Combined MRI And CT Perfusion To In Vivo Evaluate The Role Of Endothelial Progenitor Cell Involving Glioma Angiogenesis

Background and ObjectiveGlioma is the most common form of primary brain tumor that seriously threatenshuman health. There is no cure for glioma so far. In recent years, researchers fromworldwide have made great efforts on glioma targeted therapy to prolong the survivalperiod of the patients who suffer from glioma. Accumulating evidence suggests thatendothelial progenitor cells (EPCs) play a key role in the development and infiltration ofgliomas. Thus, it has been considered that EPCs may be good vehicles for deliveringanti-angiogenesis genes for tumor therapy. Some research groups have succeeded in usingthis new therapeutic strategy within animal models. However, the role of EPCs in gliomaangiogenesis has been controversial. The capability of EPCs involving in gliomamicrovascluar has also been confused. Quantitative study from different research groupreported that the proportion of glioma microvascular angiogenesis containing EPCs rangingfrom0%-50%. Additionally, limited of in vivo tracking means has restricted the researchersto evaluate the survival, migration and distribution of EPCs in the host tissues. Thesedrawbacks also limit the development of EPCs as a vector or target for glioma therapy.Presently, the advances in magnetic resonance imaging (MRI) equipment provide anew way to track transplanted cells. MRI is an ideal tool because it is a non-invasivetechnique, and can track and/or monitor the temporal and spatial migration of transplantedcells in vivo. To be visualized on the MRI, cells need to be magnetically labeled beforetransplantation. Magnetic iron-oxide nanoparticles (IONPs) have been widely used as MRIcontrast agents to label cells. These nanoparticles have a high relaxation rate, and they canshorten T2relaxation time, which results in a low signal in targeted tissue to visualize thelesion.CT perfusion (CTP) imaging has been wildely used to study the hemodynamic ofhuman brain function. CTP is able to obtain the microcirculation information through the perfusion and vascularlize level of brain tissue. CT perfusion imaging can be a non-invasivemethod to in vivo quantitative analysis of tumor volume blood flow (CBF), cerebral bloodvolume (CBV), mean transit time (MTT) and permeability surface (PS) parameters,reflecting the tumor microvascular function and activity.In this study, we intented to employ both MRI and CT perfusion technology to in vivoquantitative analysis the glioma angiogenises that EPCs involved in. Our work will help todemonstrate the relationship between EPCs and glioma angiogenesis.Our work may asloprovide useful support for the future application of EPCs as a vector or target foranti-angiogenic therapy for glioma.Materials and Methods1. EPCs were isolated from bone marrow obtained from healthy SD rats. Firstly,MNCs were isolated using Ficoll gradient centrifugation, and the cells were cultured inendothelial condition medium to harvest pure EPCs.2. EPCs were labeled with two classes of magnetic contrast agents Resovist andGd-DTPA, respectively. Both Resovist and Gd-DTPA were set up concertrations rangingfrom0-75ug/ml to study the efficiency and toxity to label EPCs. Next, the labeled cellswere scaned by3.0-T MRI to analysis the signal intensity on different sequences. At last,cross compared between Resovist and Gd-DTPA was employed to study the labelingcharacteristic and superiority-inferiority of each contrast agent.3. EPCs labeled with25ug/ml Resovist were transplanted into rat`s glioma models bytail vein injection.3.0-T MRI was employed to in vivo track the transplanted EPCs in thehost tissue at different stages. CT perfusion was used to study the hemodynamic of gliomaangiogenesis that EPCs involved in. Immunohistochemistry and laser confocal microscopywere used to indentify the structure and fuction of glioma microvascular that EPCs involedin.Results1. EPCs isolated from bone marrow using gradient centrifugation combinedconditional cultured have the ability to self-renewal, and could be induced into ECs. EPCsexpressed the surface markes CD133, CD34and VEGFR2at the early stage. Afterconditional cultured, the intensity of CD133expression was change lower, while CD34and VEGFR2expression were enhanced. At this stage, EPCs expressed the endothelial markersCD31and vWF. EPCs have same fuction as ECs, which could uptake of DiI-ac-LDL andcombinding of FITC-UEA-1, and have the ability to angiogenesis in vitro. EPCs couldhome to glioma in vitro. The cell migration ability was related to the total amount of C6glioma cells.2. Both Resovist and Gd-DTPA could be used to label EPCs. The optimalconcentrations of Resovist labeling were25-50ug/ml. The optimal concentration ofGd-DTPA was25ug/ml. The T2sequence on MRI was the best way to detect Resovistlabeled EPCs, while T1sequence to detect the Gd-DTPA labeled cells. We suggest thatResovist should be the best for labeling EPCs due to its high labeling efficiency and lowtoxicity to the cells. Gd-DTPA contrast agents could be a secondary choice for labelingEPCs, especially in cell intra-foci administration and other hemorrhage model.3. EPCs specifically homed to gliomas and could be reliably tracked by1.5-T MR asearly as24hours after transplantation, causing a signal loss on T2-weighted images. Thelabeled cells were mainly located at the periphery of the tumor. Low signal intensitybecame more significant, and the dark areas were enlarged as time went on. The results ofPrussion blue stain showed that the number of blue stain cells at the rim of tumor washigher than that at other non-targeted tissue, such as spleen, kidney and lung tissues, whichwas consistent with MRI findings. Using CT perfusion imaging to analysis thehemodynamic of glioma angiogenesis demonstrated little variability, and the results of CTperfusion were consistent with MVD analysis. Quantitative analysis showed that EPCsenhancing the rim of tumor angiogenesis approximately15.09%to20.06%. The results ofLaser confocal microscopy demonstrated that EPCs mainly integrated into the caecum orbranch of microvacular. EPCs interconnected with tumor microvascular in situ to formdisorderly vascular network by involving in form new blood vessels to the branch spot.ConclusionsExogenous EPCs mainly homing to the rim of glioma, involving in tumor angiogenesisby integrated into the caecum and branch spot of tumor microvascular. Quantitativeanalysis showed that120hours after transplantation of EPCs,the hemodynamic of tumoredge was changed about15.09%to20.06%. MRI and CT perfusion imaging could be powerful tools to track the migration and fuction of EPCs in host tissues. MRI and CTperfusion aslo could be a powerful method for in vivo monitoring of clinical stem cellstherapy.