Study On The Significance Of M2Macrophages In Infantile Haemangiomas And Squamous Cell Carcinomas

Macrophages are white blood cells which differentiated from monocytes. As monocytes migrate from peripheral blood into the tissue, they differentiate and are referred to as resident macrophages. Macrophages can be phenotypically polarized by the different microenvironment, showing different phenotype and function. Cell polarity is a fundamental feature of various cell type, it is essential for the function of numerous cells. Cell polarity refers to the asymmetry in cell shape which results from asymmetrical signaling molecules distribution within a cell to serve a directional cell division. A highly polarized cell is characterized by the elongated shape and many intracellular signaling molecules including cytoskeletal proteins and lipids showing asymmetric distribution and cyto-architecture reconstruction. The extravasation and migration of macrophages depend on cell polarity, which determines the macrophage chemotaxis. Macrophages can be polarized by microenvironment to mount specific M1or M2functional types. During the development of tumor, the macrophage polarity could shift from M1to M2functional program, meanwhile, their functions accordingly change from inhibiting tumor growth to promoting tumor angiogenesis, growth and metastasis.Based on the above considerations, we proposed that different phenotype macrophages possibly played roles in the pathogenesis of infantile haemangioma (IH), one of the most common benign tumors of infancy, and squamous cell carcinoma (SCC). We investigated the expression status and functional significance of M2macrophages in IH tissue and explored the probable mechanism. Meanwhile, the interaction between SCCs and mononuclear macrophages was also evaluated. Our studies could provide evidence on searching novel therapeutic targets of IH and SCC. This study was divided into two parts as follows:Part Ⅰ Expression of M2-polarized macrophages in infantile haemangioma and the correlation with proliferation and angiogenesisObjective:To data, the pathogenesis of infantile haemangiomas (IHs) is still far from clear despite they are common vascular tumors distinctive for their perinatal presentation, rapid growth during the first year of life and subsequent slow involution. This study was designed to determine the role of M2-polarized macrophages in IHs.Methods:M2-polarized macrophages were initially identified in20specimens of IHs by both immunochemistry and immunofluorescence for CD68and CD163. Then the immunopositive M2-polarized macrophages in different phases of IHs were quantified, and further analyzed for their correlations with the expression levels of Ki67, vascular endothelial growth factor (VEGF), macrophage colony-stimulating factor (M-CSF).Results:The data showed that the infiltrating macrophages in proliferative IHs were predominantly CD68+/CD163+, thus of the M2-polarized phenotype, whereas the density of these cells was significantly decreased in the involuting IHs. Moreover, we also found that the high density of M2-polarized macrophages in proliferative IHs was closely correlated with the over-expression of M-CSF, one of the cytokines considered to induce macrophages to polarize toward an M2phenotype. Furthermore, the infiltrating M2-polarized macrophages probably contributed to the proliferation and angiogenesis of haemangioma endothelial cells, as evidenced by their close correlations with the immunoreactivities of Ki67and VEGF.Conclusions:Taken together, our findings indicate that the infiltrating M2-polarized macrophages may contribute to the progression of IHs through its function of promoting the angiogenic process.Part II Squamous cell carcinoma induce phenotypic and genomic changes in macrophagesObjective: Tumor associated inflammation is a major driving force in the progression of squamous cell carcinoma (SCC), the most common caner in head and neck. The present study is aim to investigate that whether SCC cells could affected the inflammatory environment by interaction with monocytes.Methods:Monocytes (THP1) stained with PKH26were exposed to SCC cells (Cal27) and adenoid cystic carcinoma (ACC) cells (SACC83), followed by FACS analyses and detection of monocyte-to-marcophage differentiation, polarization, attachment and gene expression by real time PCR. Changes induced by co-culture were compared with that observed under classical differentiation and polarization conditions. Human squamous cell carcinoma in nude mice model was used to analyze in vivo microenvironment of the monocyte-to-macrophage differentiation and polarization. Immunohistochemical staining was performed to evaluate the expression level of CD68in SCC and ACC(as a control for non-inflammatory cancer).Results:In vitro studies revealed that THP1cells co-cultured with Cal27had more significantly altered gene expression with up-regulation of both M1and M2 macrophage markers (IL-1β, IL-6, TNF-a, and MMP-9) than that co-cultured with SACC83. Compared to SACC83, Cal27induced more THP1cells to attach and differentiate into macrophages. THP1that co-cultured with Cal27expressed higher levels of CXCR4and CCR-2than with SACC83, moreover, more extensive chemokine SDF-1α and CCL-2was detected in the co-cultured Cal27rather than SACC83cells. The co-culture system containing AMD3100and RS504393significantly suppressed THP1cells attachment and differentiation. The studies in vivo showed THP1cells may contribute to the progression of SCC and SCC cells could induce THP1cells attachment and differentiation.The immunohistochemical results revealed that much more expression of CD68+macropahge in SCC tissues than that in ACC.Conclusions: Compared to non-inflammatory cancer, SCC, as an inflammatory tumor, showed enhanced abilities in recruiting and inducing differentiation of monocytes, which indicated that inflammation in such tumor was the nature of itself rather than induced by external factors.