No abstract

We have previously labeled cyclic RGD peptide c(RGDyK) with fluorine-18 through conjugation labeling via a prosthetic 4-[18F]fluorobenzoyl moiety and applied this [18F]FB-RGD radiotracer for alphav-integrin expression imaging in different preclinical tumor models with good tumor-to-background contrast. However, the unfavorable hepatobiliary excretion and rapid tumor washout rate of this tracer limit its potential clinical applications. The aims of this study were to modify the [18F]FB-RGD tracer by inserting a heterobifunctional poly(ethylene glycol) (PEG, M.W. =3,400) between the 18F radiolabel and the RGD moiety and to test this [18F]FB-PEG-RGD tracer for brain tumor targeting and in vivo kinetics. [18F]FB-PEG-RGD was prepared by coupling the RGD-PEG conjugate with N-succinimidyl 4-[18F]fluorobenzoate ([18F]SFB) under slightly basic conditions (pH=8.5). The radiochemical yield was about 20-30% based on the active ester [18F]SFB, and specific activity was over 100 GBq/micromol. This tracer had fast blood clearance, rapid and high tumor uptake in the subcutaneous U87MG glioblastoma model (5.2+/-0.5%ID/g at 30 min p.i.). Moderately rapid tumor washout was observed, with the activity accumulation decreased to 2.2+/-0.4%ID/g at 4 h p.i. MicroPET and autoradiography imaging showed a very high tumor-to-background ratio and limited activity accumulation in the liver, kidneys and intestinal tracts. U87MG tumor implanted into the mouse forebrain was well visualized with [18F]FB-PEG-RGD. Although uptake in the orthotopic tumor was significantly lower (P<0.01) than in the subcutaneous tumor, the maximum tumor-to-brain ratio still reached 5.0+/-0.6 due to low normal brain background. The results of H&E staining post mortem agreed with the anatomical information obtained from non-invasive microPET imaging. In conclusion, PEGylation suitably modifies the physiological behavior of the RGD peptide. [18F]FB-PEG-RGD gave improved tumor retention and in vivo kinetics compared with [18F]FB-RGD.

SUMMARY Plasminogen activator inhibitor-1 (PAI-1) paradoxically enhances tumor progression and angiogenesis, however the mechanism supporting this role is not known. Here we provide evidence that PAI-1 is essential to protect endothelial cells (EC) from FasL-mediated apoptosis. In the absence of host-derived PAI-1, human neuroblastoma cells implanted in PAI-1 deficient mice form smaller and poorly vascularized tumors containing an increased number of apoptotic EC cells. We observed that knockdown of PAI-1 in EC enhances cell-associated plasmin activity, and increases spontaneous apoptosis in vitro. We further demonstrate that plasmin cleaves FasL at Arg144 - Lys145, releasing from the surface of EC a soluble pro-apoptotic FasL fragment. The data provide a mechanism explaining the pro-angiogenic activity of PAI-1. SIGNIFICANCE PAI-1, the central regulator of plasmin generation is a predictor of poor clinical outcome in cancer patients. We previously reported that PAI-1 has a pro-angiogenic function but the mechanism has remained poorly understood. Here we describe a mechanism for the pro-angiogenic function of PAI-1 by providing evidence that PAI-1 protects EC from Fas/FasL-mediated apoptosis. We demonstrate that in the absence of PAI-1 in EC, there is an increase in plasmin generation and the release by plasmin of a soluble FasL fragment. This plasmin-generated soluble FasL activates Fas and is a potent inducer of apoptosis in EC. Our results suggest that PAI-1 could be a target for anti-angiogenic and anti-vascular therapies.

The cyclic pentapeptide EMD 121974 may become a treatment option specific to brain tumors. Because of its antiangiogenic effect, its use may be especially indicated after tumors are removed surgically.