Dual Targeting VEGF And PFKFB3 Induces Tumor Vascular Normalization In Glioblastoma And MRI Evaluation

Background and PurposeDespite the great advances in various diagnostic and therapeutic approaches,the prognosis of glioblastoma（GBM）patients remains dismal with a median survival of approximately 14.5～16.6 months.It has been recognized that normalizing tumor vasculature by anti-angiogenic agents such as monoclonal humanized vascular endothelial growth factor（VEGF）antibody bevacizumab（BEV）is a promising strategy to remodel tumor microenvironment（TME）for enhancing anti-tumor therapeutic efficacy.Anti-angiogenic therapy（AAT）-induced tumor vascular normalization（TVN）offers a window of opportunity for optimally combining other anti-cancer therapies to integrate the anti-tumor effect.However,the normalization window is transient.An urgent need exists to develop a novel therapeutic strategy to reinforce the TVN effect induced by AAT thus gaining a durable therapeutic synergy.Monitoring TVN noninvasively is conducive to guide anti-tumor therapy administration during the normalization window for enhancing treatment efficiency.The quantitative parameter K^trans of dynamic contrast-enhanced magnetic resonance imaging（DCE-MRI）measures vascular functionality by characterizing the distribution of Gd-based contrast agent（GBCA）between intra-and extravascular spaces and the degree of leakage from blood vessels.This parameter is recommended by the Quantitative Imaging Biomarkers Alliance of Radiology Society of North America as the endpoint of AAT evaluation.Intravoxel incoherent motion-MRI（IVIM-MRI）is a non-GBCA-based advanced imaging modality attracting more and more attention due to its ability to characterize tissue microcirculation and diffusion simultaneously.As different quantitative MRI techniques,combining the advantages of DCE-MRI and IVIM-MRI contributes to providing more information in tumor vessel functionality.Exploring the value of DCE-MRI and IVIM-MRI for monitoring tumor response and TVN process is of important clinical implications to accelerate the translation of TVN strategy.In this study,we conducted a dual therapy by inhibiting VEGF and glycolytic activator PFKFB3 to investigate whether dual targeting VEGF and PFKFB3 could enhance the TVN effect in orthotopic patient-derived xenograft（PDX）of GBM.Moreover,DCE-MRI and IVIM-MRI were performed to evaluate the dynamic process of tumor response after treatment and the correlations between MRI parameters and TVN indicators were analyzed.Materials and Methods1.The GEO dataset GSE39221 was used to analyze the expression level of PFKFB3 in U87-MG GBM tissues before and after BEV treatment.Then,224 orthotopic GBM PDX models were established,of which 172 PDX models were randomly divided into four treatment groups including BEV monotherapy,3PO（PFKFB3 inhibitor）monotherapy,BEV+3PO dual therapy,and control groups.The PDX models were analyzed with a 7.0T preclinical MRI scanner,western blotting,and histology to measure tumor size,PFKFB3expression,cell proliferation and apoptosis,and survival for evaluating the anti-tumor effect of different therapies on GBM.Histology and ¹H-magnetic resonance spectroscopy（¹H-MRS）were conducted at baseline before treatment,2 days,5 days,8 days,14 days,and 25 days after treatment to evaluate morphological characteristics of blood vessels,tumor hypoxia,lactate dehydrogenase-A（LDHA）expression,and the concentration of tumor metabolites.Doxorubicin hydrochloride（DOX）was used and 52 PDX models were divided randomly into four treatment groups（DOX,DOX+3PO,DOX+BEV,DOX+BEV+3PO）for evaluating chemotherapeutic drug delivery and efficacy.The microarray of angiogenesis cytokines,immunoblotting,and bioinformatics was used to evaluate therapy-associated molecular profiling.2.For the 172 PDX models,DCE-MRI and IVIM-MRI were performed at baseline before treatment,2 days,5 days,8 days,14 days,and 25 days after treatment to monitor the dynamic alteration and heterogeneity of tumor response by tumor region segmentation and histogram analysis.The imaging-histology correlations were analyzed to investigate the value of MRI quantitative parameters to TVN evaluation.Results1.GEO dataset showed that PFKFB3 level in U87-MG GBM tissues was increased significantly after BEV monotherapy.This result was consistently verified in the GBM PDX models.Western blotting further revealed that PFKFB3 inhibitor 3PO effectively inhibited the high expression of PFKFB3 induced by BEV monotherapy.Compared with BEV monotherapy,the combined treatment of BEV+3PO significantly prolonged the survival of tumor-bearing mice（68.5 days vs.81 days,P（27）0.05）,delayed tumor growth,promoted tumor cell apoptosis,and inhibited cell proliferation.Histology analysis demonstrated that the anti-tumor synergy of dual therapy targeting VEGF and PFKFB3 was benefited from the sustained TVN effect.The pericyte coverage index（PCI）and collagen IV expression were significantly increased at day 2 after dual therapy and lasted by day 25.This normalization window was longer than that induced by BEV therapy alone（day 2～day 14,P（27）0.05）.The dual therapy-induced TVN synergy remodels anoxic and acidic TME characterized by decreased pimonidazole staining and reduced LDHA production（P（27）0.05）.¹H-MRS further revealed that the tumor metabolites（choline,lipid,and lactate）were significantly reduced（P（27）0.05）.Additionally,the synergistic effect of TVN induced by BEV+3PO treatment prominently improved the drug delivery and efficacy of DOX.The enrichment of DOX in the tumor area in the BEV+3PO group was more remarkable than that in the BEV monotherapy group（P（27）0.05）.The inhibitory effect of tumor growth and the survival of tumor-bearing mice in the BEV+3PO+DOX group were significantly prolonged（P（27）0.05）.Mechanistically,multiple proangiogenic cytokines and anti-normalization signaling pathways were downregulated after dual therapy.Strikingly,dual therapy downregulated Tie1 expression,which is the critical regulator of TVN,in both tumor cells and endothelial cells.2.Tumor response after treatment monitored by DCE-MRI and IVIM-MRI showed that BEV+3PO promoted a more homogeneous tumor response characterized by improved vascular functionality with increased blood flow and decreased vascular permeability in tumor core and tumor rim.DCE-MRI parameter K^trans,IVIM-MRI parameters f and D^*had significant correlations with tumor microvascular density and TVN indicators（P（27）0.0001）with K^trans having the highest correlation with microvessel density（r（28）0.7202,P（27）0.0001）.The correlation coefficients between D^*and PCI,collagen IV expression,and tumor hypoxia were 0.6365,0.6628,-0.5446,respectively,higher than those between K^trans and corresponding indicators（r（28）-0.5214,-0.5781,0.4656,respectively）.The correlation between K^trans and D^*was relatively weak（r（28）-0.4499,P（27）0.0001）.Conclusions1.Dual targeting VEGF and PFKFB3 strategy enhances the anti-tumor effect and TVN synergistically in the orthotopic GBM PDX model,thereby improving TME remodeling and offering a durable window of opportunity to improve the efficacy and drug delivery of chemotherapy.This strategy overcomes the limitations of the current TVN effect which may be an alternative promising strategy to fill gaps in current TVN approaches.2.The IVIM-MRI parameter D^*has much potential as a non-GBCA-dependent imaging biomarker complementary to the DCE-MRI parameter K^trans to evaluate the TVN effect.This may accelerate the clinical translation of TVN strategy and provide valuable metrics for guiding therapeutic decisions within the TVN window.