Magnetic Labeling Endothelial Progenitor Cells With USPIO And In Vivo Magnetic Resonance Tracking Of Angiogenesis In Rat Cerebral Glioma

BACKGROUNDS AND OBJRCTIVESGliomas represent the most frequent neoplasm of the central nervous system. High-grade gliomas are characterized by rapid growth, high aggressiveness and recurrence rate. The phenotype of tumor is closely related with the microvessel and angiogenesis which plays a pivotal role in tumor growth and recurrence. Endothelial progenitor cells (EPCs) primarily located in the bone marrow after borning. In some cases they can be mobilized into the peripheral blood, and then homed to target tissues and involved in physiological or (and) pathological angiogenesis. The purpose of this research is to detect the distribution of magnetically labeled endothelial progenitor cells (EPCs) by MRI and explore the role of of EPCs in tumor angiogenesis in rat brain C6 glioma.METHODSSprague–Dawley (SD) rats weighing about 90g to 110g were prepared and bilateral femur and tibia were taken off for marrow collection. Lymphocyte separation medium was used to isolate the mononuclear cells, and the attachment growth method was used to purify endothelial progenitor cells. Cells were identified by immunohistochemisty and immunofluorescent staining with surface marker CD133, VEGFR-2 and CD34, Dil-labeled acetylated low-density lipoprotein (DiI-ac-LDL) and FITC-labeled ulex europaeus agglutinin-1 (FITC-UEA-1). Poly-lysine (PLL) was used as the transfection agent (TA) to help USPIO particles enter into endothelial progenitor cells in vitro with the concentration of 25ug/ml. Magnetically labeled cells and un-labeled cells were injected intravenously into the rat bearing glioma through the tail vein and MR imaging was performed 24 hours,48 hours,3 days and 7 days later.Then the tumor was removed and examed by Prussian blue staining and immunohistochemical techniques for the expression of endothelial cell markers using anti-mouse CD34,anti-mouse CD105,anti-mouse CD138 and KDR1 (VEGF receptor 2) antibodies. Fe-PLL-labeled cells were also incubated with quantum dots for 45⁶0 minutes, Double-labeled cells were injected intravenously into the rat bearing glioma through the tail vein. MR imaging was performed 2 days and 4 days later. Texas-red labeled tomato lectin were injected intravenously through the tail vein 5¹0 minutes before euthanasia to delineate the vessels. Tumors were collected and divided for frozen section ,then were observed under the fluorescent microscope.RESULTSMononuclear cells can be induced and differentiated into endothelial progenitor cells in vitro. USPIO-PLL can label EPCs effeciently, and Prussian blue staining showed that the iron particles were deposited in the cytoplasma.The labeling efficiency was more than 99%.EPCs can be labeled with USPIO and QDs successfully. Migration and incorporation of labeled EPCs into the neovasculature appeared as low signal intensity at the tumor periphery both on T₂WI and T₂* map sequences, especially on T2* map sequence. Prussian blue staining has shown iron-positive cells at the sites of tumor corresponding to low signal intensity on MRI. Prussian blue staining and immunohistochemical results suggested that magnetically labeled EPCs could transform into endothelial cells. QDs labeled endothelial cells were at sites corresponding to the sites with vessels delineated by texas red-labeled lectin. CONCLUSIONSEPCs incorporated into the neovasculature and participated in tumor angiogenesis of glioma in situ and can be tracked with MRI sensitively.