Integrin αvβ6-targeted MR molecular imaging of breast cancer in a xenograft mouse model

Background: The motif RXDLXXL-based nanoprobes allow specific imaging of integrin alpha(v)beta(6), a protein overexpressed during tumorigenesis and tumor progression of various tumors. We applied a novel RXDLXXL-coupled cyclic arginine-glycine-aspartate (RGD) nonapeptide conjugated with ultrasmall superparamagnetic iron oxide nanoparticles (referred to as cFK-9-USPIO) for the application of integrin alpha(v)beta(6)-targeted magnetic resonance (MR) molecular imaging for breast cancer. Methods: A novel MR-targeted nanoprobe, cFK-9-USPIO, was synthesized by conjugating integrin alpha(v)beta(6)-targeted peptide cFK-9 to N-amino (-NH2)-modified USPIO nanoparticles via a dehydration esterification reaction. Integrin alpha(v)beta(6)-positive mouse breast cancer (4 T1) and integrin alpha(v)beta(6) negative human embryonic kidney 293 (HEK293) cell lines were incubated with cFK-9-AbFlour 647 (blocking group) or cFK-9-USPIO (experimental group), and subsequently imaged using laser scanning confocal microscopy (LSCM) and 3.0 Tesla magnetic resonance imaging (MRI) system. The affinity of cFK-9 targeting alpha(v)beta(6) was analyzed by calculating the mean fluorescent intensity in cells, and the nanoparticle targeting effect was measured by the reduction of T2 values in an in vitro MRI. The in vivo MRI capability of cFK-9-USPIO was investigated in 4 T1 xenograft mouse models. Binding of the targeted nanoparticles to alpha(v)beta(6)-positive 4 T1 tumors was determined by ex vivo histopathology. Results: In vitro laser scanning confocal microscopy (LSCM) imaging showed that the difference in fluorescence intensity between the targeting and blocking groups of 4 T1 cells was significantly greater than that in HEK293 cells (P < 0.05). The in vitro MRI demonstrated a more remarkable T2 reduction in 4 T1 cells than in HEK293 cells (P < 0.001). The in vivo MRI of 4 T1 xenograft tumor-bearing nude mice showed significant T2 reduction in tumors compared to controls. Prussian blue staining further confirmed that alpha(v)beta(6) integrin-targeted nanoparticles were specifically accumulated in 4 T1 tumors and notably fewer nanoparticles were detected in 4 T1 tumors of mice injected with control USPIO and HEK293 tumors of mice administered cFK-9-USPIO. Conclusions: Integrin alpha(v)beta(6)-targeted nanoparticles have great potential for use in the detection of alpha(v)beta(6)-overexpressed breast cancer with MR molecular imaging.

Automated summary

Nanoprobes based on RXDLXXL motifs can specifically image integrin alpha(v)beta(6), a protein overexpressed in various tumors. The integration of cFK-9-USPIO in MRI imaging showed significant potential in detecting breast cancer overexpressing alpha(v)beta(6).