Abstract
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Objectives Validation of the angiogenesis PET tracer 68Ga-NODAGA- E[c(RGDyK)]2 targeting integrin αVβ3: comparison of tracer uptake (% ID/g) with gene expression of integrin β3 (ITGB3), angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2) in human neuroendocrine xenograft tumors (NCI-H727) in nude mice. 68Ga-NODAGA- E[c(RGDyK)]2 is a promising candidate for validating tumor-angiogenesis, because RGD peptide targets integrin αVβ3 which are markers of neo-angiogenesis. In addition, the Angiopoietin proteins (ANG-1 and ANG-2) have an important role in vascular development and angiogenesis. ANG-2 has been established as an antagonist inhibiting ANG-1 activation of its receptor Tyrosine Kinase (TIE-2). Binding of ANG-1 to TIE-2 has an important stabilizing effect on the vasculature, whereas binding of ANG-2 to the same receptor destabilizes vessels. ANG-2 promotes vascular sprouting and destabilizes blood vessels in presence of Vascular Endothelial Growth Factor (VEGF-A). In absence of VEGF-A ANG-2 contributes to vascular regression. ANG-2´s unique role in angiogenesis during development, growth and metastasis has been investigated in various human cancers and makes ANG-2 an attractive therapeutic target.
Methods 68Ga-NODAGA- E[c(RGDyK)]2 was synthesis using a 50 mCi 68Ge/68Ga generator and 2 nmol NODAGA- E[c(RGDyK)]2 actetate resulting in a radiochemical purity of 89-99%. Female NMRI nude mice were inoculated subcutaneously on both flanks with 1x107 human lung bronchus carcinoid cells (NCI-H727). Some weeks after inoculation, at a mean tumor volume of 332 mm3, mice were enrolled in the study (n= 10 tumors). PET scans were performed using a small animal PET scanner with a scan time of 15 minutes. Quantification of mean tracer uptake (% ID/g) in the xenograft tumors was performed using commercial image analysis software by drawing 3D regions of interest covering the whole tumor volume. Total RNAs were isolated from tumors using a column based separation kit obtaining 0.30- 0.45 µg/µl RNA. Real-time quantitative PCR was performed using a commercial kit and TaqMan chemistry for the QPCR quantification of genes of interest (GOI) and three optimized housekeeping genes. The relative gene expression levels were correlated to the tracer uptake values (% ID/g) as were the GOIs mutually.
Results Strong correlations were found between 68Ga-NODAGA- E[c(RGDyK)]2 uptake and h-ITGB3 (r=0.711; p=0.021), h-ANG-1 (r=0.851; p=0.002) and h-ANG-2 (r=0.838; p=0.003), respectively. Also strong correlations were found between genes pairwise: h-ITGB3 versus h-ANG-1 (r=0.665; p=0.036), versus h-ANG-2 (r=0.812; p=0.004), versus h-TIE-2 (r=0.945; p=<0.0001) furthermore h-ANG-1 versus h-ANG-2 (r=0.940; p=<0.0001).
Conclusions 68Ga-NODAGA-E[c(RGDyK)]2 is a promising candidate as companion diagnostic for Angiopoetin-directed therapies.
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