Published online Jun 28, 2019. doi: 10.3748/wjg.v25.i24.3030
Peer-review started: February 25, 2019
First decision: March 20, 2019
Revised: April 3, 2019
Accepted: May 18, 2019
Article in press: May 18, 2019
Published online: June 28, 2019
Processing time: 123 Days and 23.6 Hours
Hepatocellular carcinoma (HCC) ranks second in terms of cancer mortality worldwide. Molecular magnetic resonance imaging (MRI) targeting HCC biomarkers such as alpha-fetoprotein (AFP) or glypican-3 (GPC3) offers new strategies to enhance specificity and help early diagnosis of HCC. However, the existing iron oxide nanoparticle-based MR molecular probes singly target AFP or GPC3, which may hinder their efficiency to detect heterogeneous micro malignant HCC tumors < 1 cm (MHCC).
We hypothesized that the strategy of double antibody-labeled iron oxide nanoparticles which simultaneously target AFP and GPC3 antigens may potentially be used to overcome the tumor heterogeneity and enhance detection rate for MRI-based MHCC diagnosis, including the sensitivity and specificity.
The main objective of the current research was to synthesize an AFP/GPC3-double antibody-labeled iron oxide MR molecular probe and to assess its impact on MRI specificity and sensitivity at the cellular level. The preliminary in vitro data could help to optimize the key factors of MRI molecular probe design including labeled biomarkers and hydrodynamic size for future in vivo experiments.
The double antigen-targeting MRI probe for MHCC anti-AFP–USPIO–anti-GPC3 (UAG) was developed by simultaneously conjugating alpha-fetoprotein (AFP) and glypican-3 (GPC3) antibodies to a 5 nm ultra-small superparamagnetic iron oxide nanoparticle (USPIO). At the same time, the singly labeled probes of anti-AFP–USPIO (UA), anti-GPC3–USPIO (UG), and non-targeted USPIO (U) were also prepared for comparison. The physical characterization including morphology (transmission electron microscopy), hydrodynamic size, and zeta potential (dynamic light scattering) was conducted for each of the probe. The antigen targeting and MR imaging ability for these four kinds of USPIO probes were studied in the GPC3-expressing murine hepatoma cell line, Hepa1-6/GPC3. First, AFP and GPC3 antigen expression in Hepa1-6/GPC3 cells was confirmed by flow cytometry and immunocytochemistry. Then, the cellular uptake of USPIO probes was investigated by Prussian blue staining assay and in vitro MRI (T2-weighted and T2-map) with a 3.0 Tesla clinical MR scanner. The sensitivity and specificity were evaluated based on the cellular uptake of four kinds of USPIO probes at the same dosage of iron concentration.
The in vitro data showed that the double antibody-conjugated probe UAG had the best specificity in targeting Hepa1-6/GPC3 cells expressing AFP and GPC3 antigens (vs other USPIO probes including single antibody-labeled and unlabeled USPIOs). The iron Prussian blue staining and quantitative T2-map MRI analysis showed that, compared with UA, UG, and U, the uptake of the double-targeting UAG probe demonstrated a 23.3% (vs UA), 15.4% (vs UG), and 57.3% (vs U) increased Prussian stained cell percentage and a 14.93% (vs UA), 9.38% (vs UG), and 15.3% (vs U) reduction of T2 relaxation time, respectively. Such bi-specific probe might have the potential to overcome tumor heterogeneity with enhanced sensitivity and HCC specificity. Meanwhile, the coupling of two antibodies did not influence the magnetic performance of USPIO and the relatively small hydrodynamic size (59.60 ± 1.87 nm) of the double antibody-conjugated USPIO probe makes it a viable candidate for use in MHCC MRI in vivo, as they are slowly phagocytosed by macrophages. AFP and GPC3 were chosen based on clinical considerations. However, the cytoplasmic expression of AFP raised the complexity of the study in terms of probe internalization. The exact internalization route for such cytoplasmic antigen-targeted probes and whether the secreted AFP proteins contribute to probe internalization require detailed studies in the future.
The iron Prussian blue staining assay and in vitro MRI results confirmed that the bi-specific probe presents enhanced targeting efficiency and MRI sensitivity to HCC cells than singly- or non-targeted USPIO. Therefore, it implies that the multi-targeting strategy may be potentially applied in MRI probe design to enhance the malignant tumor recognition and MRI detection efficiency of MHCC for animal experiments and early clinical diagnosis.
The current research utilized monoclonal antibodies against AFP and GPC3 to ensure the specificity and purity. In future in vivo studies or investigation of cytoplasmic targeting by USPIO, small antibody fragments possessing smaller molecular weights might be more effective. In addition, to further reduce non-specific uptake of USPIO by the reticuloendothelial system or mononuclear phagocytic system, surface modifications, such as hydrophilic PEG coatings for the USPIO, could be also considered in the in vivo experiments.