Metformin Inhibits M2-like Polarization Of Macrophage And Synergizes The Effect Of CDK4/6 Inhibitors Against Tumor Metastasis

IntrodutionTumor-associated macrophages(TAMs)with high plastic and multiple functions are the major component of stromal cells in tumor microenvirontment.TAMs could be affected by tumor microenvironment and polarized into distinguished subsets with totally different characteristics and biological functions.The M1-like(Classical polarization)TAMs display anti-tumor effect while the M2-like(Alternative polarization)TAMs accelerate tumor progression.The M2-like TAMs produce and secrete the abundant anti-inflammatory cytokines,immunosuppressive factors and various cytokines that can promote tumorigenesis,growth,migration,and enhance the formation of lymphatic and blood vessels,resulting in promoting angiogenesis and extracellular matrix decomposition and improving the motility of tumor cells.Moreover,the direct contaction between macrophages and tumor cells can improve the invasion and metastasis of tumor cells into blood vessels.Therefore,M2-like TAMs play a key role in promoting tumor metastasis and are important targets for tumor therapy.At present,three main directions for TAM-centred approaches to anti-cancer therapy are summarized and include:inhibition of macrophage recruitment to,and/or survival in,tumours;functional re-education of TAMs to an anti-tumour mode;and modulation of macrophage phagocytic activity.Current interventions for TAM proliferation and recruitment,such as CSF-1R inhibitors and CCL2 antibodies,can significantly reduce the growth and metastasis in various tumors such as breast cancer,glioma,etc.But there are prone to relapse after treatment or drug withdrawal.Bisphosphonates(e.g.chlorophosphates,zoledronic acid,etc.)can inhibit tumor growth through macrophages depletion,but complete regression of tumors using bisphosphonate alone has not been achieved.Inhibiting the CD47-SIRPa signaling axis with anti-CD47 antibodies is proved to restore the ability of macrophage to phagocytose cancer cells and prime a cytotoxic CD8+ T cell response,leading to a significant suppression in tumor size and metastasis in several cancer models.One potential challenge in the clinical translation of anti-CD47 therapies is the ubiquitous expression of CD47 on red blood cells and hematopoietic stem cells which may serve as an antibody sink to reduce efficacy while also causing transient anemia.Currently there are some preclinical studies on drugs that interfere with the polarization process of M2-like TAM.For example,all-trans retinoic acid can exert an effect on anti-osteosarcoma metastasis through inhibiting the M2-like polarization of macrophages;4-HPR plays a role in the prevention of colon cancer through the inhibition of M2-like polarization.These findings not only clarify the role of M2-like TAM in tumorigenesis,especially metastasis,but more importantly,confirm the potential value of TAMs as targets for anti-tumor therapy.Although TAMs play an important role in the tumor development,given that TAMs are non tumor cells,the TAM-centred approaches to anti-cancer therapy can only play the role of adjuvant therapy.In clinical applications,the TAM-based strategies should be combined with the chemotherapy targeting tumor cells in order to achieve the best therapeutic effect.However,under which conditions and what kind of chemotherapy drugs combined with can achieve good therapeutic effect are still the problems for the clinical application of TAMs as therapeutic targets.It has been reported recently that chemotherapeutic drugs may affect the function of TAMs in tumor treatment including the promotion of M2-like polarization which in turn weaken the anti-tumor effects of chemotherapeutic drugs.Under such circumstances,the combined use of TAM-based treatments with chemotherapeutic drugs can greatly improve the therapeutic effect of chemotherapeutic drugs.ObjectiveThe aim of this study was to investigate the effect of metformin on the inhibition of M2-like macrophage and synergy with CDK4/6 inhibitor against tumor metastasis.The current study included three chapters:1)To investigate whether and how metformin had the effect on M2-like polarization of macrophage.2)To confirm the inhibition effect of metformin on M2-likemacrophage from the biological function and evaluate the effect of metformin against tumor metastasis in vivo.3)To explore the promotion effect of CDK4/6 inhibitor on M2-like polarization and access the possibility of metformin synergy with CDK4/6 inhibitor against cancer metastasis in vivo.Chapter 1 The inhibition effect of metformin on M2-like polarization and the mechanism of inhibitionMethodsIn the study,mouse cell line RAW264.7,Bone-marrow derived macrophage(BMDM)and human cell line THP-1 were used as cell models to evaluate the influence of metformin on M2-like polarization.(1)SRB assay was used to detect the proliferation effect of metformin on RAW264.7.(2)Flow cytometer was performed to analyze the cell surface antigen F4/80,CD206 and CD86,and then determined the influence of metformin on macrophages polarization.(3)Real time PCR assay was used to analyze the mRNA levels of M2-like macrophages markers.(4)Wester blot was employed to detect the expression level of AMPKal and p-AMPKal.(5)Small-interfer RNAs were applied to knockdown the specific AMPKal and flow cytometer was performed to analyze the cell surface antigen CD206.Results(1)RAW264.7 was exposed to serial concentrations of metformin and metformin didn't cause significant growth inhibition in the concentration from 0.25 to 4.0 mM.(2)Significant upregulation of CD206 was observed when RAW264.7 were treated with IL-13 from 3.28±1.47%to 20.91 ± 3.10%(p<0.01),which was greatly reduced by metformin from 20.91 ± 3.10%to 7.90±1.48%(vs.IL-13,P<0.01).(3)IL-13 induced CD206 expression in BMDMs from 27.33 ± 7.71%to 55.09±7.14%(p<0.05)which was reduced in a concentration-dependent manner by 0.5 mM,1 mM and 2 mM metformin from 55.09 ± 7.14%to 45.61 ± 5.07%(vs.IL-13,p=0.10),40.52±3.89%(vs.IL-13,p<0.05)and 31.21±7.19%(vs.IL-13,p<0.05)respectively.Meanwhile,M2 marker genes,including Mrcl,Pparg,Retnla,Argl,Ccl24,Ccr2,Chil3 and Mgl2 were decreased by metformin compared with IL-13 treated group.(4)Significant upregulation of CD206 was observed when THP-1 were induced by PMA from 2.61 ± 0.14%to 34.12±2.61%(p<0.001),which was greatly reduced by metformin from 34.12±2.61%to 12.27 ± 5.85%(vs.PMA,p<0.01).Similarly the upregulation of M2 marker genes,including MRC1 and CLEC7A were restrained by metformin,for example the relative mRNA level of MRC1 was up-regulated to 4.33 ± 0.48 by PMA(p<0.01),which was significantly down-regulated to 1.95 ±1.52 by metformin(vs.PMA,p<0.05)and the relative mRNA level of CLEC7A was decreased from 6.50±0.21(P<0.001)to 2.96 ± 1.63(vs.PMA,P<0.05).(5)The percentage of CD86 positive cells went up(p<0.05)when the cells were treated with LPS or IFN-y.However,there was no significant difference between RAW264.7 cells incubated with LPS or IFN-? and the ones treated with metformin and LPS or IFN-?(?>0.05).(6)Metformin triggered a significant AMPKal phosphorylation and there was a further increase when combined treated with IL-13 in 8 h,which remained elevated in 24,48 and 72 h.(7)AIRCA,another AMPK activator,was found to prevent the M2-like polarization stimulated by IL-1.3 as also from 21.53 ± 0.35%(p<0.01)to 10.95 ± 1.38%(vs.IL-13,p<0.05).(8)The effect of metformin in M2-like polarization inhibition was abolished when AMPK?1 was silenced.Chapter 2 The anti-metastasis effect of metformin through M2-like polarization inhibitionMethodsIn the study,the conditioned medium derived from BMDM was co-incubated with Lewis lung cancer cell line LLC or mouse pulmonary micro-vascular endothelial cell MMVEC in order to evaluate biological function of metformin suppression on M2-like polarization in vitro.(1)SRB assay was used to detect the proliferation effect of conditioned medium on LLC and MMVEC.(2)Flow cytometer was performed to evaluate the apoptosis of conditioned medium in LLC.(3)Transwell assay was employed to detect the migration ability of LLC and MMVEC cultivated with different conditioned medium.(4)Wood healing assay was applied to investigate the effect of conditioned medium on MMVEC motility.(5)Tube formation assay was conducted to evaluate the tube formation ability of MMVEC cultivated with different conditioned medium.(6)Real time PCR assay was used to analyze the mRNA levels of metastasis and angiogenesis related cytokines in BMDM.In the study,LLC subcutaneous model,LLC intravenous model and 4T1 orthotopic model were established to investigate the anti-metastasis role of metformin and the macrophage depletion model established by the clodronate liposomes was used to evaluate the effect of macrophages on the anti-metastasis action of metformin in vivo.(1)C57BL/6 mice were used to construct LLC subcutaneous model and LLC intravenous model,on the basis clodronate liposomes were used to remove macrophages in order to evaluate the role of metformin against lung metastasis of LLC cells,and investigate the effect of macrophages on metformin anti-tumor metastasis.(2)Balb/c mice were used to establish 4T1 ortho topic model to evaluate the role of metformin against lung metastasis of 4T1 cells.(3)Immunofluorescence and immunohistochemistry were performed to detect the M2 macrophage in the tumor region.(4)Immunofluorescence was applied to analyze the maturation of vessels in tumor region.(5)ELISA assay was used to detect the secretion of CCL2 in serum.Results(1)The conditioned medium had no effect on the proliferation of LLC and MMVEC.(2)The conditioned medium did not cause apoptosis in LLC.(3)The number of migrated LLC cells incubated with conditioned medium of BMDMs treated with IL-13 was remarkably increased to 3.36 ± 1.09(p<0.05).Meanwhile,those incubated with conditioned medium of BMDMs treated with IL-13 and metformin significantly decline to 1.38 ± 0.46(vs.IL-13,p<0.05).(4)Wound healing assay revealed that conditioned medium from IL-13 challenged macrophages promoted MMVECs migration,which was abrogated by metformin.(5)Conditioned medium from IL-13 treated macrophage significantly promoted tube formation of MMVECs,while the conditioned medium from macrophage with combined treatment of IL-13 and metformin didn't have this effect.(6)The upregulation of metastasis related cytokine genes,including MMP9,Mmp1O,Mmp14,Ccl7,Il1b and angiogenesis related cytokine genes,including Fgf1,Ccl2,Cxcl2,Ednl,Igfl was restrained by metformin.(7)In LLC subcutaneous model,the number of metastasis was strongly reduced in metformin group and clodronate group(p<0.05)while the treatment of metformin did not further affect metastasis under the elimination of TAMs(p>0.05).(8)In LLC intravenous model,the number of metastasis was not affected by metformin(p = 0.26).(9)In 4T1 orthotopic model,the number of metastasis was strongly reduced in metformin group(p<0.05).(10)Less F4/80+ TAMs expressed CD206 in metformin treated LLC tumors(p<0.001).(11)Less CD206 was expressed in metformin treated 4T1 tumors(p<0.05).(12)Double staining of CD31 and the matural marker ?-smooth muscle actin(?-SMA)revealed that more a?-SMA+ covered tumor vessels in tumor tissue from metformin treated mice(p<0.01).(13)Metformin could obviously decrease the expression of CCL2 in serum(p<0.05).Chapter 3 The synergistic anti-metastasis effect of metformin and CDK4/6 inhibitor ribociclibMethodsIn the study,mouse cell line RAW264.7 and Bone-marrow derived macrophage(BMDM)were used as cell models to evaluate the influence of ribociclib on M2-like polarization.4T1 orthotopic model was established to investigate the synergistic anti-metastasis effect of metformin and ribociclib.(1)SRB assay was used to detect the proliferation effect of ribociclib on RAW264.7.(2)Flow cytometer was performed to analyze the cell surface antigen F4/80 and CD206,and then determined the influence of ribociclib on macrophages polarization.(3)Real time PCR assay was used to analyze the mRNA levels of M2-like macrophages markers.(4)4T1 orthotopic model was conducted to evaluate the synergistic anti-metastasis effect of metformin and ribociclib.(5)Immunohistochemistry was performed to detect the M2 macrophage in the tumor region.Results(1)Ribociclib increased the mRNA level of Mrcl induced by IL-4(p<0.05,vs.IL4).(2)RAW264.7 was exposed to serial concentrations of ribociclib and ribociclib did not cause significant growth inhibition in the concentration from 0.63 to 10 ?M.(3)Significant upregulation of CD206 was observed when RAW264.7 were treated with 1.25 ?M,2.5 ?M and 5 ?M ribociclib as increasing from 4.08 ± 0.73%to 16.86 ± 1.60%(p<0.01),23.11 ± 5.74(p<0.05)and 25.09±0.98%(p<0.001)respectively.Additionally,ribociclib increased the percentage of CD206+ cells induced by IL-4 from 21.05±2.52%(p<0.01)to 26.13±4.16%(vs.IL-4,p?0.15),28.90±1.04%(vs.IL-4,p<0.05)and 41.49 ± 1.87%(vs.IL-4,p<0.01)repectively.(4)Ribociclib up-regulated the mRNA level of Ccl2 to 6.50 ± 0.21(p<0.05),and increased the mRNA level of both Ccl2 and Mgl2 induced by IL-4 respectively,from 3.41±1.68(p<0.05)to 5.59 ± 2.19(vs.IL-4,p<0.01)and from 10.55 ± 1.18(p<0.05)to 34.30 ± 4.51(vs.IL-4,p<0.05).(5)Ribociclib increased the percentage of F4/80+CD206+ cells in a concentration-dependent manner(1.25 ?M,2.5?M and 5 pM),from 23.57 ± 1.50%to 35.61 ± 3.37%(p=0.09),38.91±2.73(p<0.05)and 46.63 ± 10.03(p<0.05)respectively.(6)In 4T1 orthotopic model,the number of metastasis was strongly reduced in metformin group(p<0.05),and had a further decrease when combined treated with ribocilib(p<0.05).(7)Less CD206 was expressed in metformin treated 4T1 tumors(p = 0.09).Significant upregulation of CD206 was observed in ribociclib treated 4T1 tumors(p<0.01)which was greatly reduced by metformin(p<0.01).ConclusionIn this study,we find that metformin is able to block the M2-like polarization of macrophages partially through the activation of AMPK.Further,metformin can be proved to inhibit macrophage M2-like polarization in biological function,which plays an important role in metformin inhibiting metastasis of Lewis lung cancer and breast cancer.Meanwhile,the molecule-targeted drug ribociclib,a CDK4/6 inhibitor is able to promote the M2-like polarization of macrophages.And we prove that the M2-like polarization inhibitor metformin can synergize with ribociclib in suppressing tumor metastasis in vivo.Our study illustrates that metformin is a small molecule compound that effectively inhibits M2-like polarization,suggesting that metformin has an anti-metastasis effect,which further expands the application of metformin in anti-tumor therapy.According to the experimental data,we confirm that drug can have a synergistic anti-metastasis effect through the intervention of M2-like polarization.