Evaluation of ⁶⁸Ga-labeled iNGR peptide with tumor penetrating motif for microPET imaging of CD13-positive tumor xenografts

Objectives To evaluate the efficacy of 68Ga-labelled iNGR peptide containing CendR tumor penetrating motif as a new molecular probe for microPET imaging of CD13-positive tumor xenografts.

Methods NGR and iNGR peptides both conjugated with NOTA were synthesised. CD13 of HT1080 and HT29 cells was confirmed by western blot and immunofluorescence. NGR and iNGR peptides were labeled by 68Ga and assayed by HPLC and stability tests. In vitro affinity to cancer cells of the two peptides was compared by competitive cell binding assays. In vivo imaging of ⁶⁸Ga-NGR and ⁶⁸Ga-iNGR was compared by mricoPET imaging of the same nude mouse bearing HT1080 and HT29 tumors. The in vivo CD13 affinity was verified by competitive PET imaging of the same mouse with or without cold NGR blocking. The mechanism of the theoretically improved performance of iNGR via NRP1 was verified by comparison imaging by using blockade NRP1 antibody. Tumor uptake was measured by ROI method and quantified by SUV.

Results CD13 was overexpressed in HT1080 cells but not in HT29 cells. Both probes exhibited high radiochemical purity and high stability in mouse serum or saline. The CD13 binding affinity of iNGR was comparable to that of NGR.The uptake of ⁶⁸Ga-iNGR in HT1080 cells increased with incubation time, and the maximum uptake ratio was about 1.78±0.04% in HT1080 cells after 2h incubation, about 2-fold higher than that of ⁶⁸Ga-NGR. In vivo imaging showed that the HT-1080 tumors were of higher contrast to HT29 tumors at all time points after injection of ⁶⁸Ga-iNGR or ⁶⁸Ga-NGR. The HT1080 tumor uptake of ⁶⁸Ga-iNGR was significantly higher than that of ⁶⁸Ga−NGR. Moreover, tumor ⁶⁸Ga-iNGR uptake could be completely blocked by cold NGR and partly blocked by NRP1 antibody. Immunohistochemistry confirmed CD13 and NRP1 overexpression in HT1080 tumor neovasculature.

Conclusions ⁶⁸Ga-iNGR demonstrated higher tumor uptake and better tumor retention than ⁶⁸Ga-NGR through NRP1, indicating the CendR motif modification is a promising way for improving NGR peptides performance.

Research Support This work was supported by the National Natural Science Foundation of China (Grant Nos. 81230033, 81227901, 81090270, 81371594, 81401442), the National Basic Research Program of China (973 Program) (Grant No. 2011CB707704).