3.2.1 Q-PCR was used to detect mRNA levels of M2 markers CD206,CD163,IL-10 and M1 markers iNOS and TNF-?of macrophages treated with GST-hS100A6.3.2.2 Immunofluorescence was used to detect CD206 protein level of macrophages treated with GST-hS100A6.4.The effects of S100A6-treated macrophages on angiogenesis,migration and proliferation of HUVEC cells and their mechanisms4.1 Preparation of conditioned medium:Macrophages were treated with GST-hS100A6(final concentration of 30?g/ml)for 12 hours,washed three times with PBS and then incubated for 24 hours with fresh serum-free medium.The medium was collected and centrifuged at 1000rpm for 3 min.The supernatant was collected and named as A6-M?-CM.4.2 Capillary formation assay was used to detect the effect of A6-M?-CM on angiogenesis.4.3 Q-PCR and Western blot were used to detect the mRNA and protein levels of pro-angiogenic factors CCL2,IL-6 and VEGFA in macrophages treated with GST-hS100A6.4.4 Wound healing assay and Transwell assay were used to detect the effect of A6-M?-CM on HUVEC cell migration.4.5 MTT assay was used to detect the effect of A6-M?-CM on HUVEC cell proliferation.5.The effect of macrophages treated with S100A6 on the proliferation,migration and invasion of CRC cells and their mechanisms5.1 MTT assay was used to detect the effect of A6-M?-CM on CRC cell proliferation.5.2 Transwell assay was used to detect the effect of A6-M?-CM on CRC cell migration.5.3 Western blot was used to detect the protein levels of E-cadherin and N-cadherin of CRC cells treated with A6-M?-CM.6.Animal experimentsTo verify in vitro results including the effect of S100A6 on recruiting and activating macrophages and angiogenesis,the adenovirus expressing S100A6(Ad-S100A6)and the adenovirus expressing S100A6 interfering siRNA(Adsi-S100A6)were used to infect SW480 cells respectively to up-regulate or knock down S100A6 expression.A total of 2×10~6 cells were subcutaneously injected to nude mouse to develop tumors.Tumors were collected 30 days later,embedded in paraffin,and sectioned.Immunohistochemistry was used to detect the expression of S100A6,common macrophage marker CD68 and microvessel biomarker CD31.Results1.The recombinant proteins GST-hS100A6 and GST were successfully prepared.2.THP-1 cells were successfully induced into macrophages.3.The role of S100A6 in recruiting and activating macrophages and their mechanisms3.1 The migrated cell number of GST-hS100A6 group was higher than that of the control in vitro(p<0.05).In subcutaneous xenograft tumors,the CD68 level of S100A6 overexpression group or S100A6 knock-down group was higher or lower,respectively.These results suggested that S100A6 can recruit macrophages.3.2 The protein levels of RGAE,JAK2,STAT3,p-JAK2(Tyr1007/1008),and p-STAT3(Tyr705)were increased in macrophages treated with GST-hS100A6(p<0.05),suggesting that the RAGE/JAK2/STAT3signaling pathway was activated by S100A6.3.3 The migrated cell number of FPS-ZM1+GST-hS100A6 group was less than that of GST-hS100A6 group(p<0.001);The migrated cell number of XL019+GST-hS100A6 group was less than that of GST-hS100A6 group(p<0.01).FPS-ZM1 inhibited the upregulation of RAGE and activation of JAK2/STAT3 pathway mediated by GST-hS100A6;XL019 inhibited the activation of JAK2/STAT3 pathway mediated by GST-hS100A6.These results suggested that the RAGE/JAK2/STAT3 signaling pathway is involved in the macrophages recruitment by S100A6.3.4 The mRNA levels of M2 markers CD206,CD163,and IL-10 of GST-hS100A6 group were higher than those of the control,while the mRNA levels of M1 markers iNOS and TNF-?were no significant difference compared with their control.Meanwhile GST-hS100A6 up-regulated protein levels of CD206 in macrophages.These results suggested that S100A6 can induce the polarization of macrophages into M2 phenotype.4.The effect of macrophages treated with S100A6 on angiogenesis,migration and proliferation of HUVEC cells and their mechanisms4.1 The capillary formation assay showed that GST-hS100A6 had no significant effect on angiogenesis(p>0.05),while the branch number and length of branches of A6-M?-CM group were higher than those of control(p<0.05),suggesting that S100A6-activated macrophages can promote angiogenesis.In subcutaneous xenograft tumors,overexpression of S100A6 resulted in higher level of CD31,while knock-down of S100A6resulted in lower level of CD31.These results suggested that S100A6-activated macrophages can promote angiogenesis.4.2 The mRNA and protein levels of CCL2,IL-6 and VEGFA in macrophages of GST-hS100A6 group were higher than those of the control,suggesting that S100A6 can stimulate macrophages to produce angiogenesis factors.4.3 GST-hS100A6 had no significant effect on HUVEC cell migration(p>0.05),while the healing rate and the migrated cell number of A6-M?-CM group were higher than those of the control(p<0.01),suggesting that S100A6-activated macrophages can promote HUVEC cell migration.4.4 GST-hS100A6 and A6-M?-CM both had no significant effect on HUVEC cell proliferation.5.The effect of macrophages treated with S100A6 on the proliferation,migration and invasion of CRC cells and their mechanisms.5.1 The migrated and invaded cell number of were higher than those of the control,while the proliferation ability of A6-M?-CM group was not significantly changed compared with the control,suggesting that S100A6-activated macrophages can promote CRC cell migration and invasion.5.2 The protein level of N-cadherin in CRC cells treated with A6-M?-CM was higher than that of the control,while the protein level of E-cadherin in CRC cell treated with A6-M?-CM was lower than that of the control,suggesting that S100A6-activated macrophages can induce the EMT of CRC cells.These results suggest that S100A6-activated macrophages can promote CRC cell migration,invasion and EMT.The promotion of EMT is one of mechanisms that S100A6-activated macrophages promote CRC cell migration and invasion.Conclusion1.S100A6 can recruit macrophages,and the underlying mechanism involves the activation of RAGE/JAK2/STAT3 signaling pathway.2.S100A6 induces polarization of macrophages into M2 type.3.S100A6 itself has no significantly effects on angiogenesis,but S100A6-activated macrophages promote angiogenesis,its mechanism involves that S100A6 can stimulate macrophages to produce angiogenesis factors CCL2,IL-6 and VEGFA.4.S100A6-activated macrophages promote tumor cell migration,invasion and EMT,the underlying mechanism involves the activation of macrophages.