Effect Of CD133 Positive Hemopoietic Progenitor Cells On Human Colorectal Carcinoma Cell Line

BACKGROUND & OBJECTIVEColorectal cancer (CRC) is the second worldwide leading cause of cancer death in the world. Metastasis is one of the basic characteristic of malignant tumors and is the main cause which affects the therapeutic efficacy and leads to the death of cancer patients. Metastasis is a sequential process, contingent on tumor cells breaking off from the primary tumor, travelling through the bloodstream, and stopping at a distant site. At the new site: the cells establish a blood supply and can grow a life-threatening mass.Kaplan et al demonstrate that bone marrow-derived haematopoietic progenitor cells that express vascular endothelial growth factor receptor 1 (VEGFR-1; also known as Flt-1) home to tumour-specific pre-metastatic sites and form cellular clusters before the arrival of tumour cells. Preventing VEGFR-1 function using antibodies or by the removal of VEGFR-1⁺ cells from the bone marrow of wild-type mice abrogates the formation of these pre-metastatic clusters and prevents tumour metastasis, whereas reconstitution with selected Id3 (inhibitor of differentiation 3)-competent VEGFR-1⁺ cells establishes cluster formation and tumour metastasis in Id3 knockout mice. They set up an ingenious experiment to track the movements of various cell populations as tumour cells metastasized in the lungs of live mice. The mice were irradiated to kill off all their bone-marrow cells, which were then replaced by bone-marrow cells tagged with green fluorescent protein; this made the cells easy to find under a microscope. Once the new bone-marrow cells were established, the mice were injected in the skin with lung carcinoma or melanoma cells, each marked with red fluorescent protein. The tumour cells were expected to form a primary tumour in the skin, and then to metastasize to the lungs. But the green bone-marrow-derived cells appeared in the lungs on days 12-14 after injection of the red cells-well before any of the tumour cells had arrived in the lung. The red tumour cells turned up only on day 18 post-injection, and by day 23 micrometastases had formed, with more than 95% of the tumour cells being found in exactly the same sites as the bone-marrow-derived cells. Mice were injected intraperitoneally with anti-VEGFR-1 antibody, it eliminated the initiating clusters and completely prevented metastasis, whereas anti-VEGFR-2 antibody did not prevent the formation of VEGFR-1⁺ clusters but limited metastatic progression. The hematopietic stem cells can now be obtained from peripheral or umbilic cord blood, as well as from bone marrow. The umbilic cord blood has more advantages than the bone marrow, with the sufficient donors and without the side effects, such as graff-versus-host disease(GVHD).CD133 is a 120-kDa five-transmembrane-domain glycoprotein expressed on normal primitive haematopoietic, endothelial, neural and epithelial cells. Expression of CD133 mRNA is significantly increased in peripheral blood mononuclear cells in solid cancers regardless of tumour type, particularly in patients with bone metastasis; overall survival was higher in patients with low or undetectable CD133 mRNA expression, although it was not ascertained whether the mononuclear cell fraction also contained CD133⁺ tumour cells as well as CD133⁺ haematopoietic progenitor cells. In this study, we aim to clarify the possible role of CD133⁺ haematopoietic progenitor cells from cord blood in the proliferation, invasion and metastasis of CRC line SW480. It will be helpful to understand the molecular basis of CRC, and establish a new target for early metastatic diagnostic markers and novel therapeutic strategies.METHODS1,The identification and cultivation of CD133⁺ haematopoietic progenitor cells from cord bloodAll human umbilical CB samples were obtained from the Obstetrics and Gynecology Department of Southern Hospital after informed consent. CD133⁺ haematopoietic progenitor cells were isolated from human umbilical CB samples (total of 20 samples, 2 lost). Each pooled mononuclear cells obtained from CB samples after Ficoll density gradient centrifugation separation. CD133⁺ haemato -poietic progenitor cells were positively isolated using the mini-MACS immunomagnetic separation system, according to manufacturer's instructions. After CD133⁺ haematopoietic progenitor cells isolation, the purity of the isolated cell populations was determined by fluorescein-activated cell sorting with CD34-R-phycoerythrin-conjugated mouse anti-human monoclonal antibody, CD133-phycoerythrin anti-human monoclonal antibody (Miltenyi Biotec) and immunocytochemistry.2,The effects of CD133⁺ haematopoietic progenitor cells on human tumor cells SW480The treatment group is the co-culture of CD133⁺ haematopoietic progenitor cells and SW480, the control group used the same culture medium without CD133⁺ cells. The effect of CD133⁺ haematopoietic progenitor cells on proliferation in SW480 cell was measured by MTT assay, the migration abilities of SW480 cell were assayed in Transwell cell culture, Cell adhesion assay was carried out in a 96 microplate well precoated with fibronection. The expression of VEGFR-1,MMP-2,E-Cadherin were analyzed by western blot.3,Using whole-body visualizing orthotopic animal model in colon cancer to demonstrate the effection of CD133⁺ haematopoietic progenitor cells in vivoThe metastatic ability evaluated by whole-body visualizing orthotopic animal model. The effection of CD133⁺ haematopoietic progenitor cells on in vivo tumor growth was assessed by tail vein injection of SW480/EGFP⁺/CD133⁺ and SW480 /EGFP⁺ cells .RESULTSThe main results and findings are as follows:1,The identification and cultivation of CD133⁺ haematopoietic progenitor cells from cord bloodIsolated CD133⁺ haematopoietic progenitor cells (purity over 80% as determined by flow cytometry) were characterized by flow cytometry and immunofluorescence staining for hematopoietic stem cells markers (CD133, CD34) and then cultured in undifferentiation conditions.2,The effects of CD133⁺ haematopoietic progenitor cells on human tumor cells SW480 in vitroCD133⁺ haematopoietic progenitor cells could significantly improve the growth ability of SW480 cell, not only the ability of proliferation(n=60,Z=-6.106,P=0.000), but also the morphology. The morphology of the cells was remarkably changed. Irregular nucleus, double nucleus and polymorphic nucleus appeared in the treatment group, and the cells shaped as polygon-like and leptosomatic. In the cell adhesion assay, OD₄₉₀ of the treatment group was 0.11±0.01, the control group was 0.05±0.01, (n=60,Z=-3.679,P=0.000) . The expression of VEGFR-1,MMP-2,E-Cadherin protein could be up-regulated in vitro .3,The effection of CD133⁺ haematopoietic progenitor cells on human tumor cells SW480/EGFP⁺ in vivoSW480/EGFP⁺ cells stably expressed high-levels of enhanced green fluorescent protein (EGFP) and EGFP had no effect on cell proliferation. Visualizing orthotopic animal model developed with tail vein injection was built with metastasis rate. After injection, animals had metastasis within 2 weeks in the treatment group, and subsequently, 4 weeks after injection, 67% of animal had metastasis in the treatment group. In control group ,only one animasl had metastasis (n=6, Breslow test, P=0.045) .The whole-body visualizing orthotopic animal model was successfully validated by pathological detection. The expression of VEGFR-1, MMP-2 protein could be up-regulated in vivo, but E-cadherin protein could be down-regulated.CONCLUSION1. CD133⁺ haematopoietic progenitor cells are effective in increasing tumor cell proliferation and invasion. Hematopoietic progenitor cells may play an important role in the invasion and mesatasis of colorectal neoplasms.2. CD133⁺ haematopoietic progenitor cells may involve in direct action to mediate proliferation, invasion and metastasis of CRC. The expression of VEGFR-1,MMP-2 protein could be up-regulated in co-transplantation and two-ways regulation the expression of E-cadherin protein. These data will be helpful to elucidate the molecular mechanism of CD133⁺ haematopoietic progenitor cells and provide new clues.