Colorectal cancer (CRC) such as adenocarcinoma occurred with high incidence worldwide belonging to a malignant tumor, which is diagnosed without major symptom at an early stage. Almost 60% of CRC occurs in the developed countries.1 Since early diagnosis of CRC improves the outcome of therapeutic treatment for CRC,2 it is urgent to develop a reliable and early CRC diagnostic agent. Moreover, CRC is always diagnosed occurring in late stage with metastasis, but the current diagnosing standard, colonoscopy with the histopathologic examination, is insufficient to detect the early tumor and metastasis.
Epidermal growth factor receptor (EGFR) overexpresses in the tissues of CRC by 97% as a cell membrane protein participating in cell proliferation.3,4 Literature has indicated that EGFR is a tumor target of CRC.5–7 A previous study has developed EGFR-targeted therapeutic antibodies against CRC such as cetuximab8 which is a chimeric monoclonal antibody. Besides, cetuximab is also utilized as a targeting probe carrying various diagnostic and therapeutic agents.9–11 The CRC diagnosis based on the use of cetuximab is proven a promising strategy for noninvasively tracking tumor locations and monitoring the therapeutic effects.
Compared to the gold standard, colonoscopy with histopathologic examination, in diagnosing CRC, the noninvasive nuclear imaging techniques such as single-photon emission computerized tomography/computer tomography (SPECT/CT) or positron emission tomography/computer tomography (PET/CT) provide higher sensitive diagnostic resolution and wide-screen for whole tissues in body specimen,12–14 suggesting that early tumor diagnosis and metastasis diagnosis by nuclear imaging techniques are feasible. Based on the concept of nuclear imaging methodology, this study aimed to evaluate the noninvasive nuclear imaging technique for diagnosing EGFR-positive CRC using a radioactive isotope-chelated-cetuximab, which may provide an evidence for the consequent utilization of EGFR-specific anti-tumor therapy.
In this study, we intended to label cetuximab with 111indium (111In) (half-life = 2.83 days, r-ray = 0.2454 MeV) through diethylene triamine penta acetic acid (DTPA) chelator. The optimal labeling ratio was calculated and experimentally investigated. The cell binding of cetuximab-DTPA to EGFR-positive HCT-15 cells was investigated. The reliability of nuclear imaging diagnosis using 111In -cetuximab was demonstrated in the HCT-15-induced tumor xenografts which carrying small (50 mm3) and large (250 mm3) tumors individually. Furthermore, the biodistribution of 111In-Cetuximab in tumor xenografts was also investigated.
2.1. HCT-15 culture and tumor xenograft model
Human colorectal carcinoma cells (HCT-15) were purchased from the American Type Culture Collection (ATCC) and cultured in F12K medium with 10% of fetal bovine serum. HCT15 is an EGFR-positive colorectal cancer cell line with KRAS mutation15 which model is similar to a previous study utilizing HCT116 for EGFR-nuclear imaging.10 All cells were incubated at 37 °C and 5% CO2. Male nude mice were purchased from BioLASCO Taiwan Co., Ltd, Taiwan. The 5-week-old mice were housed in a 12 h-light cycle at 22 °C. The animal studies were approved by the institutive ethical review committee in Institute of Nuclear Energy Research, Taiwan, which followed the NIH guidelines on the care and welfare of laboratory animals. HCT-15 cells (2 × 106) were subcutaneously (s.c.) inoculated into the right leg of nude mice. Tumors were established for 7 days as the small tumor model (50 mm3), and 30 days as the large tumor model (250 mm3) before the tumor imaging.
2.2. Conjugation and measurement of cetuximab-DTPA
To create cetuximab-DTPA, cetuximab was incubated with P-SCN-Bn-DTPA (w/w 1:10, Macrocyclics, Dallas, TX, USA) in carbonate-bicarbonate buffer (pH 9.0) at room temperature for 2 h. The cetuximab-conjugated DTPA was purified using G-25 column, whereas the second ml was collected. The cetuximab and cetuximab-DTPA were added with an equal volume of sinapinic acid (20 mg/ml in 50% acetonitrile/0.5% TFA) and dried on a steel plate. The molecular weights of antibodies were analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS, UltraflexIII, Bruker Daltonics GmbH, Germany).
2.3. Cetuximab-DTPA binding assay
Each HCT-15 cells (2 × 106 cells) were treated with 1 μg/ml of cetuximab-FITC for 30 min at room temperature. The 20-fold of cetuximab or cetuximab-DTPA higher than cetuximab-FITC was added simultaneously for investigating the competing capacity. After the reaction, the medium was removed and washed using phosphate-buffered saline (PBS) for three times. The cells in PBS buffer were analyzed using FACSCalibur Flow Cytometer (BD Bioscience, USA).
2.4. The labeling of 111In-cetuximab in vitro
First, the cetuximab-DTPA was mixed and incubated with 111In by 1:1 molar ratio. The specific radioactivity (SRA) of 111In was calculated as following formula: SRA (Bq/g) = λN = 0.693/half-life of 111In (seconds) x 6.03 × 1023/MW = 1.51351 × 1016. Therefore, 10 mCi of 111In = 3.7 × 108 Bq/1.51351 × 1016 = 2.445 × 10−2 μg. According to the calculated results, the cetuximab-DTPA incubated with 10 mCi of 111In was 2.445 × 10−2 μg/111 × 156176 = 34.4 μg by 1:1 molar ratio. Since the conjugated ratio between cetuximab and DTPA was measured 1:6, we expected that 5.73 μg of cetuximab could be labeled with 10 mCi of 111In molecules. In experiments, we incubated 3, 6, 12, 24, 48, 96, 192, 384, and 768 μg of cetuximab-DTPA with 10 mCi of 111In, respectively, for 30 min and 24 h in PBS buffer, pH7.4. This experiment was performed just once for reason of as low as reasonably achievable (ALARA), however, the labeling rate was measured every time when applying in animal nuclear imaging. The labeling rate >80% was acceptable as performed in the tumor xenografts. The labeling efficiency was measured using instant thin layer chromatography (iTLC) on the silica gel impregnated glass fiber sheets (PALL corporation, USA), whereas PBS was used as the mobile phase. Then, the sheets were measured using a radioactive scanner (AR-2000radio-TLC Imaging Scanner, Bioscan, France).
2.5. Nuclear imaging and biodistribution in HCT-15-induced tumor xenografts
The HCT-15-induced tumor xenografts were intravenously injected with 111In -cetuximab (n = 3) or 111In alone (n = 3) by 1 mCi of radioactivity for each mouse. A Nano-SPECT/CT (Mediso Medical Imaging Systems, USA) was utilized to detect and image the tumors in the tumor model in vivo. For investigating the biodistribution of 111In-cetuximab in EGFR-positive HCT-15-induced xenografts, the organs were harvested and measured the radioactivity using a gamma counter (1470 WIZARD, PerkinElmer, USA) after the agents and 111In injection for 48 h. The percent injected dose per gram of tissue (%ID/g) was utilized to represent the radioactive intensity in each collected organ.
2.6. Statistical analysis
Statistical analysis was performed using GraphPad Prism V5.01 software (GraphPad Software, Inc., California, USA). All analysis data with more than two groups were performed by ANOVA followed by posthoc analysis with Bonferroni's test. Student's t-test was used to compare two groups. The significance difference was acceptable as p < 0.05.
3.1. Cetuximab specifically bound to EGFR-overexpressed HCT-15 cells
To investigate EGFR expression in colorectal HCT-15 cancer cells, cetuximab was labeled with fluorescent FITC, and then the agent was purified using a G-25 column. HCT-15 cells were treated and incubated with none, 1 μg/ml of cetuximab-FITC, or 1 μg/ml of cetuximab-FITC plus 10 μg/ml of cetuximab, respectively, for 30 min at room temperature. We figured out that the fluorescent intensity in the cetuximab-FITC group was higher than that in other groups (Fig. 1A and B), revealing that cetuximab specifically bound to HCT-15 cells which overexpressed EGFR.15
3.2. Cetuximab-DTPA bound to HCT-15 cells
Cetuximab was labeled with 111In through DTPA chelator, therefore, we first conjugated cetuximab with DTPA. We added excess DTPA to cetuximab by 10-fold for maximally conjugating DTPA to available amide groups of cetuximab. MALDI-TOF MS results showed that the molecular weight 152,153Da of cetuximab shifted as 156,176Da (Fig. 2A), indicating that the conjugated ratio was approximately 1:6 between cetuximab and DTPA. In order to investigate the binding capacity of cetuximab-DTPA to HCT-15 cells, 10 μg/ml of cetuximab-DTPA was added with 1 μg/ml of cetuximab-FITC as a competitor. We found that cetuximab-DTPA reduced the binding capacity of cetuximab-FITC to HCT-15 cells (Fig. 2B and C), revealing that cetuximab-DTPA still had the binding capacity to EGFR on HCT-15 cells.
3.3. The optimal labeling ratio of cetuximab-DTPA with 111In
The theoretical labeling concentration of cetuximab-DTPA with 111In is 5.73 μg of cetuximab-DTPA to 10 mCi of 111In described in Methods and Materials. We intended to evaluate the accurate labeling ratio experimentally, 3, 6, 12, 24, 48, 96, 192, 384, 768 μg of cetuximab-DTPA were incubated with 10 mCi of 111In, respectively. We found that the amount of cetuximab-DTPA over 48 μg resulted in >80% labeling efficiency (Fig. 3A and B), indicating that the optimal labeling concentration was 48 μg of cetuximab-DTPA to 10 mCi of 111In corresponding to the theoretical calculation. The labeling efficiency of 111In-cetuximab was measured >80% in 24 h (Fig. 3A), revealing that 111In -cetuximab was stable.
3.4. 111In-cetuximab nuclear imaging and biodistribution
Since 111In-cetuximab was created, we applied 111In-cetuximab to detect the EGFR-positive tumors in the HCT-15-induced xenografts. The 111In-cetuximab was injected in the tumor models implanted with a small tumor (50 mm3) and large tumor (250 mm3) and consequently imaged using a Nano-SPECT/CT device. We found that 111In-cetuximab accumulated in the mouse liver and tumor in 24 h poster injection, including small and large tumors (Fig. 4A and B). Otherwise, 111In accumulated in kidney majorly. The radioactivity of 111In-cetuximab in the tumor was higher in small or large tumor model compared to control group. Next, the biodistribution of 111In-cetuximab was investigated compared to that of 111In. The results were consistent with the SPECT/CT images showing the higher radioactivity in tumors compared to other organs in the 111In-cetuximab-injected large tumor xenografts (Fig. 5B). Otherwise, 111In majorly distributed in the kidney (Fig. 5A). The tumor to muscle ratio of 111In -cetuximab was measured 7.5-foldwhich was higher than that of 111In group measured as 3.1-fold (Fig. 5C), indicating that 111In-cetuximab specifically bound to EGFR-positive tumors as a reliable diagnosing agent. Meanwhile, the sum of radioactivity in 111In group was higher than that in the 111In-cetuximab group (Fig. 5D). The result indicated that 111In labeled with cetuximab through chelator DTPA was easily excreted out the mice better than free 111In, suggesting that this labeling method may not lead to accumulation of 111In metal in mice.
EGFR overexpresses in a variety of cancers, including CRC,3,16 head and neck cancer,17 lung cancer.18,19 Therefore, diagnosing the EGFR-positive tumor is an important issue for selecting the adequate therapy. In this study, we intended to label cetuximab with radioactive 111In and to optimize the labeling condition for creating 111In-cetuximab serving as a diagnostic imaging tool for CRC. Our results demonstrated that 111In-cetuximab specifically targeted to EGFR-positive HCT-15-induced tumors, and the optimal labeling concentration was 48 μg of cetuximab with 10 mCi of 111In. This research provided the manufactured condition for EGFR nuclear imaging agent, 111In-cetuximab.
Targeting to EGFR based on its specific antibody is useful and potential for developing the diagnostic methodology for EGFR-positive tumors. Since utilization of radiolabeled cetuximab has been applied for diagnosing tumors such as CRC20 or head and neck tumor,21 to investigate and optimize the labeling ratio between cetuximab and isotope was needed. Although the labeling efficiency was limited around 80% when using 48 μg of cetuximab incubated with 10 mCi of 111In, the EGFR-positive tumor was clearly imaged and diagnosed. The higher amount of cetuximab with 10 mCi of 111In led to increased labeling efficiency, however, the unlabeled cetuximab played as a competitor blocking the binding of 111In-cetuximab similar to a study published by Nayak et al. using co-injection of cetuximab with 86Y-labeled cetuximab.22 Therefore, finding the optimal radio-labeled rate is necessary for improving the radio-imaging signals. Previously, Shih et al. have utilized 100 μg of cetuximab to label with 10 mCi of 111In, resulting in similar radio-labeled rate (˜80%).10 Although the radio-labeled rate >80% is acceptable for a nuclear imaging application, the extra unlabeled cetuximab may reduce the radioactive signals in real practice, leading to lower resolution of nuclear imaging. According to their data, they have declared that 111In-cetuximab leads to highest radioactive signals in 72 h after injection by tail vein. However, we not only detected the HCT-15-derived large tumors in 24 h but also detected the small tumors, implying the optimal labeling ratio was significant for nuclear imaging. Therefore, in order to obtain a better and higher radioactive imaging, we suggested that the labeling ratio: 48 μg of cetuximab with 10 mCi of 111In was adequate.
Currently, cetuximab has been labeled with 111In,10, 2389Zr,24, 2564Cu,26,27 and 99mTc.28 The half-life of 99mTc is 6 h, which is not enough applied in antibody-based nuclear imaging such as cetuximab with the apparent imaging signals after 24 h injection. 89Zr and 64Cu are PET isotopes having higher resolution than SPECT imaging such as images derived from 111In.2964Cu (half-life: 12.7 h) is potential for applying not only in diagnostic imaging but also for targeted radiotherapy due to the additional β− particles emitting. Because the half-life of circulation of antibody in the biologic body was over 63 h, and the highest imaging intensity of antibody appeals after 48 h injection, 89Zr (half-life: 3.3day) and 111In (half-life: 2.8day) are more suitable to label with cetuximab. No matter what radioactive isotopes are selected to label with cetuximab, the optimal labeling ratio is equivalent to the result demonstrated in this study.
111In-cetuximab was demonstrated to diagnose an early small tumor and advanced large tumor in this study. Due to the labeling of isotope was through DTPA chelator, further radiotherapy using β−-emitted yttrium-90 (90Y through DTPA) chelating is feasible.30–3290Y-cetuximab combined with external irradiations had been demonstrated to reduce tumor size in a 3D cell assay.33 Moreover, 111In-cetuximab may be used to diagnose the prognosis of chemotherapy, tumor metastasis, and the cetuximab-derived resistance. This study addressed and evaluated the correct labeling ratio, nuclear imaging, and biodistribution of 111In-cetuximab.
Beside tumors, 111In-cetuximab was also highly accumulated in the liver which also overexpresses EGFR.34 The biodistribution results indicated the equal radioactive intensity between tumor and liver in a large tumor model in 48 h. Since existed location of the colon is distinguished from the liver, the nuclear imaging of CRC using 111In-cetuximab may be not interfered by that from the liver. However, the acute toxicity in the liver is needed to be monitored in the performance of 111In-cetuximab, particularly in the patients with liver diseases. Moreover, for detecting the tumor metastasis in the liver, this nuclear imaging technique based on 111In-cetuximab is inadequate. Other techniques are needed for assisting the diagnosis of tumor metastasis in liver.
In conclusion, we evaluated that 111In-cetuximab was useful for detecting an EGFR-positive tumor, and optimized the labeling ratio between cetuximab and radioactive 111In. The theoretically labeling ratio was equivalent to the experimental result, which may be applied in another labeling pair of antibody and isotope. We suggest that optimal labeling of 111In-cetuximab can be used to diagnose EGFR-positive CRC.
This study was supported by the grant ARA010201 from Atomic Energy Council of Republic of China, and the grant 104-03 from Cheng Hsin General Hospital.
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