Effects Of Tumor-associated Macrophages On The Biological Behaviours Of Cutaneous Malignant Melanoma And The Fundamental Study On Mechanism

Backgrand and ObjectiveCutaneous malignant melanoma is one of the most fatal skin tumors which withworse prognosis, the morbidity and mortality of CMM are increasing year by year. Thecases of melanoma have been confirmed by our hospital were increaseing nearly threeyears. The follow-up data showed that the patients not only with poor prognosis, butalso with shorter survival period compared with the other cutaneous tumors,which isseriously threaten patient’s life.Skin melanoma cell have the feature of early invasionand metastasis, and the feature is closely associated with poor prognosis ofoutcome.There are many factors and links involvod in the process of invasion andmetastasis of tumor, including that immunocytes, cell signal transduction pathways,various cytokines in tumor microenvironment.Tumor-associated macrophages are themacrophages that infiltrating in the tumor stroma, which are the largest number ofinflammatory cells in most solid tumors.TAMs have been found to play an importantrole in tumorigenesis, angiogenesis, matrix remodeling, and metastasis.However therole and mechanism of TAMs still haven’t a unified definition in diverse cancers.Researches show that TAMs have a dual function of promoting or inhibiting tumor cellgrowth. It has greatly influenced the function of TAMs in the following three aspects:Quantity, Distribution and Phenotype. Phenotype could be the most important factoramong these.Classic or M1macrophages that are activated by Interferon-γ or lipopolysaccharide, which are supposed to be antitumorigenic cell. In contrast,alternative or M2macrophages that are activated by interleukin-4（IL-4）, interleukin-13（IL-13） or immune complexes, which are supposed to be protumorigenic cell. However,the effect of TAMs in the proliferation, invasion and metastasis of malignant melanomacells is still not clear.But there is no correlated study whether there are some relatedproteins took part in the process. In recent years, as for the effect of tumor cell signalingpathways in the development of tumor has been more acquainted, it is found that Signaltransducer and activator of transcription （STAT）-3is an important member of STATsfamily and which is the focus of many types of carcinogenic way, regulates theexpression of related target genes involved in Vascular endothelial growth factor（VEGF）. Researchers show that VEGF is one of the most important angiogenesis factor,could be directly regulated by STAT3signaling pathway.VEGF can promote thegeneration of tumor angiogenesis, which was one of the important factors that lead toinvasion and metastasis of tumor cells. TAMs might play an important role in thedevelopment and progression of cutaneous malignant melanoma.Whether TAMs mightaffect the biological behaviour of malignant melanoma by regulation the expression ofVEGF and STAT3protien were still evolving.Human monocytic U937cells can be induced to differentiate to macrophages whentreated with phorbol12-myristate13-acetate （PMA） in vitro.The expression of CD68was evaluated by flow cytometry.LPS or IL-4was added into the cells after treatmentwith PMA for48hours. Macrophages were induced into M1phenotype with LPS, M2phenotype with IL-4respectively. After cultured for48hours, the culture supernatantwere collected. The concentration of IL-12p70, IL-10was determined using an ELISAkit. Human malignant melanoma cel11ine A375was co-cultured with differentlyactivated macrophages in the transwel1. To study the effect of differently activatedmacrophages on the human malignant melanoma cell, we took MTT method andTranswell test. Immunohistochemistry was took to investigate the infiltration and significance of tumor associated macrophages in cutaneous malignant melanoma skintissue as well as the relationship with STAT3, VEGF protien expression andclinicopathologic feature in cutaneous malignant melanoma tissue. Further identifies thefunction of TAMs in the invasion and metastasis of cutaneous malignant melanoma, andtried to find out the possible mechanism of TAMs in cutaneous malignant melanoma.Methods1The induced and phenotype identify of macrophagePMA was used to induced U937cells to macrophages. The expression of CD68was identified as the specificity surface antigen of macrophage which was evaluated byflow cytometry. Macrophages were induced into M1phenotype with LPS, M2phenotype with IL-4respectively. The culture supernatant were collected, IL-12p70andIL-10level were detected by ELISA to indentify the phenotype of macrophages.2Effects of TAMs on the biological behaviours of human malignant melanoma cel11ine A375The human malignant melanoma cel1s were seeded in Transwell lower chambers（0.4μm pores） culture24hours. Macrophages were seeded in Transwell upper chambers（0.4μm pores）. The human malignant melanoma cel11ine A375were co-cultured withdifferent active macrophags without direct cell-to-cell contact in a24-well transwellplate. The propagation of A375in each group was detected by a way of using MTTevery24hours for3days. A375cells those co-cultured with the different activemacrophags were resuspened with serum free medium and seeded in Transwell lowerrooms（0.8μm pores） culture24hours. The invasion and migration capability of A375ineach group were measured by the numbers of the tumor cells that had migrated throughMatrigel-coated or without Matrigel-coated membranes respectively. With these experiments we can observe the role of different active macrophage in A375cellproliferation, invasion and migration ability.3The infiltration and significance of Tumor associated macrophages in cutaneousmalignant melanoma skin tissue and the relationship with STAT3and VEGFprotien expressionWe use Immunohistochemistry to investigate the infiltration and significance ofTumor associated macrophages as well as the relationship with STAT3, VEGF protienexpression, and analysis the clinicopathologic feature in cutaneous malignant melanomatissue.Result1The induced and phenotype identify of macrophages1) Macrophages were induced by PMA, and the expression of CD68was evaluated byflow cytometry. The overexpression of CD68was evaluated.2) The macrophages which were stimulated by LPS or IL-4respectively showedIL-12p70^highIL-10^lowand IL-12p70^lowIL-10^high, indicated that we had succeeded ininducing M1macrophages and M2macrophages.3) The IL-12p70and IL-10secretion lever of unstimulated macrophages that wereinduced by PMA separately was detected closely to M2macrophages.2Effects of TAMs on the biological behaviours of human malignant melanomacel11ine A3751) M2macrophages which were induced with IL-4promoted A375cell proliferation,invasion and migration, while M1macrophages had the opposite effects.2) Unstimulated macrophages that were induced by PMA were more inclined to M2cell phenotype. Unstimulated macrophages could be discovered may enhance A375cell proliferation, invasion and migration. 3The infiltration and significance of Tumor associated macrophages incutaneous malignant melanoma skin tissue and the relationship with STAT3and VEGF protien expression1) The infiltration of TAMs was closely correlated with the expression of STAT3andVEGF protein in cutaneous malignant melanoma, they were also correlated withClark grading and lymphatic metastasis in cutaneous malignant melanoma.2) TAMs might play an important role in the invasion and metastasis of malignantmelanoma by regulation the expression of VEGF and STAT3protien, which mightbe a new potential target for therapy of it.ConclutionMacrophages could be differently activated successfully into M1or M2phenotypein vitro which could bring out the possibility of relevant researches. M2macrophagemay enhance A375cell proliferation, invasion and migration, while M1macrophagehad the opposite effects. The infiltration of TAMs was closely correlated with theexpression of STAT3and VEGF protein in cutaneous malignant melanoma, they werealso correlated with Clark grading and lymphatic metastasis in cutaneous malignantmelanoma.TAMs might play an important role in the invasion and metastasis ofmalignant melanoma by regulation the expression of VEGF and STAT3protien, whichmight be a new potential target for therapy of it. We speculated that TAMs may beexisted as M2cell phenotype and play an supporting role in cutaneous malignantmelanoma.TAMs might play an important role in the invasion and migration ofmalignant melanoma by regulation the expression of VEGF and STAT3protien.