Tumor Associated Vascular Endothelial Cells Correlate With The Tropism Of MSC For Tumor

Objective Isolate, culture and identification of breast tumor associated vascular endothelial cells and normal breast associated vascular endothelial cells. And explore their biological differences.Methords (1) Collect the fresh breast tumor tissue and normal breast tissue in aseptic conditions, tissues are mechanically minced and subsequently digested enzymatically with collagenase. Tumor-associated vascular endothelial cells and normal breast associated vascular endothelial cells in the resulting single-cell suspension are labeled with Abs against CD31antigens and separated by capture with magnetic beads, and then endothelial cells were primary cultured in vitro and observated morphology with microscope.(2) Tumor-associated vascular endothelial cells and normal breast associated vascular endothelial cells were analyzed by flow cytometry using phycoerythrin(PE)-conjugated monoclonal antibody (mAb) to CD34, CD31, CD105, vWF. Isotype-matched mouse mAbs were used as negative control.(3) Real-time quantitative PCR was used to detecte different gene expression between normal breast associated vascular endothelial cells and breast tumor associated vascular endothelial cells.(4) In vitro angiogenesis model was used to verificate capability of two endothelial cells.Result (1) Breast tumor associated vascular endothelial cells and normal breast associated vascular endothelial cells can be obtain and culture in vitro, and they have some differences in morphological.(2) The expression of molecular tumor specific markers of tumor associated vascular endothelial cells were higer than the normal breast associated vascular endothelial cells.(3) The angiogenesis-inducing ability of tumor associated vascular endothelial cells was stronger than normal breast associated vascular endothelial cell and HUVECs.Conclusion High purity of Fresh tissues associated vascular endothelial cells can be easily obtained and cultured in vitro by immunomagnetic separation technology, and they have some differences in morphological and biological properties. Objective Mesenchymal stem cells derived from the mesoderm are a population of multipotent cells, because of its low immunogenicity, tumor homing properties, as well as its easy separation, amplification, can be genetically modified and other advantages, have become the most ideal cell carrier for gene therapy. However, it is lack of systematical investigation and expanded understanding on the tumor homing of mesenchymal stem cells. This study aims to investigate the tumor associated vascular endothelial cells involve in mesenchymal stem cells homing to tumor.Methord (1) A Modified Boy den chamber assay was used to investigate the in vitro migration ability of MSCs to fresh breast tumor tissue and normal breast tissue.(2) The result of immunofluorescence showed that microtubule organization center (MTOC) of MSCS becomes polarized in the direction of migration. This indicated that changes in morphology correlated with enhanced migration.(3) Using collagenasedigestion and difference trypsin digestion to obtain the primary breast tumor associated fibroblasts and primary tumor cells. And a Modified Boyden chamber assay was used to investigate the in vitro migration of MSCs to different cells obtained from breast tumor tissue.(4) co-cultured mesenchymal stem cells stained with Phalloidin were observe by confocol microscope’Result (1) Compared with the normal tissue, the tumor tissue promote migration ability of mesenchymal stem cell in vitro.(2) Tumor tissue causes mesenchymal stem cells Tubulin.(3) The migration of MSCs across the membrane were obviously more than the other groups (p<0.05).(4) Cytoskeleton of mesenchymal stem cell co-cultured with tumor vascular endothelial cells changed markedly. Conclusion Tumor associated vascular endothelial cells involve in mesenchymal stem cells homing to tumor. Objective Human mesenchymal stem cells (MSCs) are increasingly being considered in cell-based therapeutic strategies for the delivery of therapeutic genes to tumors. However, the signals required for their homing and recruitment to tumor site in vivo are not yet fully understood. In this study, we aimed to investigate the distinct molecular signals underlying the homing of MSCs to tumor sitesMethord (1) RayBio(?) Human Cytokine Antibody Array was used to screen the levels of various factors in the cell-free culture supernatants.(2) IL6levels were probed by ELISA on the culture supernatants at the indicated time points.(3)The effect of IL6on proliferation of mesenchymal stem cells were detected by MTT.(4) A Modified Boyden chamber assay was used to investigate the in vitro migration ability of MSCs to IL6.(5) We investigated the effect of IL6on morphological and cytoskeletal changes in MSCs by immunofluorescence staining with phalloidin.(6) Western-blot was used to analyze the cell signal pathway of mesenchymal stem treatment with IL6.(7) SiRNA interference technology was used to block the expression of IL6in TECs.(8) Mesenchymal stem cells were labeled with CellTrackerTM dye In vitro and the positive rate was analyzed by Flow cytometry.(9) Mesenchymal stem cell homing to TECs through IL6was validated with mouse ear model in vivo.Results (1) Human Cytokine Antibody Array showed that the expression of IL6protein in the culture supernatants of tumor vascular endothelial cells co-cultured with mesenchymal stem cells was3.6times higher than that in tumor vascular endothelial cells, and it is8.92times higher than that in the culture supernatants of mesenchymal stem cells.(2) The secretion of IL6were significantly increased in culture supernatant of tumor vascular endothelial cells co-cultured with mesenchymal stem cells.(3) MTT showed that interleukin6did not significantly promote the proliferation of mesenchymal stem cells.(4) The migration ability of mesenchymal stem cells treatment with IL6was significantly enhanced in vitro.(5) Confocal laser scanning microscopy found that polarity and actin cytoskeleton of mesenchymal stem cell treatment with10ng/ml IL6changed obviously.(6) IL6increased the expression of phosphorylation STAT3in mesenchymal stem cells and up-regulate the expression of MMP3, MMP9which are closely related to cell movement.(7) Anti-IL6neutralizing antibody can effectively decrease the transmembrane ability of mesenchymal stem cell.(8) IL6/SiRNA can effectively inhibit the expression of the target protein and, inhibit the activation of STAT3in tumor-associated vascular endothelial cells, result in the decreasing of migration ability of mesenchymal stem cells.(9) The positive rate of endothelial cells staining with DiO and mesenchymal stem cells staining with CellTracker TM dye were96.53±3.21%,98.71±4.67%.(10) Mouse ear model support the evidence that IL6is critically involved in the migration capacity of MSCs, acting through the IL6/STAT3axis.Conclusion IL6/STAT3axis may be involved in recruitment of expanded MSCs to tumor site.