Primary liver cancer, also known as hepatocellular carcinoma (HCC), is the fifth most common neoplasm and the third leading cause of cancer-related deaths worldwide [1
]. Symptoms of HCC at early stage are usually atypical, and thus, HCC patients often present with symptoms at an advanced stage. Only 10%–20% of HCC are diagnosed early enough for appropriate surgical treatment [4
]. The poor prognosis of this disease is largely due to the lack of effective and accurate early diagnostic methods, causing most patients to be diagnosed at the late stages, which seriously limits treatment options. Therefore, highly sensitive and accurate molecular imaging techniques that allow early HCC detection are urgently needed.
Currently, the most commonly used positron emission tomography (PET) probe for tumor imaging is 18
F-FDG). However, the use of 18
F-FDG-PET in the detection of HCC is rather limited, and it was reported that 18
F-FDG could even miss 30%–50% of HCC lesions in the liver [7
]. Another PET probe commonly used for detection of HCC is 11
C-labeled acetate, which was reported to show higher sensitivity than 18
]. It plays a complementary role to 18
F-FDG in both HCC cell lines and human HCC detection, being able to detect HCC tumors with low 18
F-FDG uptake only. But similar as 18
C-labeled acetate is a largely nonspecific probe for HCC imaging, as it typically enters the tricarboxylic acid cycle as a substrate for β
-oxidation in fatty acid synthesis [10
]. Development of molecular probes suitable for imaging other HCC associated biomarkers is thus considered as a promising strategy whereas a largely unexplored field.
The contextual complexity of understanding HCC is defined by the functional involvement of several signaling cascades (epidermal growth factor, insulin-like growth factor, RAS, WNT-β
catenin, etc.) as well as multiple risk factors (such as hepatitis B and C viral infection and alcohol abuse) [1
]. Among them, epidermal growth factor (EGF) signaling is one of the most thoroughly evaluated signaling cascades in human HCC development. EGF is demonstrated to control proliferation, differentiation, and cell survival and is overexpressed in a wide range of solid tumors including HCC [13
]. The growth factor receptor (EGFR) is a receptor tyrosine kinase that regulates a number of key processes, including cell proliferation and differentiation, tissue homeostasis, and tumorigenesis [15
]. Dysregulation of EGFR expression is associated with several key features of cancer, such as autonomous cell growth, apoptosis inhibition, invasion, and metastasis [17
]. Overexpression of EGFR has been frequently detected in a wide range of human tumors, including non-small-cell lung cancer, gastric cancer, breast cancer, as well as liver cancer [19
]. In HCC, there is increasing evidence demonstrating a correlation between EGFR overexpression and tumor aggressiveness, metastasis formation, therapy resistance, and poor prognosis of this disease [15
]. Functional involvement of EGFR in HCC development was best demonstrated by the observation that EGFR inhibitor, Gefitinib, can significantly reduce HCC incidence in a genotoxic animal model of HCC [23
]. Taken together, EGFR represents an attractive target for small molecules or antibodies in applications such as tumor-targeted imaging and therapy.
Several anti-EGFR affibody molecules (ZEGFR
) with high affinities (in nanomolar range) have been reported recently. Among them, the affibody molecule, ZEGFR:1907
, has been shown to specifically bind EGFR with no cross-binding to other growth factor receptors [24
], as well as fast tumor targeting and excellent tumor-to-normal tissue contrast on EGFR-expressing xenografted epithelial cancer models [25
]. Affibody molecules are small (approximately 7
kDa), engineered proteins with 58-amino acid residues and a three-helix bundle scaffold structure [24
]. Its small molecular weight, high stability, high binding specificity, and affinity make it an excellent probe for tumor-targeted imaging in vivo
]. In this study, we hypothesized that EGFR targeted affibody probes can be promising molecular probes for HCC detection. Two types of affibody-based probes, 64
for PET, and Alexa680-ZEGFR:1907
for near-infrared fluorescent (NIRF) imaging (), were evaluated and compared for molecular imaging of three type of HCC xenograft models. It is expected that the EGFR targeted NIRF probe can not only image HCCs noninvasively but also provides a tool for image-guided therapy, whereas the PET probe can find more broad applications for clinical cancer imaging.
Schematic structure of affibody-based PET and NIRF probes. Different probes were used in various imaging studies (64Cu-DOTA-ZEGFR:1907 for PET and Alex680-ZEGFR:1907 for optical imaging).