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.
Colorectal cancer (CRC) is the third most common cancer worldwide and accounts for the second largest number of cancer-related deaths (1). In recent years, the mortality among younger patients in both Europe and the United States has been high, despite the statistical decline at the whole-population level (2, 3). Newly diagnosed patients also showed metastasis in follow-up (4). Although CRC screening may play a greater role in the future, CRC imaging remains the primary method used to determine disease stage (5).
Colonoscopy has a high sensitivity and specificity for detecting CRC; as such, it is the gold-standard diagnostic method (6). However, its high cost means that it is unavailable to large populations in developing countries, resulting in delayed detection and treatment. Therefore, noninvasive detection using established molecular markers is an inexpensive alternative. Various biomarkers are available for this purpose; these include carcinoembryonic antigen (CEA), growth differentiation factor 15 (GDF15) (7, 8), and ephrin (Eph) receptors (9-11).
Ephrin (Eph) receptors are the largest subfamily of receptor tyrosine kinases (RTKs), and their expression is upregulated in lung, prostate, colon, pancreatic, esophageal, ovarian, thyroid, bladder and tongue cancer, as well as in hepatocellular carcinoma, glioma, melanoma, neuroblastoma and leukemia (11-16). These receptors regulate tissue development and maturation by controlling cell adhesion, migration and proliferation, which are involved in a multitude of physiological and pathological processes (17-20). The Eph family is divided into two subclasses, EPHA and EPHB, based on their affinity for binding either ephrin-A or ephrin-B ligands. Human Eph receptor type-A 2 (hEPHA2), an oncoprotein that drives pathogenesis of several tumors (21, 22), is a 130 kDa glycoprotein (9, 10) that binds to its ligand (ephrin-A) to trigger bidirectional signaling in hEPHA2-expressing (forward signaling) and ephrin-expressing (reverse signaling) cells (9, 23). Ephrin-dependent signaling plays a role in cancer cell growth, migration, and invasiveness via the RAS and AKT pathways, integrin-mediated adhesion, and epithelial-to-mesenchymal transition (10, 24, 25). Studies show that stage I and II CRC expresses hEPHA2 and its ligand ephrin A1 at much higher levels than stage III and IV CRC. This suggests that hEPHA2 plays a vital role during the early stages of oncogenesis (4, 26, 27). Hence, hEPHA2 may be a prominent biomarker for early-stage CRC. Indeed, several hEPHA2-targeting agents such as monoclonal antibodies (28, 29) and peptides (30, 31) have been studied to assess their therapeutic and diagnostic potential.
We evaluated a monobody produced from a scaffold protein derived from the tenth human fibronectin type III domain (Fn3) and an artificial binding protein with high affinity and specificity for its target (32-34). Monobodies can be fused to reporter proteins such as Renilla luciferase variant 8 (Rluc8) and enhanced green fluorescence protein (EGFP) without loss of binding activity (35). Previously, we screened EPHA2-targeting monobodies named E1 and E10, which are similar in terms of amino acid sequence; the only variation is that E1 has four amino acids in the DE loop and E10 has six. E10 has similar affinity for mEPHA6 and 8, but E1 has high affinity only for hEPHA2 (32, 36). Because successful treatment of cancer is highly dependent on detection at an early stage, and because EPHA2 is expressed at high levels by early stage CRC. In this study, we examined the utility of the E1 monobody as a diagnostic probe for CRC in a mouse model and we extended our study to imaging of clinical specimens of cancerous and non-cancerous patient colorectal tissue.
Materials and Methods
Cell lines. Human CRC cell lines HCT116, SW620, SW480, SW116, Colo205, HCT15, HT29, CaCo2 and DLD1; the prostate cancer cell line PC3; and the cervical cancer cell line HeLa were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown at 37°C/5% CO2 in RPMI 1640 (LM011-01) or DMEM (LM-001-05) containing 10% fetal bovine serum (FBS) (S 101-07; Welgene, Daegu, Republic of Korea) and 1% penicillin-streptomycin (4433, Sigma-Aldrich, St. Louis, MO, USA). All cells were subcultured under aseptic, contamination-free conditions, and observed under a microscope before every experiment to confirm absence of mycoplasma.
Purification of proteins by affinity chromatography. Expression vectors pETh-E1-Rluc8, pETh-Fn3(DGR)-Rluc8, and pETh-E1-EGFP have been described elsewhere (35). E. coli BL21Star (DE3) (C600003; Invitrogen, Waltham, MA, USA) cells were transformed with these plasmids as described previously (35), and E1-Rluc8, Fn3(DGR)-Rluc8, and E1-EGFP proteins were purified using a His trap FF column (175255015 GE Healthcare Biosciences, Pittsburgh, PA, USA) according to the manufacturer’s instructions and as described in (35).
Western blot analysis. Cell pellets (5×105) were mixed with SDS sample buffer (1052; Elpis Biotech, Daejeon, Republic of Korea) and boiled for 10 min. After centrifugation at 10,778 × g for 10 min, the proteins in the supernatants were separated in 8% SDS-PAGE gels and transferred to a nitrocellulose membrane (1620115; Bio-Rad, Hercules, CA, USA). The membrane was blocked for 1 h at 23°C with 5% skim milk (2010; Bio prince, Chuncheon, Republic of Korea) in 1 × PBS containing 0.1% Tween-20 (PBS-T), and then washed twice with PBS-T (10 min each). Next, the membrane was probed for 16 h at 4°C with an anti-hEPHA2 (1:2,000 dilution; 398832; Santa Cruz Biotechnology, Santa Cruz, CA, USA), followed by a horseradish peroxidase-conjugated secondary antibody (1:5,000 dilution; 205719; Abcam, Boston, MA, USA) for 1 h at 22°C. Each sample was prepared as triplicate and loaded to two different gels. β-actin was used as a control and was detected by an anti-β-actin antibody (1:2,000; 8224; Abcam). The proteins were visualized using an LAS-3000 image reader (Fuji Film, Tokyo, Japan). EPHA2 expression was quantified using image J software (National Institutes of Health, Bethesda, MA, USA) and relative expression was calculated by dividing with the corresponding beta actin signal.
Flow cytometric analysis. Cells were detached from culture dishes with a cell scraper and resuspended in 1 × PBS containing 1% bovine serum albumin (PBSA). Cells (5×105) were then incubated for 1 h on ice with a mouse anti-hEPHA2 diluted 1:1,000 in PBSA (3035; R&D Systems, Minneapolis, MN, USA). After washing with PBSA, the cells were incubated for 30 min on ice with an Alexa 488-conjugated anti-mouse secondary antibody (1:1,000 dilution; 11001; Invitrogen, Carlsbad, CA, USA).
To detect E1 bound to cells, the same number of cells were incubated for 1 h on ice with 50 nM E1-Rluc8 or Fn3(DGR)-Rluc8 in PBSA, followed by incubation for 30 min with a FITC-conjugated anti-6×His antibody diluted 1:1,000 in PBSA (81891; Invitrogen). Fluorescence intensity was measured using a FACS Calibur flow cytometer (BD Biosciences, San Jose, CA, USA) and data were analyzed using FlowJo 10.8.1 software (Tree Star, Ashland, OR, USA). Experiments were conducted in triplicate and unstained cells and second antibody stained samples were used as control for antibody staining and unstained cells and Fn3(DGR)-Rluc8 as control for monobody.
Immunohistochemical staining of human CRC cells. Cells (1×104) were cultured for 24 h in an 8-well chamber, followed by incubation at 23°C for 2 h with a human anti-hEPHA2 (1:500; 398832, Santa Cruz Biotechnology) or 50 nM E1-Rluc8. After washing with 1 × PBS-T containing 1% BSA, the cells were incubated for 2 h in the dark at 23°C with an Alexa 555-conjugated (1:1,000 dilution; 31570; Invitrogen) secondary antibody or an Alexa 555-conjugated anti-6×His (1:1,000 dilution; MA1-21315-A555, Invitrogen) anti-6×His tag secondary antibody. After washing, the chamber slides were mounted with Prolong Gold mounting medium containing 0.2 μg/ml 4’,6-diamidino-2-phenylindole (DAPI; 36931; Thermo Fisher Scientific). The fluorescence signals were imaged using an LSM510 confocal microscope and data were analyzed using ZEN-LSM imaging software (ZEISS, Jena, Germany). Each cell was cultured on two different wells and several images from each well were captured for analysis.
Optical imaging of tumor-bearing mice using E1-Rluc8. For the animal model, male BALB/c athymic nu-/nu- mice (aged 6 weeks; Orient Company, Seongnam, Republic of Korea) were used to conduct the experiments in unique physiological condition. Three animals were included in each group. Cancer cells were transplanted subcutaneously (5×107 cells in 100 μl of 1 × PBS), as described previously (35). Animal care, animal experiments, and euthanasia procedures were performed in accordance with protocols approved by Chonnam National University Animal Research Committee (Permit Number: HCRL 16-001). Anesthesia was performed via inhalation of 2% isoflurane. Tumors reached 100-150 mm3 after 3 weeks. E1-Rluc8 (75 μg) was injected intravenously via the tail vein. On 21 days after tumor implantation, and immediately after injection of coelenterazine (100 μg in 50 μl of 1 × PBS) (10110-1; Biotum, Hayward, CA, USA), bioluminescence signals were measured using an in vivo imaging system (PerkinElmer, Waltham, MA, USA). The photon signals were quantified after area gating against tumor tissues using Living Image 4.3 software (PerkinElmer).
Immunohistochemical staining of human CRC tissues. Paraffin slides of endoscopically resected human CRC tissues obtained at an early stage of disease were kindly provided by Chonnam National University Hwasun Hospital (Jeollanam-do, Republic of Korea). We retrospectively analyzed patient cases. Due to the nature of this study, the Institutional Review Board of Chonnam National University Hwasun Hospital reviewed and approved the research protocol and waived the requirement for informed consent (CNUHH-2020-163). Three patients with early CRC underwent endoscopic submucosal dissection, and whole resected tissues (including the tumor portion and surrounding normal portion) were prepared by paraffin embedding. Prior to analysis, the paraffin was removed by washing with xylene (3×3 min at 85°C), followed by 100% ethanol (3×3 min) and 70% ethanol (3×3 min). Next, the slides were heated for 5 min in a boiling water-bath containing citrate buffer (0.1 M, pH 6.0) and then cooled under running water. After washing three times with Tris-buffered saline containing 0.1% Tween-20 (TBS-T), the slides were placed in 3% methanol for 30 min and then washed three times with TBS-T. Slides were blocked for 10 min at 23°C with TBS-T containing 1% BSA, followed by incubation for 2 h at 23°C with either an hEPHA2 antibody (1:500 dilution; 398832; Santa Cruz Biotechnology) or E1-EGFP (100 nM). After washing, tissues stained with the hEPHA2 antibody were incubated with an Alexa 555-conjugated secondary antibody (1:1,000 dilution; 31570; Invitrogen). For co-staining, tissues were co-stained with the anti-hEPHA2 (1:500) and E1-EGFP (100 nM) for 2 h at 23°C. Imaging and analysis were performed as described for cell staining (see above). Fluorescence intensity was quantified using image J software (National Institutes of Health).
Statistical analysis. Statistical analysis was performed using a two-tailed Student’s t-test or two-way ANOVA. A p-value of <0.05 was considered statistically significant. Data are expressed as the mean±standard deviation.
Results
EPHA2 expression by human CRC cell lines. In previous studies, we developed an engineered E1 monobody which targets hEPHA2-expressing cancer using PC3 cells as a positive line and HeLa cells as a negative line (35); therefore, we also used PC3 and HeLa as controls in this study. PC3 cells, but not HeLa cells, showed high expression of hEPHA2 on the cell surface. Western blot analysis of all nine human CRC cells lines with an anti-hEPHA2 revealed that all showed high expression of hEPHA2 (Figure 1A). Cell surface expression of hEPHA2 was also evaluated by flow cytometry (Figure 1B). The fluorescence intensity of CRC and PC3 cells was significantly higher than that of HeLa cells. The mean fluorescence intensity (MFI) of CRC cells ranged from 67 to 149, whereas that for PC3 and HeLa cells was 186 and 7.25, respectively. In addition, we stained CRC cells with the anti-hEPHA2 for fluorescence imaging (Figure 1C). The fluorescence intensity of HCT116 and SW620 CRC cells was similar to that of PC3, but much higher than that of HeLa cells. Taken together, the results show that CRC cells express high levels of hEPHA2 on the cell surface.
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.
Binding of the E1 monobody to CRC cells in vitro. E1-Rluc8, an E1 monobody conjugated to the Rluc8 reporter protein was shown to bind specifically to hEPHA2-expressing PC3 cells both in vitro and in vivo (35). Fn3(DGR)-Rluc8, a Fn3 harboring an RGD-to-DGR change in the FG loop, has lost binding affinity for any protein, making it a good negative control monobody. These monobody conjugates were ligated into expression vectors and purified from E. coli. The purity and enzyme activity were confirmed by SDS-PAGE and bioluminescent imaging in the presence of coelenterazine as a substrate.
First, we used flow cytometry to measure binding of E1-Rluc8 and Fn3(DGR)-Rluc8 to CRC cells (Figure 2). When used at a concentration of 50 nM, E1-Rluc8 bound to CRC cells (Figure 2A) with MFIs ranging from 17.8 to 30.5, compared with 58 and 7.28 for PC3 and HeLa cells, respectively. Noticeably, Fn3(DGR)-Rluc8 did not show any significant binding to these cells, suggesting that the E1 monobody targets hEPHA2 expressed specifically by cancer cells.
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.
Fluorescence microscopy results confirmed functional binding of E1-Rluc8 to CRC cells (Figure 2B). As seen for the antibody in Figure 1C, E1-Rluc8 bound to HCT116 and SW620 CRC cells (but not HeLa cells) at levels similar to those observed for PC3 cells.
The E1 monobody targets CRC tumor xenografts in mice. Previously, we reported in vivo targeted imaging of the E1 monobody in hEPHA2-expressing PC3 xenograft models (35). Here, we examined targeting of E1-Rluc8 in nude mice bearing HCT116 xenografts (Figure 3). E1-Rluc8 (75 μg) was injected into the tail vein and bioluminescent signals were monitored for up to 12 h. As shown previously, signals were detected in PC3, but not HeLa, cell-derived control tumors (Figure 3A). In mice transplanted with HCT116 CRC cells, E1-Rluc8 generated a bioluminescent signal; however, Fn3(DGR)-Rluc8 did not generate bioluminescence in tumor-bearing mice (Figure 3B). When the bioluminescence signal in the tumor region was quantified, we found that the E1-Rluc8 signal was strong in PC3 and HCT116 tumors. The lack of signal in Fn3(DGR)-Rluc8-injected HCT 116 tumor-bearing mice suggests that the E1 monobody is specific for tumors expressing hEPHA2 (Figure 3C).
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.
Binding of the E1 monobody to tissue from patients with CRC. Finally, we investigated binding of the E1 monobody to tissue samples obtained from patients with CRC. All CRC tumor tissues were obtained by endoscopy at our Hospital, and all samples were sectioned into several slices. All three cases were early colon cancer treated en-bloc using an endoscopic resection technique. In samples 1 and 2, the tumor had invaded the submucosa; invasion in sample 3 was limited to the lamina propria. The slices were stained separately with an hEPHA2 antibody (1:500 dilution) or with E1-EGFP (100 nM), or co-stained with the antibody and monobody. Both the antibody and the monobody (Figure 4A and B, respectively) showed a similar pattern of binding, with high fluorescence intensity in cancer regions and no signal in non-cancer regions. The quantification of signals from the tissue showed those from both the antibody and monobody were significantly higher in the cancer region compared to non-cancer regions (Figure 4C). Interestingly, the co-stained slices showed a similar pattern of fluorescence, with strong signals in the cancer region and no signal in the non-cancer region (Figure 5A and B, respectively). There was no significant difference between antibody and monobody co-stained slices (Figure 5C). Fluorescent signals in cancer and non-cancer regions were examined in corresponding slices stained with hematoxylin and eosin staining (data not shown). The fluorescence image patterns obtained for E1-EGFP were similar to those obtained for the EPHA2 antibody. Figure 6 illustrates the binding pattern of E1 monobody to tissue from patients with CRC, suggesting that the latter can detect CRC in clinical samples as well as the hEPHA2-specific antibody.
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.
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.
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.
Discussion
Detecting cancer at an early stage is a crucial factor in determining treatment success. Conventionally, the diagnosis of precancerous and cancerous colorectal lesions is made by endoscopic examination of the entire colon with broad-band visible light (6). This can be augmented by magnifying narrow-band imaging to observe the microstructure of the mucosal enteric surface. However, improved imaging techniques are required to detect flat and indistinguishable lesions and visualize underlying biological processes. Molecular imaging is a tool that can detect and visualize specific molecular targets within organs. Various targets, including the epidermal growth factor receptor or the vascular endothelial growth factor, c-MET, glutathione S-transferase, γ-glutamyltranspeptidase, cathepsin B, or endothelin A receptors, are used for CRC because all are located at the cell surface or in the cytoplasm (37). Molecular imaging endoscopy of the digestive tract has been developed and has shown promising results in preclinical and clinical tests (38). Several monoclonal antibody-based CRC targeting molecular probes entered clinical trials (39, 40). Since these molecular probes have a higher molecular weight and require 1-2 days to reach optimum fluorescence intensity, probes with a smaller molecular weight are preferred. Furthermore, the potential for immune response makes monoclonal antibodies less ideal (42, 43).
Here, we show that an hEPHA2-specific E1 monobody (lower size compared to antibody) conjugated to a reporter protein (Rluc8 or EGFP) specifically detects CRC cells in both tumor-bearing mice and human CRC tissues. This suggests that the E1 monobody is a viable alternative to the hEPHA2-specific antibody. The monobody can be expressed and purified in E. coli, making large-scale production much less costly than that of the antibody. In the present study, we used our monobody to image clinical human tissue samples, and the results confirmed the specificity of the E1 monobody for EPHA2 in human tissues. There was no nonspecific binding in normal tissue regions.
Success of tumor-targeted imaging largely comes from the specificity of targeting agents here the FN3 DGR control monobody did not bind to CRC at either the cellular or tissue level, further confirming the specificity of the E1 monobody for CRC. The backbone of the E1 monobody is derived from human fibronectin, which is a major serum protein; therefore, we expect that it will generate no (or only a very weak) immune response.
Conjugation of the E1 monobody to reporter proteins (Rluc8 or EGFP) or chemicals (Cy5.5 or Cu64-NOTA) does not lead to loss of binding activity (35, 44). Thus, it is likely that the monobody can be conjugated to therapeutic molecules such as proteins (toxins, enzymes and cytokines) and anticancer agents. At present, we are developing various E1 monobody conjugates for use as hEPHA2-targeting therapeutics.
The study has several limitations. Firstly, only three human tissue samples were included. In addition, we did not compare different stages of CRC to confirm the utility of our monobody for stage-specific diagnosis of cancer. Therefore, examination of more CRC specimens is required, as well as prostate and other cancer types, at different stages (at which EPHA2 is expressed in differing amounts).
Conclusion
Our study showed that E1 monobody has specificity for hEPHA2 at the cellular and tissue level and similar pattern of hEPHA2 antibody. The specificity of E1 monobody for hEPHA2 makes it a cost-effective agent in targeting hEPHA2 for CRC imaging and potentially in targeted therapy.
Acknowledgements
This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) of the Korean government (2020M3A9G3080282, 2020M3A9G3080330, and 2020R1A5A2031185), and by the Chonnam National University Hwasun Hospital Institute for Biomedical science (HCRI 19008).
Footnotes
Conflicts of Interest
The Authors declare no conflicts of interest.
Authors’ Contributions
All Authors had full access to the study data and take responsibility for its integrity, and for the accuracy of the data analysis. Conceptualization: A.V., Y.H., and J.J.M.; Methodology: A.V. and Y.Z.; Investigation: A.V., Y.Z., and J.S.; Formal analysis: Y.H., W.S.L., and J.J.M.; Writing – Original Draft: A.V.; Writing – Review & Editing: A.V., W.S.L., and J.J.M.; Supervision: Y.H., W.S.L. and J.J.M.
- Received December 26, 2023.
- Revision received January 26, 2024.
- Accepted February 20, 2024.
- Copyright © 2024, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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