Bi-specific T1 positive-contrast-enhanced magnetic resonance imaging molecular probe for hepatocellular carcinoma in an orthotopic mouse model

Abstract

Background: Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related mortality. HCC-targeted magnetic resonance imaging (MRI) is an effective noninvasive diagnostic method that involves targeting clinically-related HCC biomarkers, such as alpha-fetoprotein (AFP) or glypican-3 (GPC3), with iron oxide nanoparticles. However, in vivo studies of HCC-targeted MRI utilize single-target iron oxide nanoprobes as negative (T2) contrast agents, which might weaken their future clinical applications due to tumor heterogeneity and negative MRI contrast. Ultra-small superparamagnetic iron oxide (USPIO) nanoparticles (approximately 5 nm) are potential optimal positive (T1) contrast agents. We previously verified the efficiency of AFP/GPC3-double-antibody-labeled iron oxide MR molecular probe in vitro.

Aim: To validate the effectiveness of a bi-specific probe in vivo for enhancing T1-weighted positive contrast to diagnose the early-stage HCC.

Methods: The single- and double-antibody-conjugated 5-nm USPIO probes, including anti-AFP-USPIO (UA), anti-GPC3-USPIO (UG), and anti-AFP-USPIO-anti-GPC3 (UAG), were synthesized. T1- and T2-weighted MRI were performed on day 10 after establishment of the orthotopic HCC mouse model. Following intravenous injection of U, UA, UG, and UAG probes, T1- and T2-weighted images were obtained at 12, 12, and 32 h post-injection. At the end of scanning, mice were euthanized, and a histologic analysis was performed on tumor samples.

Results: T1- and T2-weighted MRI showed that absolute tumor-to-background ratios in UAG-treated HCC mice peaked at 24 h post-injection, with the T1- and T2-weighted signals increasing by 46.7% and decreasing by 11.1%, respectively, relative to pre-injection levels. Additionally, T1-weighted contrast in the UAG-treated group at 24 h post-injection was enhanced 1.52-, 2.64-, and 4.38-fold compared to those observed for single-targeted anti-GPC3-USPIO, anti-AFP-USPIO, and non-targeted USPIO probes, respectively. Comparison of U-, UA-, UG-, and UAG-treated tumor sections revealed that UAG-treated mice exhibited increased stained regions compared to those observed in UG- or UA-treated mice.

Conclusion: The bi-specific T1-positive contrast-enhanced MRI probe (UAG) for HCC demonstrated increased specificity and sensitivity to diagnose early-stage HCC irrespective of tumor size and/or heterogeneity.

Keywords: Alpha-fetoprotein; Glypican-3; Hepatocellular carcinoma; Magnetic resonance imaging; Molecular imaging; Positive contrast agent.

Figure 1

Experiment workflow. Probe treatment and T1- and T2-weighted magnetic resonance imaging were performed on day 10 after establishment of the orthotopic hepatocellular carcinoma mouse model. MRI: Magnetic resonance imaging; HCC: hepatocellular carcinoma; UA: Anti-AFP-USPIO; UG: Anti-GPC3-USPIO; UAG: Anti-AFP-USPIO-anti-GPC3.

Figure 2

Magnetic resonance imaging properties of ultra-small superparamagnetic iron oxide phantoms. A: T1- and T2-weighted images of a series of 0.9% saline water solutions containing different concentrations of the ultra-small superparamagnetic iron oxide probes as indicated by iron concentration; B: Changes in the T1- and T2-weighted signal intensities according to iron concentration, with standard deviation also illustrated; C: T1 and T2 map illustrated in pseudo color under different iron concentration; D: Linear regression fitting of the longitudinal relaxation-rate (1/T1); E: transversal relaxation-rate (1/T2) data vs different iron concentrations (with standard deviation also illustrated) for extracting the longitudinal relaxivity (r₁) and transverse relaxivity (r₂), respectively.

Figure 3

T1-weighted images of hepatocellular carcinoma-model mice administered with ultra-small superparamagnetic iron oxide probes at various time points. Columns represent livers treated with different probes, and the first row shows pre-injection (0 h) T2-weighted images as references for the tumor location indicated by yellow arrowhead. UA: Anti-AFP-USPIO; UG: Anti-GPC3-USPIO; UAG: Anti-AFP-USPIO-anti-GPC3.

Figure 4

T2-weighted images of hepatocellular carcinoma-model mice administered ultra-small superparamagnetic iron oxide probes at various time points. Columns represent livers treated with different probes. (Yellow arrowhead: tumor location). UA: Anti-AFP-USPIO; UG: Anti-GPC3-USPIO; UAG: Anti-AFP-USPIO-anti-GPC3.

Figure 5

Time-dependent increasing rates of T1- and T2-weighted T/B signal ratios. Increasing rates of T1- and T2-weighted T/B signal ratio at different time points (12, 24, and 32 h) post-injection as compared with pre-injection (0 h) of the U, anti-alpha-fetoprotein (AFP)-ultra-small superparamagnetic iron oxide (USPIO), anti-glypican-3 (GPC3)-USPIO, and anti-AFP-USPIO-anti-GPC3 probes. Data points represent averaged rates from two mice if the image quality was acceptable. A: T1-weighted T/B signal ratios; B: T2-weighted T/B signal ratios. UA: Anti-AFP-USPIO; UG: Anti-GPC3-USPIO; UAG: Anti-AFP-USPIO-anti-GPC3.

Figure 6

T1- and T2-weighted images of hepatocellular carcinoma-model mice treated with the anti-alpha-fetoprotein-ultra-small superparamagnetic iron oxide-anti-glypican-3 probe (1.6 mg Fe/kg body weight). Top (T1WI) and bottom (T2WI) rows, from left to right: T1- and T2-weighted images, respectively, at 0 h (pre-injection) and 12-, 24-, and 32-h (post-injection). (Yellow arrowhead: Tumor location).

Figure 7

Histological results of hepatocellular carcinoma tumors treated with U, anti-alpha-fetoprotein-ultra-small superparamagnetic iron oxide, anti-glypican-3-ultra-small superparamagnetic iron oxide, and anti-alpha-fetoprotein-ultra-small superparamagnetic iron oxide-anti-glypican-3 probes. UA: Anti-AFP-USPIO; UG: Anti-GPC3-USPIO; UAG: Anti-AFP-USPIO-anti-GPC3; USPIO: Ultra-small superparamagnetic iron oxide; AFP: Alpha-fetoprotein; GPC3: Glypican-3.