Oxygen Enhanced-MRI In Evaluating Hypoxia Microenvironment And Application Of CMET-spectic Probe In Treatment Of Glioma

Objective:The objective of this study was to determine whether using oxygenenhanced magnetic resonance imaging(OE-MRI)to assess hypoxia is feasible and whether historical measurements,p O2 changes,and percentage of signal intensity changes(PSIC)are correlated in an animal model of glioma.cMET is a receptor tyrosine kinase involved in tumor cell growth,invasion,metastases and angiogenesis.Overexpression of cMET is frequently observed in Glioblastomas multiforme(GBM).In this regard we assessed the effects of cMET targeting molecular probe with cMET-binding peptides(c MBPs)targeting cMET in GBM models in vitro and in vivo.Methods:1.A total of 25 Sprague-Dawley rats were used to establish C6 brain or subcutaneous glioma model.Nine rats with brain gliomas underwent OEMRI followed by histopathologic analysis to assess microvessel density and hypoxia.Another 11 rats were underwent OE-MRI and were followed for a survival analysis.Time–T1-weighted MR signal intensity(SI)curves and PSIC maps were derived from the OE-MRI data.High–regions of interests(PSIC > 10%)and low-ROIs(PSIC < 10%)were defied on the PSIC maps.To validate the PSIC map for identifying tumor hypoxia,we subjected an additional 5 rats with subcutaneous glioma to OE-MRI and p O2 measurements.2.Human GBM cell line U87,and rat astrocytes were used for the experiments.For targeting the cMET receptor,cMET targeting molecular probe with c MBP2-peptide,Den-c MBP2,was synthetized.Effects of cMET inhibition on U87 cells were determined by growth assays,scratch test,cell apoptosis assay and western blotting.Moreover,orthotopic xenogeneic mouse(BALB-C nu/nu)models of GBM were used to assess in vivo efficacy of targeting cMET.Results:1.All tumors showed regional heterogeneity on the PSIC maps.For the brain tumors,the time-SI curves for the ROIs-h showed a greater increase in SI than those for the ROIs-l did.The percentage of tumor area with a low PSIC was signifiantly correlated with the percentage of hypoxia staining and necrosis(r =0.71;P<0.05).ROIs with a higher PSIC typically had more vessels(r=0.88;P<0.05).A signifiant difference in survival was shown(logrank P = 0.035).The time-p O2 curves of the subcutaneous tumors were similar to the time SI curves of the brain tumors.PSIC was signifiantly correlated with p O2 changes(r =0.82;P<0.05).2.Immunohistochemical analysis revealed that cMET expressed high in xenogeic mouse models of GBM,especially in the margin of tumor.Reproduction ability,motility of cancer cells was reduced upon treatment with Den-c MBP.Only minor effects on rat astrocytes were detected.Treatment with Den-c MBP impairs activation of signaling intermediates in GBM cancer cells.Interestingly,Den-c MBP hardly influenced the expression of cMET,and the targeting molecular probe effectively inhibited the phosphorylation of cMET and its downstream protein.In vivo,therapy with Den-c MBP led to inhibition of orthotopic tumor growth in xenogeneic models and prolonged survival of the models.Similar to in vitro results,cMET expression was hardly increased and the phosphorylation of cMET and its downstream protein were inhibited.Conclusions:These fidings suggest that OE-MRI measurements can be used to assess hypoxia in C6 glioma models.In these models,the PSIC value was correlated with survival,indicating that PSIC could serve as a prognostic marker for glioma.And cMET with molecular probe with CMBP2-peptide may be effective in human GBM treatment and warrants further clinical evaluation.