The Effects Of CSF1 In The Interactions Between Colon Cancer Cells And Tumor-infiltrated Macrophages And The Effect Of Leptin On Human Extravillous Trophoblast Invasion And Its Mechanisms

BackgroundThe association between tissue microenvironment and disease is now a hot field in biological science. The development of innovative research field, such as oncobiology, oncoimmunology and stem cells, is dependent on the elucidation of tissue microenvironment and its regulatory mechanisms. The biological behaviors of cells are dependent on the microenvironment around. Tissue microenvironment is composed of various types of cells, stroma and interstitial fluid. A complex interaction exists between different compositions of the microenvironment, and reaches a dynamic balance. The interruption of this balance would lead to the change of cellular biological behaviors, and is involved in various physiological and pathological processes. One good example is the decidua tissue in early phase of pregnancy. Numerous immunocytes infiltration and dramatic concentration change of cytokines and chemokines are important characteristics of the decidua microenvironment, and play essential part in the maintenance of pregnancy. Another example is the tumor microenvironment, which is also characterized by unique immune status, cytokines and chemokines expression. Tumor microenvironment involves in multiple behaviors of cancer cells, including invasion, metastasis and immune escape. Therefore, demonstration of the composition and regulatory mechanisms in the tissue microenvironment would play important part in the development of new therapeutic strategies.Macrophage colony stimulating factor (CSF1)is a pluripotential cytokine which has close relationship with monocytes and macrophages. It is the major lineage modulator for the differentiation, polarization and chemotaxis of macrophages. The over-expression of CSF1 and its receptor, CSFIR, has been found in various types of cancer, including breast cancer, hepatic cancer and glioma, and showed positive association with numbers of infiltrated macrophages. The serum CSF1 concentration of colorectal patients was significantly higher than healthy controls by approximately two times, and showed association with lymph node metastasis and levels of tumor markers, including CEA and CA19-9. However, the regulatory mechanisms on the formation of high-CSF1 microenvironment in colorectal cancer, and also the roles of CSF1 in cancer development are still not fully understood. The anti-tumor immunity plays an important role in the development of colorectal cancer. The number and types of immune cells infiltrated in cancer tissue are associated with the recurrence rate of colorectal cancer patients and their prognosis. Macrophages are the most abundant immune cell population in solid tumor, representing up to 50% of all tumor mass. Under the stimulation of exogenous signals, macrophages could polarize into two different subpopulations which show anti-tumor or tumor-promoting activities. Therefore, we speculate that macrophages infiltrated in the colorectal cancer are involved in the establishment of CSF1 over-expressed microenvironment, and also are involved in CSF1-regulated cancer development. Based on the above speculations, we analyzed the expression of CSF1 in colorectal cancer patients and its regulatory mechanisms. We also explored the role of cancer-derived CSF1 in macrophages recruitment and secretion. Moreover, we found that fucoidan affected the autocrine effects of CSF1 in macrophages. This finding would provide new supplementary strategies for CSF1-based anti-tumor therapies.Decidua tissue in early phase of pregnancy shows similar biological characteristics of cancer microenvironment with multiple types of immunocytes and high invasive activity. The extravillous trophoblast (EVT) cells show similar invasive activity with cancer cells, but its invasion is tightly controlled in a spatial and temporal manner. The change of hormones concentration is a fundamental feature of decidua microenvironment, and leptin is one of the important pregnancy-related factors. Leptin concentration is significantly elevated in the early phase of pregnancy, and is an essential factor for blastocyte implantation and pregnancy maintenance. Intense leptin receptor expression is found in EVT cells, suggesting that leptin might affect their biological behaviors. In this study, we explored the effects of leptin on EVT cells invasion and its relevant mechanisms.Two classic physiological and pathological tissue microenvironment, colon cancer tissue and decidua tissue of early pregnancy, were analyzed in this study. We explored the regulatory mechanisms of high CSF1 microenvironment in colon cancer tissue, and the roles of CSF1 and leptin on multiple biological behaviors of macrophages and trophoblasts, including invasion, migration and differentiation. Our findings provide valuable evidences for understanding the composition of tissue microenvironment and their interactions.Section â…  The roles and mechanisms of colon cancer-derived macrophage colony stimulating factor in the interactions between colon cancer cells and macrophagesPurposeTumor-infiltrated macrophages were potential targets of the immune therapy for patients with colon cancer. Macrophage colony stimulating factor (CSF1) is an important regulator in the recruitment and functions of macrophages. However, its expression in primary colon cancer tissue and its roles in cancer development are largely unknown. In this study, we explored the mechanisms by which an over-expressed CSF1 colon cancer microenvironment was formed and the effects of colon cancer-derived CSF1 on the interactions between colon cancer cells and tumor-infiltrating macrophages. Moreover, we explored the regulation of fucoidan on CSF1 secretion in macrophages and the effects of CSF1 autocrine process on cytokines production and migration of THP-1-derived macrophages.Methods1. Immunohistochemistry staining was used to analyze the expression of CSF1 and CD68 (a marker of macrophages) and their association with prognosis and clinicopathological parameters of colon cancer patients.2. An indirect co-culture system between colon cancer cells and macrophages was formed using transwell chamber. qRT-PCR, ELISA and immunofluorescence were used to analyze the effect of co-cultivation with macrophages on CSF1 expression of colon cancer cells.3. qRT-PCR, ELISA and Western blotting were used to determine the effects of macrophage-derived IL-8 on CSF1 expression of colon cancer cells and its relevant signaling pathway.4. Transwell assay and qRT-PCR was performed to analyze the effects of colon cancer-derived CSF1 on recruitment and secretion of macrophages.5. A CSF1-overexpressed colon cancer xenograft model was established in nude mice to analyze the effects of cancer-derived CSF1 on development of colon cancer and infiltration of macrophages.6. The effects of fucoidan on migration and cytokines production of THP-1-derived macrophages were analyzed.7. qRT-PCR and ELISA were used to analyze the effects of fucoidan on CSF1 secretion of macrophages. The effects of CSF1 on migration and cytokines production of macrophages were also analyzed.Results1. The expression of CSF1 was significantly higher in colon cancer tissue compared with peritumoral tissue or normal tissue. Significantly higher numbers of CD68+cells were observed in both intratumoral and peritumoral stroma compared with normal colon mucosa. High expression of CSF1 and CD68 was associated with TNM stage, and was an independent prognostic factor.2. Indirect co-cultivation with macrophages promoted CSF1 expression in colon cancer cells.3. Macrophages produced high level of IL-8 compared with colon cancer cells. Macrophage-derived IL-8 promoted CSF1 expression and activation of PKC signaling pathway in colon cancer cells. The activation of PKC pathway enhanced CSF1 expression.4. Colon cancer cell-derived CSF1 promoted macrophages recruitment to colon cancer and regulated their secretion profile.5. CSF1 over-expression in colon xenograft tumor promoted macrophages infiltration and partially suppresses tumor growth.6. Stimulation with fucoidan during the differentiation process of THP-1-derievd macrophages altered their morphology, migration activity and cytokines production.7. Fucoidan inhibited CSF1 production in macrophages. Treatment with exogenous CSF1 reduced the inhibitory effects of fucoidan on migration and cytokines production of macrophages.ConclusionOur study analyzed the expression pattern of CSF1 in colon cancer tissue and the roles of over-expressed CSF1 microenvironment in the development of colon cancer. In addition, we demonstrated for the first time that CSF1 played crucial roles in the interactions between colon cancer cells and macrophages, and preliminarily explored the relevant mechanisms. The above results suggested that strategies targeting CSF1 might synchronously affect two primary components in tumor tissue:cancer cells and tumor-infiltrated macrophages. This study might provide innovative strategies for the treatment of colon cancer. Our results also suggested that exogenous intervention would modulate CSF1 expression in microenvironment, and further modulate the immune status. CSF1-based immunotherapy might play important roles in the treatment of various diseases, including cancer.Section II The effect of leptin on human extravillous trophoblast invasion and its mechanismsPurposeThe invasion of human extravillous trophoblast (EVT) cells is tightly controlled in a spatial and temporal manner while the regulatory mechanisms remain unclear. Leptin is a pregnancy-related hormone that is significantly elevated during the first trimester of pregnancy. The overexpression of leptin plays important roles in blastocyst implantation and pregnancy maintenance. However, knowledge about the effects of leptin on EVT cells invasion is limited. In this study, we investigated the possible role of leptin in the promotion of EVT cells invasion and the relevant mechanisms.Methods1. Transwell assay was performed to analyze the effect of leptin on invasion of human EVT cell line.2. qRT-PCR and Western blotting were performed to analyze the role of leptin in the MMP14 expression of human EVT cell lines. MMP14 siRNA was transfected in to human EVT cell lines and transwell assay was performed to investigate the role of MMP14 in the process of leptin-regulated EVT cells invasion3. qRT-PCR and Western blotting were performed to analyze the effects of leptin on the activation of Notch 1 signaling in human EVT cell lines.4. Notch1 siRNA was transfected in to human EVT cell lines. Western blotting was performed to investigate the effect of Notch1 signaling on MMP14 expression of EVT cells. Transwell assay was performed to analyze the role Notchl in invasion of EVT cells.5. Western blotting was performed to analyze the effects of leptin and Notchl signaling on activation of PI3K signaling pathway, and the role of PI3K pathway on MMP14 expression in EVT cells.Results1. Leptin promoted EVT cells invasion and MMP14 expression. The promotion of MMP14 expression was involved in leptin-mediated EVT cells invasion.2. Leptin activated Notchl signaling pathway in EVT cells. The activation of Notch1 pathway was involved in leptin-mediated MMP14 expression and invasion of EVT cells.3. Notchl regulated MMP14 expression by activating PI3K pathwayConclusionOur study revealed that leptin promoted invasion of human EVT cells through Notch1-PI3K-MMP14 axis. Our results would bring a better understanding about the physiologic role of leptin in the pregnancy microenvironment, and also provide evidences about its potential effects in the treatment of various invasive-related diseases, such as eclampsia and cancer.ConclusionUnique microenvironment exists in cancer and decidua tissue, showing great similarities in various biological characteristics, such as immune status and cell motility. Two classic physiological and pathological tissue microenvironment, colon cancer tissue and decidua tissue of early pregnancy, were analyzed in this study. The regulatory mechanisms of high CSF1 microenvironment in colon cancer tissue, and the roles of CSF1 and leptin on multiple biological behaviors of macrophages and trophoblasts were explored. Demonstrating the interactions between various compositions and compared them between different tissue microenvironment, would provide valuable evidences for understanding the mechanisms of various physiological and pathological processes, and might shed light on the development of new therapeutic strategies.