| BackgroundColorectal cancer is the third malignant cancer worldwide. There have been1.2million newly diagnosed cases every year, and estimating more than600thousand patients died from colorectal cancer. Coloretal carcinoma is the second malignancy and the fourth leading death of cancereous disease in China. Recent years, although the therapeutic methods of colorectal cancer has been greatly improved along with new technologies and chemotherapy drugs, the survival rate of patients has not significantly improved after surgery and postoperative chemotherapy. Recurrence and metastasis is the main cause of colorectal cancer, because more than half of colorectal cancer patients may be associated with micro-metastasis even before they have been underwent radical surgery. Therefore, it is urgent to find out key molecules to surpport evidences for interpreting mechanisms of colorectal cancer metastasis, so that the colorectal cancer patients would be curative by establishing a possible medical intervention to target specific molecules.Tumor metastasis is a complex, multi-step process of the evolution. To complete the process of metastasis, tumor cells must break away from the primary lesion, invade the extracellular matrix (ECM) and the basement membrane (BM), activated cells secrete a variety of degradation enzymes, pass through blood vessel walls and penetrate vascular extravasation into secondary parts, and then tissue clone formed. The process of tumor cell invasion is bound up with endothelial cells. Cancer cells produce collagen enzyme and tumor growth factors (TGF) which can cause endothelial cell activation directly or indirectly. Collagenase zymogen and platelet activating factor (PAF) secreted by cancer cells make cancer cells form intravascular tumor thrombus, and then, the cancer cells produce protease, the protease damage extracellular matrix and basement membrane formed by vascular endothelial cells, whose damages lead to invasion and metastasis. The cancer embolus attached on the vascular endothelium is one of the key steps in blood circulation metastasis, platelets release lectins make tumor cells and endothelial cells adhesion, vascular endothelial cell shrinkage, a gap between cells turn out, endothelial permeability increased, and then tumor cells go through blood vessel walls to enter the organization to form the metastases.Metastatic tumor cell is susceptible to seed its particular organs (target organs), and the particular organs are not the first organ, this priority keep in accordance with the ability of endothelial cells adhesion to target organs. In the process of tumor cells and endothelial cell adhesion, adhesive molecules express in a particular way. As the setup of a microvascular culture system, we found that organ specificity transfer tendency of tumor cells have different affinity with exclusive endothelial cells, they are easier to adhere to the target organs endothelia. Pauh found that metastatic organs transfer option was mediated by cancer cells and endothelial cells adhesion molecules in the blood, these factors regulated by specific organ microvascular phenotypic part, and the function is mediated both by the complexity of endothelial system and extracellular matrix (specificity of matrix molecules and growth factors, for example), they play the role through (large blood vessel) endothelial cell in nonspecific organs.When the tumor diamter is larger than2mm, it need generate new blood vessels to provide proliferation so that the tumor could obtain sufficient oxygen and nutrients, EC proliferation is also mediated by a variety of angiogenic factors. Physiologic angiogenesis are strictly spatio-temporally controlled in the specific tissues and organs, such as embrogenesis, organgenesis and wound healing, while persistent angiogenesis is the features of pathological changes, in particularly in tumor growth. In fact, angiogenesis is not only essential to tumor growth, but also a inducible factor for distant metastasis. Tumor angiogenesis is a consecutive process, tumor cells release a variety of angiogenic factors. Bioactive factor promotes morphogenesis of EC, including generation of organelles, EC proliferation and enlargement, forming pseudopodia. EC and tumor cells both release protease to hydrolyze the basement membrane of capillaries and extracellular matrix, which cause the extracellular matrix remodeling. A quarter EC of post capillary venules emmigrate to form capillary buds, one half of EC attribute to proliferate, and3/4EC give rise to differentiate and form tumor microvasculature.Angiogenesis factor is one kinds of growth factors or cytokines subclasses of skin material, these material directly or indirectly ACTS on the vascular endothelial cells, causing vascular expansion, deformation, capillary endothelial cells sprout to tumor tissue, and use a protein factor produced by the endothelial cells to closed newborn capillaries open make it form a complete tubular structure. In the process of tumor growth, metastatic of new blood vessel formation, but angiogenesis inhibitors can inhibit tumor metastasis.OI Reilly found that serum and urine from the tumor rat inhibited the endothelial cell proliferation, and restricted new blood vessels, can also reduce Le wis tumor metastasis of lung cancer. Research has shown that primary tumor metastasis can inhibit neovascularization to inhibit angiogenesis at least.Based on the research status, the experiment aimed at an important link of tumor blood vessels--the effect of the tumor cells interact with endothelial of target organs. We choose endothelial cells (HHSEC) as the main research object who is the metastasis target organ of colorectal cancer. We used SCP17, a subline of SW480with high metastasis of colorectal cancer cell lines, SCP40, a subline of S W480with low metastasis of colorectal cancer cell lines, and colorectal cancer cell lines HCT116and LS174T, to observe the interaction effect about colorectal cancer cells interactive with endothelial cell of metasis target organs. We used PC3prostate cancer (metasis target organs for bone), breast HCC1937cell lung/liver target organs (transfer) and endometrial cancer RL95-2cells (transfer target organs for lung) as tumor cell control test; and used the endothelial cells of umbilical vein endothelial cells (HUVEC) as EC control test. We established models of chicken embryos Matrigel allantois membrane angiogenesis, three-dimensional cultivation, tumor cell invating amniotic basement membrane etc. By using HE staining, immunohistochemistry and immunofluorescence, we could observe the effect that colorectal cancer cells interact with HHSEC, HUVEC on angiogenesis and tumor infiltration.Method1. Cell cultureColon cancer cell lines include of SCP17, SCP40, HCT116, LS174T, human breast cancer cell lines HCC1937, people prostate cancer cells PC3, hepatic sinus endothelial cell lines HHSEC using RPMI1640culture containing10%fetal bovine serum, human endometrial cancer cells HUVEC and Human Endometrial Cancer-1-B RL95-2culture with DMEM containing10%F12, at37℃and5%CO2, saturated humidity under the condition of subculture, cell growth in good condition when used in the experiment.2. Set up chicken embryos villi allantois membrane angiogenesis modelSelect7day age live chicken embryo, vaccination SCP174×106colorectal tumor cells with2x106targeted HHSEC endothelial cells,2×106nontargeted endothelial cells HUVEC cells in chicken embryo villus allantois membrane, observe the situation the chicken embryo villus allantois membrane angiogenesis.3. Identificate the influence of forming VM when Colorectal cancer cells and targeted endothelial cells co-culture by Matrix three-dimensional cultivation1) Conclusion Lentiviral vector carrying Green fluorescent protein has been successfully constructed and maintains high expression in endotheliocyte.2) Observe the asculogenic mimicry of tumor cell interactions of endotheliocyte mimicry by Matrix3d co-culture.4. Build tumor cell invasion and metastasis model, observing the influence of targeting endothelial cells of colorectal HHSEC.1) Tumor cells and endothelial cell of target organs co-culture dynamic visual model.Tumor cells SCP17, SCP40, HCT116, LS174T, HCC1937, PC3, RL95-2respective with targeting endothelial cells HHSEC, the non-targeting endothelial cells HUVEC stratificated in a concentration of80%Matrigel matrix cultivation, to observe the chemotaxis of tumor cells and endothelial cells and the changes of the ability to migrate.2) Set up model of tumor cells infiltrating into amniotic basement membrane.(a) Taking sterile placenta of cesarean section to preparate amniotic basement membrane, simulating the biological basement membrane in the process of invasion and metastasis of tumor.(b) Tumor cells SCP17, SCP40, HCT116, LS174T, HCC1937, PC3, RL95-2respectively co-culture with target organ endothelial cells HHEC and non-target organ endothelial cell HUVEC in amniotic basement membrane,4%formalin fixed4-12h, HE staining, according to filtrating depth of the tumor cells, and then assessment of the change of infiltrating depththe of the basement membrane.Result 1. Set up chicken embryos villi allantois membrane angiogenesis modelThree days later when the seven days of age chicken embryo allantois membrane plant the number of4x106scp17tumor cell suspension, the number of angiogenesis is significantly increased than one that didn’t plant tumor cell suspension. The number of4x106with SCP17respectively co-culture with the number of4x106targeting endothelial cells and the nontargeting endothelial cells HUVEC, the number of blood vessels increased,but the targeting endothelial cells HHSEC group increased even more. Chicken embryo allantois membrane planted targeted tumor cells and the nontargeted endothelial cells mixed suspensiol3days later accidentally see tumor formation, nucleated Red Blood Cells (NRBC) can be seen in the angiogenesis of tumor cells,and the tumor endothelial cells is formed by chicken embryos endothelial cells.2. Identificate the influence of forming VM when Colorectal cancer cells and targeted endothelial cells co-culture by Matrix three-dimensional cultivation1) Matrigel matrix3d culture of tumor cells or endothelial cells, did not see mimicry angiogenesis.2) Tumor cells and endothelial cells co-cultured in Matrigel matrix3d,VM is ivisible. Colorectal cancer tumor cells co-cultured with hepatic sinus endothelial cells HHSEC in Matrigel matrix3d for7days, blood vessels formation of mimicry is increased more about SCP40than SCP17, HCT116increased more than LS174T,but for the nonliver targeted organ cancer cells,such as HCC1937, RL95-2,there is no obvious VM generation, except the increased VM formation of PC3. Co-culture with the non-targeting endothelial cells HUVEC in Matrigel matrix3d for7days, VM formation of SCP40is less than SCP17, HCT116is less than LS174T, the non-liver targeted organ cancer cells, such as HCC1937, RL95-2, the VM are generated, but for PC3which mimicry angiogenesis is generated reduced when training with HHSEC.3. Build tumor cell invasion and metastasis model, observing the influence of targeting endothelial cells of colorectal HHSEC.1) Dynamic visualization model of tumor cells and endothelial cellsPut tumor cells andendothelial cells into layered Matrigel matrix training for3to5days, it’s visible for tumour cells breaking through the "line" into a tumor cell layer of matrix. But unfortunately the chemokines direction have not be observed,the influence of endothelial cell migration direction and migration ability of tumor cells are not clearly.2) Set up the model of amniotic basement membrane cells infiltrating tumor, according to the different infiltrating invasion ability evaluate the effects on tumor cells infiltrating ability of targeting endothelial cells.(a) The ability of invadding amniotic basement membrane is different with different tumor cells, HCC1937> PC3> SCP40/LS174T> SCP17/HCT116> RL95-2-2.(b) Colorectal cancer tumor cells and targeted endothelial cells co-culture for4-5days, staning by HE, the ability of SCP17infiltration capacity of amniotic basement membrane is stronger than SCP40, so it is LS174T that is stronger than HCT116, the non-liver targeted organ cancer cells, infiltrating ability of HCC1937is weaker,but PC3and RL95-2are enhancement. Co-culture with the nontargeting organ endothelial cells HUVEC for4-5days, the infiltrating ability of SCP40is stonger than SCP17, HCT116is stronger than LS174T too, however, the tumor cells of nonliver targeted organ, as PC3, RL95-2whose infiltration ability enhanced, except HCC1937whose infiltration ability induced.Conlusion1. Target organs endothelial cells can promote the ability of VM formation for low transfer colon cancer cell, promote tumor angiogenesis, and target organs endothelial cells promote the invasion and metastasis of tumor cells.2. Endothelial cells affect the migration ability of tumor cells, under the function of endothelial cells, the migration of tumor cells enfored, the influence of target organs endothelial cells is more obvious than the non-target organs endothelial cells on colorectal cancer cell.3. Endothelial cells enhanced VM infomation during the process of tumor metastasis,both target organs endothelial cells HHSEC and non-target organs endothelial cells HUVEC are actively promoting effect on the occurrence of VM.4. Endothelial cells enhance the invasion ability of the tumor cells, target organs endothelial cell HHSEC has a positive role in promoting invasion and metastasis of colorectal cancer.Innovation of thesis1. Compare the ability of migration and invasion about different tumor cells under the action of target organs endothelial cells and the non-target organ endothelial cell.2. Establish Matrigel matrix3d culture model, observe the process of tumor metastasis VM dynamic,and the target organs endothelial cell how to promote angiogenesis. |
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