Engineering of an EPHA2-Targeted Monobody for the Detection of Colorectal Cancer

Abstract

Background/aim: Colorectal cancer (CRC) is the third most common cancer worldwide, and is second only to lung cancer with respect to cancer-related deaths. Noninvasive molecular imaging using established markers is a new emerging method to diagnose CRC. The human ephrin receptor family type-A 2 (hEPHA2) oncoprotein is overexpressed at the early, but not late, stages of CRC. Previously, we reported development of an E1 monobody that is specific for hEPHA2-expressing cancer cells both in vitro and in vivo. Herein, we investigated the ability of the E1 monobody to detect hEPHA2 expressing colorectal tumors in a mouse model, as well as in CRC tissue.

Materials and methods: The expression of hEPHA2 on the surface of CRC cells was analyzed by western blotting and flow cytometry. The targeting efficacy of the E1 monobody for CRC cells was examined by flow cytometry, and immunofluorescence staining. E1 conjugated to the Renilla luciferase variant 8 (Rluc8) reporter protein was used for in vivo imaging in mice. Additionally, an enhanced green fluorescence protein (EGFP) conjugated E1 monobody was used to check the ability of the E1 monobody to target CRC tissue.

Results: The E1 monobody bound efficiently to hEPHA2-expressing CRC cell lines, and E1 conjugated to the Rluc8 reporter protein targeted tumor tissues in mice transplanted with HCT116 CRC tumor cells. Finally, E1-EGFP stained tumor tissues from human CRC patients, showing a pattern similar to that of an anti-hEPHA2 antibody.

Conclusion: The E1 monobody has utility as an EPHA2 targeting agent for the detection of CRC.

Keywords: Colorectal cancer; EPHA2; imaging; monobody.

Figure 1. Expression of hEPHA2 in various cancer cell lines. (A) Western blot analysis of hEPHA2 expression. Expression of hEPHA2 was normalized to that of β-actin. (B) Flow cytometric analysis. The mean fluorescence intensity of each cell was measured (see the numbers in each panel). (C) Fluorescence microscopy images. The indicated cells were stained with an anti-hEPHA2 (red). Cell nuclei were stained with 4’,6- diamidino-2-phenylindole (DAPI: blue). The experiment was done in triplicate and only representative images are displayed. Scale bar, 10 μm.

Figure 2. Binding of E1-Rluc8 (hEPHA2 targeting monobody) to colorectal tumor cells. (A) Flow cytometric analysis of cells treated with E1-Rluc8 and Fn3(DGR)-Rluc8 (non targeting monobody). The mean fluorescence intensity of each cell stained with E1-Rluc8 was measured (see numbers in each panel). Unstained cells were used as controls. (B) Fluorescence microscopy images. Cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI: blue). The experiment was done in triplicate and only representative images are displayed. Scale bar, 10 μm.

Figure 3. In vivo imaging analysis of E1-Rluc8 (hEPHA2 targeting monobody) binding to tumor xenografts. Tumor cells were transplanted subcutaneously into mice (n=3). After injection of the E1 monobody-Rluc8 conjugate via the tail vein, luminescence images were acquired after administration of coelenterazine at the indicated times. (A) Luminescence imaging of E1-Rluc8 in control mice transplanted with PC3 and HeLa cells. (B) Luminescence imaging of mice transplanted with HCT116 cells. E1-Rluc8 (left) and Fn3(DGR)-Rluc8 (right). (C) Quantitation of luminescence intensity in the tumor tissues shown in (A) and (B). *Significantly different at p≤0.05; ns: non-significant p>0.05.

Figure 4. Binding of the E1 monobody to human colorectal cancer tissues. Tumor tissues from patients with early-stage colorectal cancer were sectioned into several slices. Separate slices were stained with the anti-hEPHA2 or with E1-EGFP [E1 monobody conjugated to enhanced green fluorescence protein (EGFP)]. Fluorescence microscopic images of antibody staining (A) and E1 monobody staining (B) in cancer and non-cancer regions. (C) Quantification of fluorescence signals generated by the antibody and monobody in cancer and non-cancer regions. Representative images of each sample are displayed. Significantly different at: *p≤0.05, **p≤0.01. Scale bar, 10 μm.

Figure 5. Co-staining of human colorectal cancer tissues with antibody and E1 monobody. Tumor tissues from patients with early-stage colorectal cancer were sectioned into several slices. Cancer (A) and non-cancer (B) regions in separate tissue slices were co stained with anti-hEPHA2 and E1-EGFP E1 monobody conjugated to enhanced green fluorescence protein (EGFP). Quantification of fluorescence signals generated by antibody and El monobody co-staining in these regions (C). Representative images of each sample are displayed. **Significantly different at p≤0.01; ns: non-significant difference, p>0.05. Scale bar, 10 μm.

Figure 6. Schematic illustration of monobody targeting human colorectal cancer (CRC) tissue. Tissues slices of human CRC tissue stained with E1 monobody. (A) Colorectal tissue slices including cancerous and non-cancerous regions. (B) Slice stained with E1 monobody; E1 monobody binds to cells in the cancerous region not in the non-cancerous region.