An Optimized Integrin α6-Targeted Magnetic Resonance Probe for Molecular Imaging of Hepatocellular Carcinoma in Mice

Abstract

Introduction: Integrin α6 is an attractive diagnostic biomarker for molecular imaging of hepatocellular carcinoma (HCC) as it has an extremely high positive rate (approximately 94%) in clinical early-stage HCC. In this study, based on our previously identified integrin α6-targeted peptide, we developed an optimized integrin α6-targeted magnetic resonance (MR) probe dubbed DOTA(Gd)-ANADYWR for MR imaging of HCC in mice.

Materials and methods: The longitudinal (R₁) relaxivity of DOTA(Gd)-ANADYWR was measured on a 3.0 T MR system . The specific tumor enhancement of the agent was investigated in four distinct mouse models, including subcutaneous, orthotopic, genetically engineered and chemically induced HCC mice.

Results: The R₁ relaxivity value of DOTA(Gd)-ANADYWR is 5.11 mM^-1s^-1 at 3.0 T, which is similar to that of the nonspecific clinical agent Gadoteridol. DOTA(Gd)-ANADYWR generated superior enhanced MR signal in HCC lesions and provided complementary enhancement MR signals to the clinically available hepatobiliary MR contrast agent gadoxetate disodium (Gd-EOB-DTPA). Importantly, DOTA(Gd)-ANADYWR could efficiently visualize small HCC lesion (approximately 1 mm) which was hardly detected by the clinical Gd-EOB-DTPA.

Conclusion: These findings suggest the potential application of this integrin α6-targeted MR probe for the detection of HCC, particularly for small HCC.

Keywords: hepatocellular carcinoma; integrin α6; magnetic resonance; peptide; probe.

Graphical abstract

Figure 1

Chemical structure and characterization of DOTA(Gd)-ANADYWR. (A) chemical structure of DOTA(Gd)-ANADYWR. (B) HPLC analysis of DOTA(Gd)-ANADYWR. (C) MALDI-TOF mass spectra of DOTA(Gd)-ANADYWR. (D) Plots of R₁ (1/T1) versus different gadolinium concentration of DOTA(Gd)-ANADYWR. (E) Flowchart of the study design.

Figure 2

MR imaging with DOTA(Gd)-ANADYWR and Gadoteridol in subcutaneous HCC-LM3 tumor-bearing mice. (A) Representative coronal T2-weighted MR image before administration. The red arrowhead indicated the HCC. (B) Contrast-to-noise ratio (CNR) of tumors before and after injection of DOTA(Gd)-ANADYWR or Gadoteridol. Statistical analysis was performed using two-tailed Student’s t-test. Significance levels are given as: NS P>0.05, *P < 0.05. (C) Representative T1-weighted MR images obtained before and at different time points after intravenous administration of DOTA(Gd)-ANADYWR or Gadoteridol (The red arrowheads indicated the HCC. The red dotted circles delineated the area of interest that used for CNR analysis).

Figure 3

MR imaging with DOTA(Gd)-ANADYWR in an orthotopic HCC-LM3-luciferase tumor-bearing mouse. (A) Representative bioluminescence image in a mouse bearing an HCC lesion. (B) Representative T1-weighted MR images and corresponding rainbow pseudo color images obtained before and 30 min after DOTA(Gd)-ANADYWR injection in the same mouse (The red arrowheads indicate the orthotopic HCC-LM3 tumor xenografts and the red tailless arrowheads indicate the surgical incision sites. The red dotted circles indicated the visible HCC).

Figure 4

MR imaging with DOTA(Gd)-ANADYWR in a c-Myc transgenic mouse. (A) Representative T1-weighted MR images and corresponding rainbow pseudo color images for a c-Myc transgenic mouse bearing an HCC lesion (The red arrowheads indicated the HCC. The red dotted circles delineated the area of interest that used for CNR analysis). (B) The increase CNR values of tumor before and at different time points after injection of DOTA(Gd)-ANADYWR. (C) Integrin α6 expression was analyzed using immunohistochemistry IHC in tumor tissue (Scale bar, 50μm).

Figure 5

MR imaging with DOTA(Gd)-ANADYWR and Gd-EOB-DTPA in a DEN-induced HCC mouse. (A) Representative MR images and corresponding rainbow pseudo color images obtained before and at 5, 25 min after injection with DOTA(Gd)-ANADYWR or Gd-EOB-DTPA in a DEN-induced HCC mouse bearing an HCC lesion. (B) The corresponding gross photograph of the same lesion shown in (A) (The red arrowheads indicated the HCC. The red dotted circles indicated the visible HCC). (C) Integrin α6 expression was analyzed using immunohistochemistry IHC in tumor tissue (Scale bar, 50μm).