| The spread of tumor cells from primary site to lymph nodes is an early and common event in human malignant tumor, and the lymphatic vasculature metastasis is one of the main prognosis factors in a number of malignancies, and lymphatic metastasis is considered to relate closely to patients'poor prognosis. However, the means by which lymph node metastases arise is not fully understood, and many questions remain about their importance in the further spread. Lymphatic metastasis is a successive biological process, including proliferation of tumor cells, separation from primary site, movement, entering into lymphatic vessels, arriving at lymph node, entering into blood circulation system through lymphatic transport system, and achieving distant organ. Tumor environment provides a "premetastatic niche" for tumor cells before above procedure. Tumor cells interact with other cells.such as macrophages, fibroblasts, lymphocytes, blood endothelial cells, pericytes, lymphatic endothelial cells mast cells, extracellular matrix.etc in tumor environment, and enhance the metastatic ability. There are many articles about malignant biological behavior of these cells such as tumor associated macrophages, fibroblasts and various lymphocytes in tumor environment, however, few articles focus on crosstalk between tumor cells and other cells in tumor environment, and rare articles are reported about crosstalk between tumor cells and endothelial cells especially for lymphatic endothelial cells. the current knowledge on effects of lymphatic endothelial cells in lymphatic metastasis is possible responsible for above, however, the limitation of research methods and techniques on lymphatic endothelial cells should also be a reason. Following the progression of research and advance of techniques, and appearance of newer and specific lymphatic endothelial cells markers, the opportunity has arisen on depth research of crosstalk between tumor cells and lymphatic endothelial cells.SEMA4C is a member of the fourth sub-family in semaphorin family, there are few related articles of SEMA4C,which involves in several research fields,howerver.The research fields on SEMA4C include neural axon guidance, proliferation and migration of granule cell precursors,myogenic differentiation, potential mediator of the effects ofβ-catenin signaling on pigmentation and innervations.etc. Recently, SEMA4C is reported to express in neural stem/progenitor cells and in adult neurogenesis induced by cerebral ischemia. Although there are several research fields involved, almost no articles of SEMA4C focus on tumor initiation, development and progression are reported in NCBI. Our laboratory first finds that SEMA4C was over-expressed in the lymphatic endothelial cells in breast cancer; the expression of SEMA4C in lymphatic endothelial cells in breast cancer correlated with lymphatic metastasis and tumor lymphangiogenesis; SEMA4C can affect the locomotivity and tube formation of lymphatic endothelial cells; experiments confirm that SEMA4C promote tumor cells metastasis through tumor associated macrophages integrating into tumor lymphatic vessels and disturbing immune balance in tumor microenvironment. In additional, another research in our laboratory test the expression of SEMA4C in 6 common cancer tissues and find that SEMA4C not only expression in tumor associated lymphatic endothelial cells but also in cancer tissues, and the expression of SEMA4C in cancer tissues is also correlated to lymphatic metastasis;SEMA4C can affect directly the proliferation and invasion capability of tumor cells, and the research explored the related mechanism of SEMA4C on tumor cells primitively(all above data not published).Except for our lab rotary, a domestic researcher reported that SEMA4C was found expressed higher on epithelial ovarian cancer tissue than on border ovarian tumors and normal ovarian tissues, the expression level was correlated to pathological grade and patients'clinical phase; Among the 75 epithelial ovarian cancer cases,the 5-year accumulative survival rate of SEMA4C positive group was lower than that of SEMA4C negative group, the median survival time was also shorter than that of SEMA4C negative group.however,the Expression status of SEMA4C was not the independent prognosis factor of epithelial ovarian cancer. On the basis of prior research of our laboratory, this article mainly research the expression profile of SEMA4C in 30 kinds of different tumor cells and confirm the expression of SEMA4C on tissue array of 20 various tissues; the effect of SEMA4C on cytoskeleton and metastatic capability of tumor cells and the effect of SEMA4C on proliferation and apoptosis of lymphatic endothelial cells; and how SEMA4C promotes tumor cells chemotaxis to lymphatic endothelial cells in tumor environment through changing the cytokine secretion of lymphatic endothelial cells in coordination with affecting chemokine receptors expression on tumors cell synchronously. Verifying the possible mechanism of SEMA4C promoting lymphatic metastasis though mediating crosstalk between tumor cells and lymphatic endothelial cells. Collect Tumor cells in the exponential growth phase and Extract total RNA using Trizol reagent according to manufacturer's protocol, the RNA expression of SEMA4C in 30 kinds of human tumor cell lines were detected by fluorescence quantitative real time PCR (qPCR). Western blot analysis was used to quantify protein levels of SEMA4C in these tumor cells.500 core tissue microarray with 20 most common types of cancer (25 cases/type) and normal controls (5 cases/type) including bladder, bone, breast, cerebrum, colon, fatty tissue, fibrous tissue, head and neck, intestine, kidney, liver, lung, lymph node, mesentery, ovary, pancreas, prostate, retroperitoneum, skin, spleen, stomach, testis, thyroid and uterus was used to test SEMA4C expression profile by immunohistochemistry assay in tissues level. The mouse cornea micropocket lymphangiogenesis assay was done according to a reference article using human SEMA4C recombinant protein, which was expressed in Bac-to-Bac(?) Baculovirus Expression System. The oligonucleotides encoding the human SEMA4C-shRNA or NC-shRNA sequence and a loop sequence separating the complementary domains, were designed and synthesized and inserted into the pGCL-GFP (Shanghai GeneChem). The recombinant virus was packaged using Lentivector Expression Systems (Shanghai GeneChem). The recombinant lentivirus with GPF tag, which encoded human SEMA4C-shRNA or NC-shRNA sequence, were transected into MD-MBA-231 cells and human primary lymphatic endothelial cells. The cytokines IL-8 in conditional medium of lymphatic endothelial were tested by Elisa kit. The capability of cell motility was analyzed by transwell migration assay, Transwell migration assay was performed to compare the cell motility of MDA-MB-231 cells toward SEMA4C-knockout lymphatic endothelial cell condition medium to control lymphatic endothelial cells conditional medium. Laser cofocal microscopy imaging system was used to find the effect of SEMA4C on the cytoskeletons of tumor cells. Results1. SEMA4C mRNA expression was found in most of 30 tumor cells, the expression levels in many cancer cell lines was higher than that of immortalized normal cells such as MCF-10A, BEAS-2B cells.2. Western blot analysis confirms the results of real-time-PCR in protein level. In breast cancer lines, the expression levels of SEMA4C in higher metastatic potential MDA-MB-231 cell and MDA-MB-435s cell was higher than lower metastatic potential MCF-7. In prostate cancer lines, the expression pattern of SEMA4C was similar to breast cancer, the expression level in higher metastatic potential PC-3M-1E8 cell than that of PC-3M-2B4 cell.3.500 core tissue microarray with 20 most common types of cancer (25 cases/type) and normal controls (5 cases/type) was used to test SEMA4C expression profile in tissue level, the results of immunohistochemistry assay show that SEMA4C expression was found in most cancer tissues and normal tissues.Postive rate of SEMA4C expression had statistical significant differerence between cancer tissues and normal tissues in several tissue types, including melanoma, breast cancer, ovary caner and gastric cancer. Interestingly, the expression pattern was different in different tissue types. In melanoma, breast cancer and ovarian cancer, Positive rate of SEMA4C was higher than that of normal, on the contrary, the expression pattern was reversed in gastric cancer, the normal gastric tissues had higher Positive rate. The positive rate of SEMA4C in other tissue types had no difference in statistics between cancer and normal tissues.4. SEMA4C recombinant protein promoted lymphatic endothelial cell tube formation in vitro and lymphangiogenesis in mouse cornea micropocket model in vivo.5. Effects of the lentivirus vector shRNA targeted at SEMA4C were confirmed through western blot analysis. Elisa assay show IL-8 expression level in conditional medium of SEMA4C-knockout lymphatic endothelial cells was lower than control conditional medium. The tumor cell numbers of SEMA4C knockout group were less than control group significantly. Laser cofocal microscopy show that cytoskeletons of SEMA4C-knockout MDA-MB-231 cell rearranged. Dramatic actin network reorganization was perceived, actin polymerizing at cell edge, leading to the formation of evident peripheral ruffles. Abundance of SEMA4C mRNA in most of tumor cell is higher than that of immortalized normal cells. The expression levels were positively correlated with the metastatic capability of tumor cells in breast cancer lines and prostate cancer cells. In tissue level, the expression pattern of SEMA4C was different in different tissue types, melanoma, breast cancer and ovary caner had higher positive rate of SEMA4C than normal tissues of same tissue types.The mouse cornea micropocket lymphangiogenesis assay provided a nice model for research on angiogenesis and lymphangiogenesis in vivo. Human SEMA4C recombinant protein could promote lymphangiogenesis in the avascular cornea in vivo.Lentivirus vector shRNA of SEMA4C could silence expression of SEMA4C in tumor cells and human lymphatic endothelial cells efficiently and stablely. SEMA4C could mediate the cytokine secretion in lymphatic endothelial cells and affect the mobility of tumor cells toward lymphatic endothelial cell in vitro. At the same time SEMA4C expressed in tumor cells made the cytoskeletons of tumor cells be reorganized.In summary, SEMA4C expressed both in lymphatic endothelial cell and tumor cells in tumor environment, which played important roles synergistically, attracted tumor cells moving toward to lymphatic cell actively. SEMA4C promotes tumor lymphatic metastasis though mediating tumor cell-lymphatic endothelium crosstalk in tumor microenvironment... |
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