Development of a Peptide-based Cancer-specific Magnetic Resonance Contrast Agent Targeting CXCR4 and Validation of Its Effects in Three Cancer Cell Lines

  Objective  To construct a cancer-specific magnetic resonance contrast agent targeting C-X-C motif chemokine receptor 4 (CXCR4) on cancer cell surface and to discuss its ability to quantify the CXCR4 expression level of various cancer cells in vitro through the changes of magnetic resonance (MR) signal.

  Methods  Cellular immunofluorescence and flow cytometry assays were introduced to observe CXCR4 expression pattern and to quantify CXCR4 expression level in 3 different cancer cell lines (pancreatic cancer cell line PANC-1, breast cancer cell line MCF-7, and lung cancer cell line A549), respectively. By replacing the CXCR4 monoclonal antibody with a novel peptide, Pep12, we carried out these experiments again in the same condition, to prove its ability to bind to CXCR4 specifically. Ultrasmall paramagnetic iron oxide nanoparticle(USPIO-Np) was synthesized de novo and conjugated to Pep12 after surface modification. Dynamic light scattering (DLS) method was introduced to measure its hydro diameter, MTS assay was performed to test its cell toxicity, and 1.5T MR scan were carried out to evaluate the T2/T2^* signal changes. Prussian blue staining was introduced to observe the binding pattern of Pep12-USPIO to 3 cancer cell lines, and MR scanning of cells cultured with Pep12-UPSIO were done to evaluate its ability to quantify the CXCR4 expression level on different cancer cells by T2/T2^* signal changes.

  Results  CXCR4 expression was observed in different patterns and levels in all 3 cancer cell lines. Flow cytometry showed that the CXCR4 positive cell proportions were 18.7% in PANC-1, 2.9% in A549, and 1.7% in MCF-7, respectively. Pep12 was able to bind to all 3 cancer cell lines specifically in a CXCR4 level dependent manner (r=0.999, P=0.027). Pep12-USPIO formed stable aqueous colloid in PBS/water under room temperature. The hydro diameter was (86.60±1.48) nm. The cytoxicity of Pep2-USPIO was low. When the concentration of iron was less than 25 μg/ml, the values of △R2 and △R2^* were in line with concentration of iron (△R2:R²=0.996;△R2^*:R²=0.977). Prussian blue stain showed pep12-USPIO was bound to PANC-1, A549, and MCF-7 cells, while USPIO alone could not. PANC-1, A549, and MCF-7 cells were incubated with Pep12-USPIO/USPIO, and underwent MR scan. A significant T2/T2^* signal dropdown was observed in Pep12-USPIO-incubated cell suspension, while USPIO-incubated cell suspension only had slight T2/T2^* signal change(P < 0.01). The value of △R2/△R2^* change had positive correlation with the expression level of CXCR4 in those tumor cells(△R2:r=0.997, P=0.050; △R2^*:r=1.000, P=0.019).

  Conclusions  Pep12-USPIO is stable and hypotoxic. It can specifically bind to CXCR4-expressing cancer cells and produce MR signal change. The value of T2/T2^* change may be used for the prediction of CXCR4 expression.