The MRI Study Of The Effect Of Labeled Endothelial Progenitor Cells On The C6 Glioma Cells

BackgroundAshara and his co-workers separated a population of cells from human peripheral blood in 1997, which can differentiate to mature endothelial cells. Therefore, the cells were named endothelial progenitor cells (EPCs). Meanwhile, they confirmed that bone marrow derived endothelial progenitor cells contributed to postnatal neovasculature, a process that is referred to as vasculogenesis, in mice modle. After that, many researchs have been shown that EPCs can homing to the hot spot of neovascularization in tumor tissue and play a important role in tumor growth. Based on this property, it is expected to detect tumor invasion or satellite lesions via transplated labled EPCs by maganetic resonance imaging(MRI) scanning technology, thereby improve the level of clinical diagnosis and treatment. Howerver, in view of the EPCs homing to the tumor tissue and integrated in neovasculature, it is questionable whether the ectogenic EPCs thransplation would participated in tumor proliferation and progression, which also is prerequisite for the EPCs using as targeted MRI contrast agents. In this study, we have investigated the influence of EPCs on the brain glioma biological behavior both in vitro and in vivo to assessment the feasibility and safety of EPCs as targeted MRI contrast agents.ObjectiveApproach the influence of rat speen derived-EPCs on the proliferation and ivassion of C6 glioma cells in indirect contacted co-culture system in vitro; Observe the effect of SPIO labeled EPCs on the growth of glioma and measure the change of blood volum at different time, which would be compared with the pathological indication to comfirm the influence of EPCs on the tumor growth, microvascular morphology and density and VEGF expression; To provide the feasible and secure evidences for using EPCs as targeted MRI contrast agents in the diagnosis of glioma.Materials and methods 1.The influence of EPCs on the proliferation and invasion of C6 glioma cells in vitroHealthy SD rat speen derived-mononuclear cells were isolated by density gradient centrifugation, after 3 days cultured,non-attached cells were washed away by PBS and then attached cells were cultured for 7 days, the medium was changed every 3 days. EPCs were characterized as adherent cells were double positive for DiI-acLDL uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. Meanwhile, EPCs were also identified by detecting the expression of cell surface makers CD34 and CD31 through immunofluorescenc.Changed and collected the culture supernatant when the adherent cells covered about 60%～70% of culture flask, EPCs conditioned medium(EPCs-CM) was obtained by mixing and filtering the collected culture supernatant. According to 0～50% six different concentration of EPCs-CM in the culture medium, divided the C6 glioma cells in six group culture system. All groups cultured in 37℃,5% CO2 incubator for 36 and 48 hours, then examined the optical density value with microplate reader to reflect the proliferation of the C6 glioma cells in each group. In order to further confirm that the influence of EPCs-CM on the proliferation of C6 glioma cells, we supported C6 glioma cells with two culture medium, one was mixed EPCs-CM with conventional medium by 1:1, the other was pure conventional medium, cultured 24 hours in 37℃,5% CO2 incubator, the cell cycle distribution of C6 glioma cells was detected by flow cytometry separately. C6 glioma cells invasive model was constructed by making use of modified Boyden chamber with the formation of the artificial basement membrane using matrigel. Then added C6 glioma cells, which were suspended in EPCs-CM, to the super-chamber of experimental group, while the control group added equal volume C6 glioma cells suspended in DMEM/F12. The invasion of C6 glioma cells in the two groups was examined after incubated under the same condition. Except for that, the invasion of the C6 glioma cells was also detected with cell-matix adhesion assay.2.The MRI study on the effect of labeled EPCs on C6 glioma cells in vivoFour different group brain glioma model, named control group and A, B and C experimental group, were constructed using stereotaxic apparatus in healthy SD rat.The rat in control group were only transplated C6 glioma cells, while the A experimental group transplanted both C6 glioma cells and EPCs-CM, which accout for fifty percent of cell suspension volume, the B group transplanted equal volume P7228 labeled EPCs and C6 glioma cells, the C group transplanted equal volume P7228 labeled EPCs of B group via vena caudalis after the formation of brain glioma.The conventional MR imaging, including spin echo and gradin-echo sequence, were performed at 3, 7, 10, 15, 20 and 25 days after transplantation on each group to observe the growth of brain glioma. The tumor size of each group, which reflected the tumor growth, were calculated based on the enhanced MR imaging. The perfusion-weighted imaging was carried out with echo-planar imaging sequence, in order to evaluate the variance of blood current in the brain gliomas as well as reflected the formation of tumor microvessel between different groups, CBV and MTT maps of each glioma model were obtained after image manipulation. The C experimental group was underwent MR imaging examination at the 1, 3, 5, 7 and 9 days after transplanted labeled EPCs, observed the variant accumulation of EPCs in the tumor tissue in different scanning point and evaluated its effect on tumor growth and invasion. Tumors were excised from experimental rat of every group at each examined point via cardiac perfusion with 4% paraformaldehyde to make pathological assay. HE staining could describe the tumor morphology of different groups, prussian blue staining represent the distribution of P7228 labeled EPCs in the tumor site. Applied immunohistochemistry carried on CD34 staining of tumor microvessels, VEGF staining and employ the computer image analysis software to carry on quantitative analysis of microvessel density, morphology and tumor VEGF expression in each glioma model.Results1.The influence of EPCs on the proliferation and invasion of C6 glioma cells in vitroThe lately-isolated MNCs were small and round, parts of which started attaching after 24h. After 3 days cultured, attached cells become bigger and more lucency, some of which were fusiform. After 7 days cultured, the attached cells increased, appeared more spindle cells and were able to form"straight line"-like structure, the cells exhibited the typical cobblestone morphology after 14 days cultured. EPCs were identified as adherent cells double positive for DiI-acLDL uptake and lectin binding by fluorescent staining under a laser scanning confocal microscope, the proportion of double positive cells was 90%. Moreover, the EPCs expressed both CD31 and CD34 by immunofluorescence.The optical density value of each group after cultured 36h and 48h were increased with the increased concentration of the EPCs-CM, the optical density value of 50% EPCs-CM group(2.018±0.220), (2.388±0.448) in the two time point were significantly higher than the control group(1.163±0.103), (1.106±0.174). Flow cytometry results showed that the proliferation rate S+G2/M of EPCs-CM cultured C6 glioma cells was higher than the control group(P<0.05). The Reconstituted basement membrane assay indicated that the number passed through the basement membrane(the MTT methods results showed optical density value, 3.881±0.159 ) of EPCs-CM cultured C6 glioma cells was more than simple conventional culture medium cultured C6 glioma cells(3.532±0.116, P<0.05). The number of adherent cells on the artificial basement membrane of EPCs-CM cultured C6 glioma cells was more than the pure conventional culture medium cultured(P<0.05)after 2h cultured in 37℃, 5% CO2 incubator.2.The MRI study on the effect of labeled EPCs on C6 glioma cells in vivoThe MRI detected glioma in the right cerebral parenchyma, which showed high intensity in T2-weighted imaging as well as enhancement after 10days establishment of glioma model. The tumor showed exponential growth during 15days and 20days. The tumor size of the 4 model group co-varied with the increased tumor age and size difference between the 4 group based on 4 time point(10,15,20 and 25days)is significant(P<0.01).Although the tumor size of C experimental group was lightly bigger than other 3 group at 10 and 25days(46.714±7.325,646.171±37.600mm3), the difference was no statistically significant(P>0.05). Except for B experimental group wasn't able to reconstruct the CBV map for the influence of iron particles in the labeled EPCs, all the brain glioma revealed high perfusion compared with normal brain parenchyma in the other three model group. The rCBV were inceased with the passage of time and the rCBV of each group at different time point had significant difference (P<0.01), however ,the difference based on group was not significant(P>0.05).Pathological analysis of HE staining exhibited that the disordered and active glioma cells infiltrate peripherial normal brain tissue showed typical"fence"pattern, furthermore, there was local necrosis in the center of the bigger tumor. There were numerous blue-stained cells, which accumulated in the tumor neovasculature region, in the C group after prussian blue staining. CD34 staining showed that there were redundant heterogeneous tumor microvessel,the difference of MVD between the 4 groups at different measuring point wasn't significant(P>0.05).Besides the B group, the rCBV and MVD value of other groups all had positive correlation. The nascent tumor vasculature of all the 4 group were approximate, the diameter of which rang from 20.915μm to26.143μm. The positive cells for VEGF staining, characteristic as discovering brown particles located in cytoplasm of each group at every measuring time, there were no dramatic difference.Conclusions1.Combined Ficoll'density gradient centrifugation with adherence screen, we could gain SD rat speen derived-EPCs, which exhibited great differentiation and proliferation in vitro; EPCs could improve the proliferation and invasion of C6 glioma cells through cytokine paracrine action in vitro.2.It was suggested that the P7228 labeled EPCs might act as MRI targeted contrast agent as they can homing to the nascent tumor microvessel region and be found by 1.5 T MR sanner.3.Neither paracrine secretion of cytokine or the direct intercellular action couldn't change the C6 glioma cells'biological behavior during a certain time, which might partly related with the complicated tumor microecosystem.