Investigating The Effect Of Tvrosine Kinase Inhibitors On M2-like Polarization Of Tumor-associated Macrophages

Scientific evidence has revealed that M2-like polarized TAMs play a crucial role in angiogenesis,cancer progression,and metastasis of various types of human tumor.In tumor cells,the diverse role of macrophage to supports the tumor growth or dissemination of cancer cells.Several clinical studies have shown the correlation between the high influx of TAMs into tumor with poor clinical outcomes in the breast,prostate,ovarian,hepatocellular,lung and cervical cancers.Previously developed hypotheses have proposed that TAMs participated in antitumor responses of the body,but recently many clinical and experimental studies have demonstrated that in most of the cases TAMs increase tumor progression to malignancy.Increasing studies have shown that diverse role of TAMs either enhance or antagonize the anti-tumor efficacy of cytotoxic chemotherapeutics agents,cancer cells targeting antibodies,and immunotherapeutic agents depend on the types of treatment.Moreover,in cancers progression and metastasis,multiple roles of TAMs suggest that it is very important to develop novel strategies to target these cells.Thus,targeting to TAMs in tumor would be the promising therapeutic approach.Mostly,TAMs targeted by two main strategies,either by decreasing the density of TAMs in tumor tissue by converting the properties of the TAMs from tumor promoting to tumoricidal.However,TAMs-targeted access in clinically still far from clear.Collectively many experimental studies have revealed that the effect of novel approaches with a sudden decrease of a tumor mass to get the better therapeutic outcomes,which motivated to the further clinical setting.Because the plasticity and heterogeneity are the main features of macrophages in the tumor microenvironment,upon different stimuli,TAMs have either executed tumor preventing or tumor promoting role that depends on the polarization status of macrophages.Macrophages are mainly differentiated into two distinct phenotypes and both have been phenotypes including M1(classical activation)and M2(alternative activation)observed in the tumor.In nonmalignant tumor majority of TAMs are classically activated M1-like macrophages that are activate by interferon-γ(IFN-γ)or lipopolysaccharide(LPS),which elaborate the production of pro-inflammatory cytokines,presenting antigen and promoting tumor lysis.Conversely,M2-like polarized macrophages exhibit the protumor and pro-inflammatory activities.IL-4,IL-13,IL-10,and glucocorticoids induced the alternative activation of M2-like macrophages.M2-like polarize macrophages are further subdividing into three distinct subphenotypes such as M2a,M2b,and M2c macrophages.The polarization of these phenotype depends on the interaction between specific types of ligands to their receptors,plays a critical role to regulate inflammation,immunity and tumor suppression.The Th2 cytokines such as IL-4 and IL-13 that induce the M2a phenotype;M2b phenotype activated by immune complexes TLRs;glucocorticoid and IL-10 activated M2c phenotype macrophages.M2-like macrophages show high expression of IL-10,IL-1 decoy receptor,and IL-1Ra production,while low secretion of CCL-22,CCL-17 and expression of IL-12.IL-13 receptors induced the STAT6 signaling which further leads to transcription of M2 polarization genes like Mannose receptors(Mrcl),chitinase 3-like 3(Chil3,Ym1),resistin-like a(Retnla and Fizzl),which is indispensable for macrophage M2 polarization.TAMs,in established tumor,have an anti-inflammatory function and are similar to M2-like polarized macrophages,which enhances angiogenesis,tissue remodeling and suppressed antitumor immune responses.STAT6 is main transcription factor in IL-4/IL-13 mediated M2-like polarization and markedly regulating the balance between inflammation and allergic immune responses.STAT6 regulate the expression of macrophage M2-like associated genes such as Argl,Mrcl,and Ym1.Thus,M2-like polarized TAMs are considered promising target for adjuvant anticancer therapies.However,the interaction between TAMs and cancer cells is extremely complicated and has not clearly elucidated yet.Most importantly,whether,TAMs can increase tumor progression remains a subject of controversy.Over the past couple of decades,several new classes of anticancer therapies targeting TAMs,such as chemotherapy,targeted therapy,combination therapy and immunotherapy have been developed and extensively tested.Moreover,repolarizations of TAMs towards the Ml-like polarized macrophage,inhibition the recruitment of TAMs,suppression of TAMs M2-like macrophage and blocking the tumor-promoting activity of M2-like polarized TAMs may become the novel strategies against tumor.Targeted therapy refers to a new generation of anticancer drugs that are designed to interfere with a specific molecular target,usually a protein with a critical role in tumor growth or progression.Tyrosine kinases regulate cell proliferation,growth,migration,differentiation,and death;have therefore emerged as a promising target for cancer therapy.In this study,we investigate the effect small molecules tyrosine kinase inhibitors(TKIs)such as dasatinib,erlotinib,gefitinib,and lapatinib to check the activity of these compounds against TAMs M2-like polarization.Initially,we performed cell survival assay to check the proliferative and inhibitory effect of these entire four compounds on cell lines such as RAW264.7,BMDMs,LLCs,HepG2.Then cells were exposed to serial concentrations and selected the best concentration treatment in culture did not cause significant growth inhibition in all three cell lines.Dasatinib is an adenosine triphosphate(ATP)-based competitor that was developed as a dual inhibitor of the Src family and Abl tyrosine kinases.It is also an effective inhibitor of many of the ephrin receptors family members and of the KIT and PDGF receptors.In the present study;we showed that the desatinib effectively inhibits IL-13 induced M2-like polarization of macrophages both in Raw264.7 and DMDMs cells.Significant up-regulation of surface markers CD206 expression observed when RAW264.7 cells were treated with lOng/ml IL-13 for 72h.The surface markers expression of CD206 was greatly reduced by dasatinib with 0.03 8μM.Similarly,IL-13 induced surface markers expression of CD206 observed in BMDMs.The expression reduced in a concentration-dependent manner of dasatinib.To confirm the further role of dasatinib in M2-like polarization,mRNA levels of M2 markers assessed by quantitative real-time PCR.The mRNA levels of M2 marker,Mrcl,Yml,Fizzl,and Arg1,markedly decreased upon treatment of dasatinib with 0.038μM compared with IL-13 treated group.These result suggested that dasatinib inhibited macrophage M2-like polarization induced by IL-13.Erlotinib is another tyrosine kinase inhibitor effectively used to treats the patients with non-small cell lung cancer(NSCLC).Whose cancer has spread to other parts of the body and that has certain types of epidermal growth factor receptor(EGFR)mutations.In the present study,we investigate the effect of erlotinib IL-13 induced M2-like polarization.In vitro,erlotinib significantly inhibits the macrophage M2-like polarization with down-regulates the surface marker expression M2 markers and as well as mRNA levels of M2 genes.From this stage,the compound with best results proceeds further investigations on M2-like macrophages polarization in different cancers both in vivo and in vitro.In tumor microenvironment,alternative activation of M2-like polarization induced by IL-13.The binding of IL-13 to its receptor leads to the activation of two major signaling pathways notably STAT6 and PI3K.However,the role of IL-13 in macrophage polarization is still controversial.The establishment of TAMs and especially the role of M2-like TAMs in tumor progression is an attractive target for tumor therapy.Our study to investigate the first-time therapeutic effect of gefitinib on IL-13 induced macrophage M2-like polarization.Gefitinib,an EGFR tyrosine kinase inhibitor(TKI),is a small molecule and has been approved to treat the patients suffering from locally advanced or metastatic non-small cell lung cancer.It has good antitumor activity against several tumor cell lines and in a broad range of mouse xenograft models.In vitro experiments,we demonstrated that gefitinib significantly reduced IL-13 prompted M2-like polarization of macrophages,as determined by decreased expression of M2 surface markers i.e.CD206 and CD163.Down-regulation of mRNA levels of M2-like markers Mrcl,Argl,Ym1 and Fizz1.We further analyzed the effect of gefitinib on the angiogenesis-promoting feature of macrophages,to evaluated migrating ability of LLCs in different CM by using wound－healing assay.We found that IL-13 treated macrophages CM significantly promoted the migration of cells in 24h.Whereas combine treated,CM of IL-13 and gefitinib did not show this effect,nor did the CM from gefitinib treated-macrophages and evaluate the migrating ability of LLCs and RAW264.7 in different CM by using transwell assay.IL-13 treated macrophage CM promoted LLCs and RAW 264.7 migration,which was abrogated by gefitinib and as well as inhibition of M2-like macrophages promoted migration of cancer cells.Subsequently,gefitinib suppressed the phosphorylation of STAT6 in macrophage M2-like polarization treated with IL-13.Macrophage cells polarized majorly through the PI3K/Akt,MAPK/Erk or p38.Moreover,gefitinib reduced the Akt,Erkl/2,P38 phosphorylation.Since AMPKa is the major subunit expressed in macrophages,we further analyzed the phosphorylated protein levels of AMPKa and ACC during IL-13 induced polarization process in the presence or absence of gefitinib.In RAW264 cells IL-13 treatment did not effect on the phosphorylation of AMPKa and ACC.whereas,gefitinib triggered a significant phosphorylation of ACC and AMPKa triggered the expression of AMPKa and ACC phosphorylation in IL-13 induced macrophage M2-like polarization.In vivo,we demonstrated that gefitinib efficiently suppressed the LLCs metastasis through inhibiting the recruitment and percentage of M2-like polarization in LLCs tumor.Gefitinib extensively reduced the percentage of CD206 and CD68 and significantly reduced the expression of mRNA levels of M2 markers Mrcl,Argl,and Fizzl of Lewis lung cancer with effectively decreased the lung metastasis and altered TAMs polarization in tumor tissue.Compared to control,gefitinib showed markedly decreased the percentage of CD68’S CD206o and reduced M2-like polarization and macrophage infiltration in lungs tumor.Our study demonstrated that gefitinib effectively inhibits M2-like polarization both in vitro and in vivo and significantly reduced the Lewis lung cancer metastasis.Therefore,our study suggests that gefitinib may act as a regulator of tumor microenvironment in metastatic sites through affecting macrophage biology,and thus provides new mechanistic for the antitumor function of gefitinib.These observations highlight a novel effect of gefitinib and open new insights to improve the efficiency of lung cancer treatment that will lead toward an efficient approach for the treatment of lung cancer by inhibiting M2 polarization,in future.Hepatocellular carcinoma(HCC)is highly associated with inflammation.In HCC microenvironment,myeloid cells,including tumor-associated macrophages(TAMs)and myeloid-derived suppressor cells(MDSCs),are abundant and are often associated with poor prognosis.Given that tumor-associated macrophages,TAMs and their initiated signaling cascades are potential therapeutic targets in hepatocellular carcinoma.In the context of tumors,increased MMP-9 considered to promote invasiveness and angiogenesis,by degradation of the extracellular matrix(ECM)and basement membrane,the liberation of matrix-bound growth factors and activation of tumor-promoting agents.IL-13 documented to be the prototypic cytokine that induces alternative activation of macrophages.To establish an in vitro model of alternative macrophage activation,we incubated RAW264.7 cells with IL-13 lOng/ml.In experiments,we demonstrated that lapatinib significantly reduced IL-13 prompted M2-like polarization of macrophages,as determined by decreased expression of M2 surface markers such as CD206 and CD163.The secretion of MMP-9 by tumor-associated macrophages well documented and the link between macrophage exposure to IL-13 and MMP-9 production is less clear.Subsequently,in the case of HCCs,we exhibited that IL-13 causes increased MMP-9 production and expression of the alternative activation markers Arg1,Mrc1,Yml,IL10,Fizzl,and CCL2.Interestingly,in our experiments,while lapatinib had no effect on MMP-9 production unless the cells were activated by IL-13.Lapatinib prevented the IL-13 elicited alternative activation of RAW264.7 macrophages.The alternative IL-13 signaling involved in the activation of STAT6 that exhibits significant role in the M2-like polarization.Additionally,the Arg-1 expression has been described to be STAT6 dependent.Lapatinib dramatically reduced its expression with the passage of time.We further determined the protein expression levels of Argl and Mrcl analyzed by western blot.Lapatinib markedly decreased the expression of Mrcl and Argl induced by IL-13.These in vitro results indicated that lapatinib significantly inhibited the IL-13 induced M2-like polarization of macrophage through the STAT6-dependent pathway.Activation of PI3K/Akt pathway is associated with tumor survival,invasion,and poor prognosis in a variety of tumors.We found that lapatinib effectively decreased the phosphorylated protein levels of Akt.Next invasive assay lapatinib decreased hepatocellular carcinoma cells invasion angiogenesis and migration elicited by co-cultured with RAW264.7 conditioned medium treated with lapatinib.We investigated the impact of lapatinib on the functional macrophage-tumor cell interaction.Macrophages were treated with IL-13 and lapatinib,or both of them for 72h and the culture medium were replaced by fresh medium without serum,24h later the supernatant medium was collected as CM.To exclude the impact of CM on tumor cell survival,HepG2 cells were treated with the conditioned medium for 24h and cell proliferation was analyzed.No significant difference was found in four groups.CM from IL-13 treated macrophages significantly promoted migration of HepG2 cells in 24h,whereas CM from combined treatment of IL-13 and did not have this effect,nor did the CM from lapatinib-treated macrophage.Lapatinib decreased the cancer cell IL-13-induced expression of arginase-1 in RAW264.7 or bone marrow-derived macrophages(BMDMs).Down-regulation of mRNA levels of M2-like markers Mrc1,Arg1,Ym1,Fizz1,CCL2 and Reduction of MMP-9 expression and alternative activation of macrophages by lapatinib was confirmed using mouse bone marrow-derived macrophages(BMDMs).Results indicated that lapatinib may modulate tumor aggressiveness by regulating macrophage protease production and M2-like polarization within the tumor microenvironment.The results exhibit that lapatinib may attenuate the invasion-promoting effects of IL-13 or the interaction between macrophages and cancer in the context of a tumor microenvironment.The relevance of macrophage polarization and antitumor effects of these tyrosine kinase inhibitors and the polarization of M2 macrophages towards Ml response with minimal side effects may prove to be a powerful therapy against solid tumors.In short,targeting to TAMs in tumor is an upcoming area of research and will lead toward an efficient approach for the treatment of cancer,by inhibiting M2-like polarization of macrophages in the future.