MR Reporter Gene Imaging of Endostatin Expression and Therapy

Purpose

The aim of this study is to monitor endostatin gene expression and therapy using transferrin receptor (TfR) as reporter gene and transferrin conjugate of ultrasmall supramagnetic iron oxide nanoparticle (Tf–USPIO) as magnetic resonance (MR) reporter probe.

Procedure

A retroviral plasmid (pLP-LNCX) encoding mouse endostatin and TfR was constructed, and packaged with a titer of 4?×?10⁷colony-forming units per millimeter. MDA-MB-231 breast tumors were established in BALB/c mice by subcutaneous injection of 2?×?10⁶ MDA-MB-231 cells. Mice were intratumorally injected with recombinant retrovirus and imaged with MR using Tf–USPIO. Western blot, Prussian blue, and immunohistochemical staining were performed to validate the magnetic resonance imaging results. The antitumor effect of retro-endostatin (ES)-TfR was also evaluated by intratumoral injection of the viral vector.

Results

The expression of both endostatin and TfR genes in MDA-MB-231 cells after retroviral transfection was confirmed by Western blot and flow cytometry. Tf–USPIO conjugate binds specifically to cells stably transfected with retro-ES-TfR. After intravenous injection of the Tf–USPIO conjugate, there was a more pronounced decrease in T2 relaxation time in tumors treated with retro-ES-TfR than in tumors treated with empty retrovirus retro-LNCX. The expression of ES gene significantly delayed the growth of MDA-MB-231 tumor and reduction of microvessel density and VEGF level as compared to those without viral transfection or transfected with empty retro-LNCX vector.

Conclusions

Endostatin therapeutic gene expression was visualized successfully using TfR reporter gene and Tf–USPIO MR reporter probe, which indicates that MR reporter gene imaging may be valuable in gene therapy to evaluate therapeutic gene expression and treatment efficacy.