The Roles Of VEGFR-1 Positive Hemopoietic Progenitor Cells In Metastasis Of Breast Cancer

BACKGROUND & OBJECTIVEBreast cancer(BC) is one of the most common malignancies in the world.The incidence rate of BC in the world is increasing fast and has become the first or the second in women's malignancy.In western developed countries,BC has become the first worldwide leading cause of cancer death in women.Metastasis in BC is one of the main course which affects the therapeutic efficacy and leads to poor prognosis.It is an urgent task to cut down the metastasis programe to increase the survival rate and prolong patients' lives.Tumor metastasis is a complicated process refers to multi-genes,different steps and different stages.Though there are plenty of researches in tumor metastasis,it's not clear that how tumor metastasis arise.It's even difficult to detect the early stage metastasis.VEGFR-1(vascular endothelial growth factor receptor-1) which is also called Flt-1(Fms-like tyrosine kinase) is one member of vascular endothelial growth factor receptors.VEGFR-1 take part in some pathology and physiological functions such as vessel neogenesis,artherosclerosis,inflamm-diseases,tumorigenesis and metastasis by combined with VEGF-A,PIGF(placenta growth factor),VEGF-B through its weak PKC activity.Recent years,some researches advise that VEGFR-1 plays an important role in tumorgenesis and metastasis though its weak PTK activity.Kaplan et al reported that bone marrow-derived hematopoietic progenitor cells that express vascular endothelial growth factor receptor 1 which is known as VEGFR-1~+ hemopoietic precursor cell (VEGFR-1~+HPC) home to tumor-specific pre-metastatic sites and form cellular clusters before the arrival of tumor cells.In one way,VEGFR-1~+HPC can attract tumor cells to home to pre-metastatic sites,in the other,they can prepare proper soil for tumor cells to establish.In later researches the author found that tumor metastasis can be inhibited by specific anti-VEGFR-1 antibody.All these foundings demonstrate that VEGFR-1~+HPC play an important role in tumor metastasis and it will be a therapy target to cure malignant tumor.In our study,we aim to approach the influence of cord blood hemopoietic precursor cell in cell proliferation,adhesion and invasion in breast cancer.And then use the whole-body visualization model of breast cancer with high hepatic metastatic potential in nude mice to study the influence of VEGFR-1~+HPC in breast cancer metastasis in vivo.It will be helpful to understand the mechanism of metastasis of breast cancer and bring out a novel therapeutic strategy to cure malignant tumor.CONTENTThrough transfcction to establish whole-body visualization model of breast cancer with high hepatic metastatic potential in nude mice provide a good animal model for next study of the influence of VEGFR-1~+HPC in breast cancer.Sort CD133~+HPC though magnetic activated cell sorting,then co-culture CD133~+HPC with breast cancer cells to detect the influence of CD133 in cell proliferation,adhesion and invasion in breast cancer.Detect the amount of VEGFR-1~+HPC in nude mice in different stage with breast cancer through flow cytometry and immunohistochemistry to identify the role of VEGFR-1~+HPC in breast cancer metastasis.Detect the amount of MMP-2 and MMP-9 in nude mice in different stage with breast cancer Western blot to explore the mechanism of VEGFR-1~+HPC in promotion of breast cancer metastasis.Detect VEGFR-1 positive hemopoietic progenitor cells in human breast cancer tissues by immunohistochemistry to observe the roles of VEGFR-1 positive hemopoietic progenitor cells in human.METHODS & RESULTS1.Establish whole-body visualization model of breast cancer with high hepatic metastatic potential in nude miceThe pEGFP-N1 plasmid was transfected into human breast carcinoma cell line MDA-MB-231 to establish pEGFP-MDA-MB-231 cell line which expresses EGFP stably.Establish whole-body visualization model of breast cancer with high hepatic metastatic potential in nude mice through serial passage in vivo.The pEGFP-MDA-MB-231 cells were surgically orthotopically implanted as tissue fragments in mammary fat pad of nude mice.Emitted fluorescence was collected and imaged through LT-9MACIMSYSPULS system and images were analyzed with IPP5.0 software.Whole-body optical images visualized real-time primary tumor growth and formation of metastatic lesions.MTT assay was engineered to detect the effect of EGFP on cell proliferation.The evaluation of orthotropic animal model was carried out with traditionally pathological methods.Results show that:Transfected pEGFP-MDA-MB-231 cells stably expressed high-level of enhanced green fluorescent protein(EGFP) and EGFP had no effect on cell proliferation.Visualizing orthotropic animal model developed with surgical orthotopic implantation(SOI) in mammary fat pad was built with 100%survival rate. The metastasis rate in the first generation is rather lower which only 20%.With serial passage in vivo the metastasis rate in the third generation reach up 100%(5/5).The whole-body visualizing orthotopic animal model was successfully validated by traditionally pathological detection.2.Sort CD133~+HPC though magnetic activated cell sorting,then co-culture CD133~+HPC with breast cancer cells to detect the influence of CD133 in cell proliferation,adhesion and invasion in breast cancer in vitroSelect cord blood and sort CD133~+HPC by anti-human CD133 immunomagnetic beads though magnetic activated cell sorting.Culture CD133~+HPC in vitro in low sugar MDEM with 20%fetal bovine serum for 1～2 weeks,then co-culture CD133~+HPC with breast cancer cells.MTT and Transwell assays were engineered to detect effect of CD133~+HPC on cell proliferation,adhesion and invasion.Results show that:Cells expressed CD133 sorted by magnetic activated cell sorting can reach up to 80%.Sorted cells will not induce cell differentiation in 1～2 weeks in vitro and still remain the character of stem cell.MTT assay shows that CD133~+HPC can promote cell proliferation after 24h co-culture,the number of spots were found to be significantly altered(t=2.232,P=0.040).Then plant the co-cultured cells in 96 well cell culture plates which incubated with FN to observe the influence of CD133 in adhesion.MTT assay shows that CD133~+HPC can promote cell adhere after 24h co-culture,the number of spots were found to be significantly altered(t=2.435,P=0.027).Transwell camerula shows that the number of cells migrating though the basal membrane was counted and the result showed that the co-culture cells posses a remarkable increase in invasiveness as compared with cells cultured lonely(F=1028.685,P=0.000).All these findings show that CD133~+HPC can promote proliferation,adhesion and invasion of tumor cells in vitro.3.Detect the amount of VEGFR-1~+ HPC in liver tissues of nude mice before and after breast cancer inoculation by flow cytometry and immunohistochemistryExecute nude mice before and after breast cancer inoculation by certebrae colli dislocation,take out of the liver and made it into unicell suspension to detect the amount of VEGFR-1~+HPC by FCM.The surplus liver was made into paraffin blocks by traditional pathological means,the paraffin blocks were then cut into slice which is 3 um thick and then detect and analysis the expression of VEGFR-1 and CD133 by mmunohistochemistry to observe the location of VEGFR-1 and CD133Results show that:The number of VEGFR-1~+HPC is increasing in nude mice when the time of breast cancer inoculation prolong.FCM shows that the number of VEGFR-1~+HPC in nude mice which have breast cancer for 2 weeks,weeks and 6 weeks possess a remarkable increase as compared with the nude mice which doesn't have cancer at all(P=0.000).After inoculation of tumor tissues,as the time gone the amount of VEGFR-1~+HPC keep increasing.The number of spots were found to be significantly altered among different groups(F=66.896,P=0.000) and it is in positive correlation with the time.The result shows that VEGFR-1~+HPC may attract tumor cells to home to pre-metastasis sites and prepared proper soil for establishment of tumor cells.The results of immunohistochemistry show that VEGFR-1 and CD133 are expressed before tumor cells arrived and the location of VEGFR-1 and CD133 are coincidence with the location of metastatic tumor which is diffused near the membrane,alveolar wall and so on.4.Detect the amount of MMP-2 and MMP-9 in liver of nude mice by Western blot to explore the mechanisms of promotion of tumor metastasisExtract protein from liver of nude mice and detect density of protein by BCA means.Then detect the amount of MMP-2 and MMP-9 by Western blot.Results show that:The number of MMP-2(matrix metalloproteinases-2),MMP-9(matrix metalloproteinases-9) is increasing in nude mice when the time of breast cancer inoculation prolong.The findings show that MMP-2,MMP-9 may play an important role in promotion of breast cancer metastasis.VEGFR-1~+HPC may mobilize changes in microenvironment such as cells,extracellular matrix and so on to make a premetastasis niche for cancer cells.5.Detect the expression of VEGFR-1 and CD133 in tumor tissues and lymph nodes of breast cancer of human to verify the role of VEGFR-1~+HPC in humanCollect breast cancer tissues from nanfang hospital.All the tissues are fixed in informalin,paraffin imbedding in routine method,then cut into slices which are 3 um thick,then detect and analysis the expression of VEGFR-1 and CD133 by immunohistochemistry.Results show that:The location of CD133 and VEGFR-1 positive cells is near the edge of lymph nodes which is in concindence with the first place tumor metastasis happens.CONCLUSION1.Establishment of whole-body visualization model of breast cancer with high hepatic metastatic potential in nude mice with pEGFP-MDA-MB-231 cells provide a utility and dependable model for research metastasis of breast cancer.2.CD133~+ cord blood HPC can promote proliferation,adheration and invasion of breast cancer cells in vitro.3.VEGFR-1~+ HPC may play an important role in tumor metastasis.VEGFR-1~+ HPC home to tumor-specific pre-metastatic sites and form cellular clusters before the arrival of tumor cells.VEGFR-1~+HPC can attract tumor cells to home to pre-metastatic sites in which they prepared proper soil for tumor cells to colonize.