Inhibition Of Polarization Of M2-like Tumor-associated Macrophages By Dihydroartemisinin And Its Effects On Invasion And Growth Of Head And Neck Squamous Cell Carcinoma

Head and neck cancer as a kind of human malignancy,often occurs in the oral cavity,nasal cavity,pharynx and larynx.It ranks as the sixth most aggressive malignancy worldwide,with a five-year survival rate of only approximately 50%.Among various types of pathologies,head and neck squamous cell carcinoma(HNSCC)is the most common one,accounting for 85% to 95% of all head and neck cancers.Clinically,more than 90% of HNSCC patients die not from the direct rupture and bleeding of their primary tumor but from other diseases that spread after the tumour metastasizes.The continued growth,invasion and migration of tumors depend not only on the genetic mutations in tumour cells but also on the support and regulation of the tumor microenvironment.Therefore,we need to explore the relationship between the tumor microenvironment and a tumor,and find breakthroughs for cancer treatment.The tumor microenvironment is composed of endothelial cells,immune cells,tumor cells and other cells,of which components have complex interactions for facilitating tumor initiation and progression.Tumour-associated macrophages(TAMs)are an important type of immune cell.Their roles in the tumor microenvironment are mainly determined by M1 or M2 phenotype.Recent studies have shown that M2-like TAMs can promote cell matrix decomposition,cancer cell migration,angiogenesis and lymphangiogenesis,all of which are necessary for tumor metastasis.Clinical studies have also shown that the presence of large number of M2-like TAMs is positively correlated with a poor prognosis.Therefore,M2-like TAMs can be used as a new therapeutic target,and inhibiting the polarization of M2 macrophages is an effective method.Dihydroartemisinin(DHA)is a semisynthetic derivative extracted from Artemisia annua with modern technology.It has been shown to have antimalarial,antiviral,anti-inflammatory and antitumour effects.However,whether DHA can inhibit tumor progression and metastasis by regulating TAMs in the tumor microenvironment has not been reported.Therefore,we investigated if DHA could inhibit HNSCC thorough interrupting polarization of macrophages.We carried out experiments in three parts.Part One Induction and identification of activated M2 macrophages and their effects on HNSCCObjective: The purpose of this part is to establish a model of TAMs.For doing this,it is necessary to obtain the M2-polarized macrophages.We observe the effects of M2-polarized macrophages on the HNSCC.Finally,it was determined whether M2-polarized macrophages and TAMs have a similar effect on NHSCC in tumor microenvironment.Methods:1.To obtain M0 macrophages,200 ng/ml phorbol 12-myristate 13-acetate(PMA;Sigma,MO,USA)was added to THP-1 monocytes for 24 hours,Subsequently,the PMA-induced THP cells were then stimulated with IL-6 and IL-4 at concentration of 20 ng/ml for 24 hours to obtain M2 macrophages.2.The m0 and m2 macrophages were characterized by morphological changes after induction and stimulation.3.Flow cytometry was used to detect the expression levels of the M2 macrophage marker protein CD163,to prove the successful induction of the M2-polarized macrophages.4.qRT-PCR was used to detect the m RNA expression levels in macrophages.The levels of m RNA demonstrated that M0 macrophages were induced to M2-polarized macrophages.5.The medium of macrophages in different states was centrifuged at 4 ?in a high-speed centrifuge at 12000rpm/min for 15 min,to obtain the conditioned medium of macrophages in different statuses(CM).6.Wound-healing assay and Transwell invasion assay ware used to observe the effect of CM from macrophages of different statuses on the migration andinvasion ability of NHSCC.Results:1.There were obvious differences in cell morphologies between M0 and M2.A great number of pseudopodia were seen in M2 cells,and the morphology of M2 cells was mainly polygonal or fusiform.The cells were more adherent to dish,and readily grew in clusters.2.The flow cytometry and qRT-PCR assay results showed that the expression level of M2 macrophage marker CD163 protein was significantly higher than that of M0 macrophage,and the m RNA expression levels of CD206,IL-10,MMP9 and CCL18 in M2 macrophages were significantly increased.These findings demonstrated that the induction of polarized M2 macrophages was successful.3.Transwell experiment and wound-healing assay showed that CM from M2 macrophages(M2-CM)could promote the migration and invasion of Fadu and Cal-27 cells more efficiently than CM from M0 macrophages(M0-CM)and control conditioned medium(C-CM).Summary:1.THP-1 monocyte can be induced into M2 polarized macrophage by PMA and IL-4 or IL-6.2.In vitro,M2-polarized macrophages resembled TAMs in head and neck squamous cell carcinoma microenvironment.3.M2-like TAMs can promote the migration and invasion of head and neck squamous cell carcinoma.Part Two Study on the effect of DHA on M2-like TAMs and its mechanismObjective: The main purpose of this part is to study the effect of dihydroartemisinin(DHA)on M2 macrophages.It is necessary to observe the effect of M2-CM treated with DHA on HNSCC.To study the mechanism on M2-like TAMs with DHA.Methods:1.MTT assy was used to detect the cytotoxicity of DHA to macrophages.2.From the morphological feature analysis,the differences between M2 macrophages with DHA and M2 macrophages without DHA.3.The ELISA was used to detect the effect of DHA on M2 macrophages.4.Flow cytometry was used to detect the expression of M2 macrophage marker CD163 after DHA treatment.qRT-PCR was used to detect the m RNA expression of M2 macrophages under the effect of DHA.5.The influence of CM from M2 macrophage with or without DHA treatment on HNSCC metastasis was observed by wound-healing assay and transwell assay.6.The influence of CM from M2 macrophage with or without DHA treatment on HNSCC angiogenesis was observed by Tube formation assay.7.The protein expression levels of STAT3 and p-STAT3 in different macrophages with or without DHA treatment were determined by western blot.8.After lentivirus transfection,the number of M2-polarized macrophages and the expression level of p-stat3 were determined by flow cytometry and western blot,respectively.Results:1.According to the results of MTT and ELISA,the survival rate of macrophages did not change significantly with the concentration of DHA.Meanwhile,DMSO had no effect on the survival rate of macrophages.2.Through morphological observation of M2 macrophages,it was found that the pseudopodia of cells significantly shortened,and the shape changed partly from polygon or spindle to oval.3.According to ELISA,DHA could reduce the expression level of IL-10 in M2-CM.However,DMSO had no effect on the expression level of IL-10 in the culture medium.4.Results from wound-healing,transwell and tube formation assays showed that M2DHA-CM could improve the scratch spacing of tumor cells,and reduce the cells on the transwell compartment membrane and the numbers of capillary-like tube formation.5.Results from Western blot and flow cytometry assays showed that the expression of p-STAT3 protein was positively correlated with the numbers of M2 macrophages,and DHA inhibited the expression of p-STAT3,thereby reducing the number of M2-polarized macrophages.Summary:1.DHA reduced the numbers of M2-polarized macrophages.2.DHA inhibited the effect of M2-like TAMs on the invasion and migration of HNSCC.3.DHA inhibited the effect of M2-like TAMs on angiogenesis of HNSCC.4.DHA inhibited the polarization of M2 macrophages by preventing the phosphorylation of STAT3.Part Three Effects of DHA on M2-like TAMs and its association with growth of HNSCC in xenograft animal tumor modelsObjective: To verify,in vivo,that M2-like TAMs can promote tumors growth and angiogenesis,and DHA could inhibit the growth of HNSCC through its effect on M2-like TAMs.Methods:1.Xenograft animal tumor model of NHSCC was established in nude mice.Animals were divided into two groups which were control groups and Fadu +M2 groups.Changes in tumor volume,tumor weight and survival were compared between the two groups.2.Immunohistochemistry was used to compare the number of TAMs,tumor cells and capillaries in the two groups of nude mice.3.Xenograft animal tumor model of NHSCC was established in nude mice.Animals injected with Fadu and M2 macrophages cells were divided into two groups which were treated with or without DHA.Changes in tumor volume,tumor weight and survival were compared between the two groups.4.Immunohistochemistry was used to compare the number of TAMs,tumor cells and capillaries in the two groups of nude mice.qRT-PCR were used to detect the m RNA expression levels of VEGFA and MMP9 in xenograft tumors.Results:1.The tumor volume,tumor weight and mortality rate were all significantly greater in mice injected with M2 macrophages than those in control group,suggesting that M2-like TAMs can promote tumor progression.2.Immunohistochemistry showed that the number of TAMs is positively correlated with the proliferation of tumor cells and the growth of capillaries.3.Compared with the Fadu+ M2 group,the tumor volume and tumor weight were decreased in the DHA treatment group,with the improvement of the survival rate.4.Immunohistochemistry showed that the number of M2-like TAMs in DHA group decreased significantly,and the expression levels of Ki-67 and CD31 also decreased.Results of qRT-PCR showed that the m RNA expression levels of MMP9 and VEGFA in the tumour were downregulated.Summary:1.In the xenograft animal tumor models,M2-like TAMs can promote cells growth and angiogenesis of NHSCC.2.DHA reduced the numbers of M2-like TAMs in the xenograft animal tumors.3.In tumour microenvironment,tumor growth and angiogenesis through inhibiting M2-like TAMs.Conclusion:1.In vitro,TAMs are much alike M2-like polarized macrophages in HNSCC.THP-1 monocyte can be induced into M2 polarized macrophage by PMA and IL-4 / IL-6.2.M2-like TAMs can promote the growth,migration and angiogenesis of head and neck squamous cell carcinoma.3.DHA can inhibit the polarization of M2-like TAMs by blocking the phosphorylation of STAT3 protein,and reduce the number of M2-like TAMs.4.DHA can reduce the number of M2-like TAMs,and inhibited the growth of HNSCC cells and angiogenesis in xenograft tumor tissues.Besides inhibiting proliferation and promoting apoptosis of HNSCC cells,which has been proved by the previous studies,our experiment further demonstrated that DHA could inhibit the progression and migration of HNSCC by regulating the immune cells in the tumor microenvironment.