Tracking Of Human Peirpheral Blood Endothelial Progenitor Cells Labeled With Alkyl-PEI2k/SPIO In A Lung Carcinoma Xenograft Model By7.0T MR Imaging

BackgroundCancer is the leading cause of death in developed countries and developing countries.According to the latest global statistics, lung cancer had the highest incidence andmortality in worldwide cancers."Cancer Facts and Figures2011"published byAmerican Cancer Society shows that lung cancer ranked first as a cause of cancer deathin USA (male28%, female26%). Cancer is the primary task needs to overcome whichfrighten human life seriously. The growth and metastasis of cancer depends onneovascularization. Previous view angiogenesis via the sprouting of endothelial cellspreexisting, since endothelial progenitor cells (EPCs) was reported firstly by Asahara,more and more studies thinked that EPCs could homing to the tumor sites specifically andparticipate in blood vessels formation. EPCs are bone marrow-derived precursor cells,the source, differentiation and identification of EPCs is unclear, the role and significanceof EPCs in tumor blood vessels formation is also controversial. Taking EPCs as gene ordrug vector, initial achievements were obtained in therapy of ischemic cardiovasculardisease and anti-tumor. The emergence of molecular imaging makes the tracing of stemcells non-invasively in vivo possible. Among numerous imaging modalities, magneticresonance imaging has been used widely to trace the moleculars and cells because of itsexcellent spatial resolution and multi-sequences imaging.Objective1,Isolate and culture endothelial progenitor cells from adult peripheral blood, toestablish a mature culture system of EPCs in vitro.2, To assess the efficacy of labeling EPCs with a novel nano-materialAlkyl-PEI2k/SPIO and effect of MR imaging in vitro.3,To evaluate the role of EPCs in tumor neovascularization in a lung carcinomaxenograft model.4,To evaluate the feasibility of tracking of stem cells transplantation in vivo by using7.0Tesla MR imaging.Methods1,20fresh peripheral blood samples were collected via cubital venous access, isolated by density gradient centrifugation and cultured. Observed the morphology of EPCs underelectron microscope, and detected the expression of CD31, CD34, VEGFR-2by flowcytometry. Ac-LDL uptake and UEA-I binding assay was used to identify EPCs function.2,EPCs were labeled by co-incubated with Alkyl-PEI2k/SPIO, the labeling efficacywas determined by Prussian blue staining. Iron quantification of labeled EPCs wasmeasured by atomic absorption spectrometer. The function of EPCs after labeling wasidentified by ac-LDL uptake and UEA-I binding assay. Cells growth curve were plottedusing CCK-8assay to determine the EPCs viability and proliferation after labeling.Migration assay and tubulogenesis assay were used to determine the activity of EPCs afterlabeling. Different numbers of labeled EPCs (0,5×103,1×104,5×104,1×105,2.5×105,5×105,1×106and1.5×106) were mixed in agarose hygrogel in Eppendorf tubes,In vitroMRI study of labeled EPCs agarose hygrogel was performed, MR sequences includingTurboRARE-T2, MGE-T2*, MSME-T2map, MGE-T2*map, and the signal intensity(SI),T2values and T2*values were measured.3,A549human lung adenocarcinoma cells were injected subcutaneously to establishmouse subcutaneous xenograft models.18SCID mice were divided into3groupsrandomly.Group A, labeled EPCs were injected via tail vein7days after A549cells hadbeen inoculated. Group B, labeled EPCs were injected subcutaneously in mice with A549cells.Group C, A549cells were inoculated subcutaneously in mice without EPCs. Miceof three groups were imaged at7.0T Micro-MR, MR sequences including TurboRARE-T2,MGE-T2*. Mice were euthanized28days later after MR imaging, paraffin and frozensections of tumors were prepared, and HE staining, Prussian blue staining andimmunofluorescence were performed.Results1,Mononuclear cells were isolated from human peripheral blood and incubated undercertain conditions, EPCs differentiated gradually and demonstrated the typicalcobblestone-like shape. The expression of EPCs surface markers were detected by flowcytometry, CD31(52.77%), CD34(10.53%), VEGFR-2(65.85%). Immunofluorescenceshowed that EPCs could uptake Dil-ac-LDL (red fluorescence) and bind of FITC-UEA-I(green fluorescence).2,After labeling with Alkyl-PEI2k/SPIO, Prussian blue staining showed that labelingefficacy was almost100%. Cellular iron content was6.062±0.050pg/cell detected by atomic absorption spectrometer. EPCs had the ability of uptake Dil-ac-LDL (redfluorescence) and binding of FITC-UEA-I (green fluorescence) after labeling.Absorbance of labeled or unlabeled EPCs were determined by CCK-8assay, there were nostatistical differences between labeled and unlabeled EPCs (P>0.05). Migration assayshowed that the number of labeled EPCs migrated (80.6±8.0) was comparable to that ofunlabeled EPCs (77.6±4.6), no statistical significance between labeled and unlabeled EPCs(P=0.39). Tubulogenesis assay showed that the number of tube-like structures formed bylabeled EPCs (9.0±1.0) was comparable to that of unlabeled EPCs (9.4±1.5), no statisticaldifferences were found (P=0.615). In vitro MR imaging showed signal intensity (SI) andrelaxation times were reduced with the increase of labeled EPCs. Paired t-test analysisdemonstrated that both SI and relaxation times had significant differences between T2andT2*imaging.3,Severe combined immunodeficiency mouse subcutaneous xenograft models wereestablished successfully by inoculation of A549cells suspension (A group=5,B group=5,Cgroup=6). In group A, schistic or linear hypointense regions at the tumor margins wereobserved on MRI At day7or8. The hypointense regions extended into the inner oftumor on MRI followed by. In group B, a focal central region of low signal intensity wasobserved at the early days. With the growing of tumor, the low signal intensity regiondispersed within tumor. However, no exact low signal intensity observed during MRscanning. At day28, tumor in group B were significantly larger than group A and C(P<0.01). However, no significant statistical differences were found between group Aand C (P=0.199). HE staining showed that tumor stroma thickened significantly,abundant microvessels were seen throughout tumors.Prussian blue staining showedPrussian blue positive cells at sites corresponding to MRI in group A and B. None ofPrussian blue positive cells was found in group C. Anti-human CD31positive cells wereshowed in group A and B by immunofluorescence. However, there were only anti-mouseCD31positive cells in group C.Conclusion1,EPCs can be obtained from human peripheral blood with high viability andproliferation by density gradient centrifugation and culture adherently under certainconditions.2,Alkyl-PEI2k/SPIO labeled EPCs efficiently without significant effects on cells activity.3,Human peripheral blood-derived EPCs involve in tumor neovasculature in lungcarcinoma subcutaneous xenografts.4,Stem cell transplantation can be track of non-invasively in vivo by7.0TeslaMicro-MR.