Nuclear Medicine and Biology 42 (2015) 505–512

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Targeting cancer stem cells with an 131I-labeled anti-AC133 monoclonal antibody in human colorectal cancer xenografts☆ Juntao Lang a,b, Xiaoli Lan a, Yu Liu a, Xueyan Jin a, Tao Wu a,c, Xun Sun a, Qiong Wen a, Rui An a,⁎ a b c

Department of Nuclear Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology; Hubei Province Key Laboratory of Molecular Imaging, Wuhan 430022, China Department of Nuclear Medicine, Zhongshan Hospital, Fudan University, Shanghai, 200032, China Department of Nuclear Medicine, The Second People's Hospital of Wuhu, Wuhu 241000, China

a r t i c l e

i n f o

Article history: Received 28 September 2014 Received in revised form 21 December 2014 Accepted 3 January 2015 Keywords: cancer stem cells monoclonal antibody radioimmunoimaging single positron emission computed tomography (SPECT)

a b s t r a c t Introduction: Cancer stem cells (CSCs) are a subpopulation within a tumor, which possesses the characteristics of selfrenewal, differentiation, tumorigenicity, and drug resistance. The aim of this study was to target the colorectal CSC marker CD133 with an131I-labeled specific monoclonal antibody (AC133 mAb) in a nude mouse xenograft model. Methods: Colorectal adenocarcinoma cells (LoVo cell line) were separated into CD133(+) and CD133(−) cells by magnetic activated cell sorting. CD133(+), CD133(−), and unsorted LoVo cells were cultured and then implanted subcutaneously into the lower limbs of nude mice (n = 5). AC133 mAb was labeled with 131I by the iodogen method. Results: The radiolabeled compound, 131I-AC133 mAb, showed high stability, specificity, and immunoactivity in vitro. Obvious accumulation of 131I-AC133 mAb was seen in nude mice bearing xenografts of CD133(+) and unsorted LoVo cells, but no uptake was found in mice bearing CD133(−) xenografts or specifically blocked xenografts. Biodistribution analysis showed that the tumor uptake of 131I-AC133 mAb was 6.97 ± 1.40, 1.35 ± 0.48, 6.12 ± 1.91, and 1.61 ± 0.44% ID/g (n = 4) at day 7 after injection of 131I-AC133 mAb in CD133(+), CD133(−), unsorted LoVo cell and specifically blocked xenografts, respectively. The results of immunofluorescence, autoradiography, and western blotting further verified the specific binding of 131I-AC133 mAb to CD133(+) tumors. Conclusions: This study demonstrates the possibility of targeting CSCs with a radiolabeled AC133 mAb in colorectal cancer xenografts based on in vitro, ex vivo, and in vivo experiments. Our findings suggest a new method for imaging CSCs non-invasively. © 2015 Elsevier Inc. All rights reserved.

1. Introduction Colorectal cancer is one of the most common malignant tumors of the digestive system. It is the third leading gastrointestinal malignancy in China [1], and the incidence rate of colorectal cancer is the third highest in the western world [2]. Intensive research and investigation of various new medicines, techniques, and strategies have been conducted for colorectal cancer, which has resulted in a decline of its mortality rate [3]. However, mortality caused by colorectal cancer is still the second highest because of drug resistance, recurrence, and metastasis [4]. Recently, some researchers have suggested that the reason for these phenomena may be related to the existence of cancer stem cells (CSCs) in the remaining malignant tumor after treatment [5–7]. CSCs, also called tumor-initiating cells [8], are a small population within the tumor, which possesses the characteristics of self-renewal and differentiation, and can cause relapse and metastasis by giving

☆ Funding support: This study was supported by National Natural Science Foundation of China (No.81071178). ⁎ Corresponding author at: No.1277, Jiefang Ave., Wuhan, Hubei Province, 430022, China. Tel./fax: +86 27 85726877. E-mail address: [email protected] (R. An). http://dx.doi.org/10.1016/j.nucmedbio.2015.01.003 0969-8051/© 2015 Elsevier Inc. All rights reserved.

rise to new tumors. After Bonnet et al. [9] first isolated CSCs from acute myeloid leukemia in 1977, there has been accumulating evidence showing that CSCs are not only present in hematological tumors but also in some solid malignancies such as breast [10], brain [11,12], colorectal [13], and prostate cancers [14], melanomas [15], head and neck carcinoma [16], and hepatocellular carcinoma [17]. CSCs are highly tumorigenic [10,12–14,16,18,19], and as few as 100 brain CSCs are capable of initiating tumors in non-obese/severe combined immunodeficient mice, which phenotypically resemble the original tumor from the patient [12]. Furthermore, CSCs are highly resistant to chemotherapy [20–25] and radiotherapy [26–30], which may be responsible for tumor recurrence and metastasis after therapy [5–7]. The concept of CSCs [31] provides a new approach for the diagnosis and treatment of malignant tumors, and has gradually become a hot topic in oncological research. The identification of specific CSC biomarkers provides the possibility for targeting CSCs in vivo. Currently, many molecular markers have been found in CSCs such as CD44, CD133, nestin, Musashi, and aldehyde dehydrogenase 1. Of all the markers, CD133 is the one of the most intensively studied biomarkers, and it is referred to as a “molecule of the moment” [32]. CD133 is a glycosylated five-transmembrane domain polypeptide with a molecular weight of 120 kDa [18,33], which shows restricted expression within plasma membrane protrusions. It was first detected in hematopoietic

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stem cells [18]. Some recent studies [11,13,34,35] show that CD133 is expressed in a variety of human tumors, resulting in its consideration as a new marker of CSCs. Collins et al. found that CD133(+) and CD44(+) prostate cancer cells with high expression of integrin α2β1 are capable of proliferation, self-renewal, and multilineage differentiation in vivo [14]. In another study, CSCs expressing CD133 showed the abilities of self-renewal, differentiation and tumorigenicity in brain tumors, but CD133(−) cells did not exhibit such abilities [12]. O’Brien et al. [13] and Ricci-Vitiani et al. [19] also showed that CD133(+) colorectal cancer cells have the same stem cell potential, and as few as 262 CD133(+) colorectal cancer cells can reproduce the original tumor in immunodeficient mice, whereas CD133(−) and CEA(+) cells cannot form a tumor. Other researchers further showed that CD133(+) colorectal CSC are relevant to drug resistance [20,36,37], recurrence, and metastasis [5,38]. All of these studies suggest that CD133 is an important biomarker of CSCs. In the majority of studies related to CD133 and CSCs, AC133 and its monoclonal antibody (AC133 mAb) are the most extensively used factors (8, 9). AC133 is a glycosylated epitope in the extracellular portion of CD133, which shows specific expression in CSCs (29) and loss of expression after CSC differentiation (34). To date, monitoring and imaging CSCs in vivo are challenging, but they are of great significance. Limited research has been performed on imaging of CSCs in vivo. Tsurumi et al. [39] targeted CSCs with dye-labeled AC133 mAb for optical imaging in a mouse xenograft model. Jin et al. labeled several anti-CD133 monoclonal antibodies with 125I and examined the biodistribution and intratumoral distribution of CD133 in HCT116 xenograft-bearing mice [40]. To the best of our knowledge, no comprehensive investigations have been performed to monitor colorectal CSCs in vivo by radioimmunoimaging. Therefore, in our study, we aimed to target CD133 in colorectal CSCs with an131I-labeled AC133 mAb using a nude mouse xenograft model. 2. Materials and methods 2.1. Cell lines The human colorectal adenocarcinoma cell line LoVo with a 50% expression rate of cell surface CD133 [41] was obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/ F12 (1:1) (Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (Hyclone) and 1% penicillin and streptomycin (Sigma) at 37 °C in a humidified 5% CO2-containing incubator. Sorted cells (see below) were maintained in serum-free DMEM/F12 (1:1) containing 20 ng/ml epidermal growth factor (Sigma) and 10 ng/ml basic fibroblast growth factor (Sigma). AC133.1 hybridoma cells were purchased from the American Tissue Culture Collection (HB12346) and cultured in DMEM containing 10% fetal bovine serum at 37 °C in a humidified 5% CO2-containing incubator. 2.2. Cell separation and identification LoVo cells were isolated by magnetic activated cell sorting (MACS, MiltenyiBiotec, Germany) according to the manufacturer's instructions. Briefly, the cells were harvested from culture dishes by trypsinization (0.25% trypsin-EDTA) and prepared as a single cell suspension. After incubating the single cell suspension with CD133 microbeads (MiltenyiBiotec) at 4 °C for 30 min, CD133(+) LoVo cells were sorted twice with MS columns (MiltenyiBiotec), and CD133(−) LoVo cells were isolated using LD columns (MiltenyiBiotec). After MACS, the cells were immediately analyzed for CD133 expression by flow cytometry and direct immunofluorescence staining. 2.3. Antibody purification The mouse ascites method was used to produce the AC133 mAb, and purification was performed with Protein G beads (GE Healthcare Life

Sciences). The concentration of the purified AC133 mAb was determined by a Bradford protein assay kit (Beyotime Institute of Biotechnology, China). 2.4. Monoclonal antibody labeling and purification AC133 mAb was labeled with 131I by the iodogen method [42]. Glass vials coated with 50 μg iodogen (Sigma) in dichloromethane were prepared and dried with N2 gas. AC133 mAb (50 μg/25 μl), 131I-NaI (55.5 MBq/25 μl, no carrier and reducer, Beijing Atom High Tech, Beijing, China), and phosphate-buffered saline (PBS, 50 μl of 0.2 M, pH 7.4) were added to the iodogen-coated vial. After reacting for 8–10 min at room temperature, the labeled AC133 mAb was purified by ultrafiltration (Amicon Ultra-4 centrifugal filter, 10 kDa) [43], and the labeling yield was calculated. The radiochemical purity of the labeled AC133 mAb was determined by instant thin layer chromatography on a γ-scanner. To verify stability, the 131I-AC133 mAb was incubated in PBS (pH 7.4) for 1, 3, 5, 7, and 10 days [44]. 2.5. In vitro experiment: cell binding assays The binding ratios of unsorted LoVo, CD133(+), and CD133(−) cells with 131I-AC133 mAb were checked in quadruplicate. The specific blocking experiment was performed by incubating LoVo cells with a 100-fold excess of unlabeled AC133 mAb before addition of 131I-AC133 mAb. Cells (1 × 106) were suspended in 1 ml ice-cold PBS and incubated with 37 KBq131I-AC133 mAb at 37 °C. At 15, 30, 60, 120, and 240 min, 1 × 10 5 cells in 0.1 ml were analyzed for radioactivity as ‘T’ by a γ-counter. Then, the cells were washed twice with PBS, and the binding radioactivity was measured as ‘B’. The binding ratio was defined as: binding ratio = B/T. 2.6. In vivo experiment: SPECT/CT imaging and biodistribution study The animal experiments were approved by the Institutional Animal Care and Use Committee of Huazhong University of Science and Technology. A total of 1 × 106 unsorted LoVo, CD133(+), or CD133(−) cells were subcutaneously injected into the left lower limbs of 4–5-week-old BALB/ c-nu mice weighing 18–20 g (Beijing HFK Bioscience). When the diameter of the tumors reached 10 mm after 3–4 weeks, the tumor-bearing mice were intravenously injected with 14.8–18.5 MBq131I-AC133 mAb (20–25 μg) for imaging. In specific blocking experiments, a 40-fold excess of unlabeled AC133 mAb (1 mg) was used to block the binding of the tracer at 30 min before injection of 131I-AC133 mAb. At 3 days before imaging, 1‰ of iodine was added to the drinking water to block thyroid uptake of free 131I. At 1, 3, 5, and 7 days after tracer injection, in vivo imaging was performed by SPECT/CT (Siemens Symbia T6, Germany). The imaging conditions were as follows: SPECT/CT equipped with a parallel hole, high-energy collimator with a matrix of 256 × 256 and zoom of 3.2. The SPECT projection data were acquired at 45 s per projection. After imaging, the mice were sacrificed, and blood, tumor, brain, thyroid, liver, spleen, kidney, stomach, small intestine, colorectal intestine, lung, heart, muscle, and bone were extracted and weighed. The amount of radioactivity in each organ was measured with a γ-counter. The radioactivity uptake in tissues was expressed as the percentage of the injected dose per gram (% ID/g) after correcting for radioactive decay. 2.7. Ex vivo experiments: immunofluoresence, autoradiography, and western blotting After imaging, unsorted LoVo, CD133(+), CD133(−), and specifically blocked tumors were excised and fixed in 4% paraformaldehyde for 4 h and then soaked overnight in 25% sucrose. Sections (5 μm thick) were prepared with a cryostat. Kidney, liver, and muscle were also excised and subjected to the same protocol. CD133 expression was detected by immunofluoresence. The prepared tissue sections were incubated with fluorescein isothiocyanate-labeled goat anti-mouse IgG (Beyotime Institute of Biotechnology) at room temperature for 1 h. After washing

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Fig. 1. Flow cytometry analysis and immunofluorescence staining analysis of CD133 expression with anti-CD133/2-PE. A, flow cytometry analysis. After positive sorting, the ratio of CD133(+) cells higher than unsorted LoVo cells(P b 0.001). After negative sorting, there was rare CD133(+) cell in sorted negative cells. B, Immunofluorescence staining analysis of CD133 expression on unsorted LoVo cells, sorted positive cells and sorted negative cells with antibody against CD133/2 and nuclear counterstaining (DAPI), red fluorescence shows the expression of CD133. In unsorted LoVo cells, a part of cells shows high expression of CD133, while others were rare expression. Sorted positive cells were universal high expression of CD133, but there was rare CD133 expression in sorted negative cells (×400).

with PBS (0.1 M), 6-diamidino-2-phenylindole dihydrochloride (Beyotime Institute of Biotechnology) was applied, followed by incubation at room temperature for 1 h. Then, the sections were washed three times and mounted with 50% glycerol. Images were obtained under a fluorescence microscope. Autoradiography was performed on a Cyclone Plus Phosphor Scanning System (PerkinElmer, USA). After sacrificing the mice, the tumor tissues were removed immediately and ground completely. After centrifugation (400 g, 4 °C) for 5 min, the supernatant was collected to quantify the protein using the Bradford protein assay kit. Protein (40 μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then electroblotted onto a polyvinylidenedifluoride membrane (Millipore, USA). The membrane was incubated with AC133 mAb (1:300) (MiltenyiBiotec) overnight at 4 °C. β-actin was used as a control. Each experiment was performed in triplicate. 2.8. Data analysis Data are expressed as the mean ± standard error. All statistical data were analyzed with the independent samples t-test and Kruskal–Wallis test using SPSS 18.0 (PASW Statistics, Chicago, IL, USA). P b 0.05 was considered statistically significant. 3. Results

cytometry and immunofluorescence suggested successful isolation of CD133(+) and CD133(−) cells from LoVo cells. 3.2. Radiolabeling and cell binding assay The radiolabeling efficiency of 131I-AC133 mAb was 68.98 ± 11.95% (n =4), and the specific radioactivity was 92.4 ± 14.0 MBq/nmol (n = 4). After purification, the radiochemical purity of 131I-AC133 mAb was 84.36 ± 0.45% (n = 4). The radiochemical purity decreased to 69.92 ± 1.37% (n = 4) after incubation in PBS at 37 °C for 7 days, demonstrating the stability of 131I-AC133 mAb. In the cell binding experiment, the cell binding ratios of 131I-AC133 mAb to CD133(+) and unsorted LoVo cells increased with time and reached a peak at 2 h after incubation. The highest cell binding ratio of CD133(+) cells was 70.01 ± 6.02% (n = 4), which was significantly higher than the that of unsorted LoVo cells (30.52 ± 1.14%, n = 4), CD133(−) cells (2.39 ± 0.26%, n = 4), and the specifically blocked cells (2.73 ± 0.25%, n = 4) (P b 0.001, t = 12.813–22.299). The data are shown in Fig. 2. 3.3. In vivo SPECT/CT imaging The SPECT/CT images obtained after injection of 131I-AC133 mAb at the different times are shown in Fig. 3. Obvious uptake of the radiolabeled

3.1. Expression of CD133 in LoVo cells After MACS, CD133(+) and CD133(−) cells were identified by flow cytometry and immunofluorescence. The flow cytometry results showed that the percentage of CD133(+) cells in positively sorted LoVo cells was 86.26 ± 4.31% (n = 4), which was significantly higher than that in the negatively sorted subpopulation (2.06 ± 0.93%, n = 4) (P b 0.001, t = 38.162) and unsorted LoVo cells (47.22% ± 6.00%, n = 4, P b 0.001, t = 10.571) (Fig. 1A). The results of direct immunofluorescence on cell smears after MACS are shown in Fig. 1B. LoVo cells showed varied expression of CD133. Abundant CD133 expression could be seen in only specific areas of the cells, and other cells showed some or little expression of CD133. All sorted CD133(+) cells had high expression of CD133 in their membranes, whereas such expression was rare in negatively sorted cells. The results obtained from flow

Fig. 2. The binding ratio of 131I -AC133 to LoVo cells. The labeled mAb gradually combined with LoVo cells, and reached the peak at 120 min. The binding ratio of LoVo cells was significantly higher than CD133(−) cells, the binding can be blocked by abundant unlabeled mAb.

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tracer was seen in the tumor areas and gradually accumulated over time in CD133(+) tumor- and unsorted LoVo tumor-bearing mice, whereas almost no tracer could be seen in the tumor area of mice

with CD133(−) and specifically blocked tumors. These images verified that specific uptake of 131I-AC133 mAb existed in CD133(+) tumors in vivo (Fig. 3).

Fig. 3. SPCECT/CT imaging of tumor models. A, the tracer gradually accumulated in LoVo tumors. B, there was intense accumulation in sorted CD133(+) tumors. C, the accumulation in CD133(−) tumors in rare. D, the radioactivity accumulation in LoVo tumors could be blocked by unlabeled mAb.

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3.4. Biodistribution study In unsorted LoVo and CD133(+) tumors, there was a gradual increase of radioactivity in tumors, but there was decreasing accumulation in other organs. At day 7, the tumor uptakes in CD133(+) tumors and unsorted LoVo were 6.97 ± 1.40% ID/g (n = 4) and 6.12 ± 1.91% ID/g (n = 4), respectively, which were significantly higher than those in CD133(−) tumors (1.35 ± 0.48% ID/g, n = 4, P b 0.05) and specifically blocked tumors (1.61 ± 0.44% ID/g, n = 4, P b 0.05) (Fig. 4). The ratio of % ID/g in the tumor and blood was the highest (0.95 ± 0.21, n = 4) at day 7 in unsorted LoVo tumor-bearing mice and 1.06 ± 0.34 (n = 4) in CD133(+) tumor-bearing mice. There was no significant radioactivity accumulation in other vital organs (Table 1).

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The results obtained from western blotting (Fig. 6C) were consistent with those from immunofluorescence, which showed abundant CD133 expression in unsorted LoVo, CD133(+)and specifically blocked tumors. Little or no CD133 expression was observed in CD133(−) tumors and muscle, respectively. 3.6. Autoradiography Fig. 6A and B show the autoradiographic images obtained in this study. The radioactivity accumulation in LoVo tumors was higher than that in the muscle, kidney, and liver. Obvious radioactivity accumulation was seen in CD133 (+) tumors and unsorted LoVo tumors, but little radioactivity accumulation was observed in CD133(−) tumors. After blocking with unlabeled AC133 mAb, there was little radioactivity accumulation in LoVo tumors.

3.5. Immunofluorescence and western blot analyses 4. Discussion Immunofluorescence analysis of the tumor sections showed that there was intense fluorescence on the apical membranous portion of the cells in LoVo, CD133 (+), and specifically blocked tumors (Fig. 5). However, there was no fluorescence in CD133(−) tumors. Little fluorescence was detected in the liver and kidney, and no fluorescence was found in muscle tissue.

Fig. 4. A, The biodistribution of 131I-AC133 mAb in LoVo cell line tumor on 1, 3, 5, and 7 day after injection. The biodistribution was counted as percentage of the injected dose per gram (% ID/g). B, biodistribution in tumors xenografts 7 days after injection of imaging agent. The radioactivity accumulation of LoVo tumors and CD133(+) tumors were significantly higher than CD133(−) tumors and specific blocking tumors. There was high radioactivity accumulation in blood, but rare accumulation in muscle.

In this study, 131I-AC133 mAb was specifically taken up by CD133(+) colorectal CSCs at a much higher binding ratio than that in CD133(−), unsorted, and specifically blocked LoVo cells. Moreover, in the SPECT images of tumor xenografts, obvious radioactivity could be seen in the tumor area of CD133(+) and unsorted LoVo tumorbearing mice, but not in CD133(−) or specific blocked tumor-bearing mice. Based on these in vitro, ex vivo, and in vivo results, we verified that 131I-AC133 mAb could bind specifically to CD133(+) colorectal CSCs, and 131I-AC133 mAb could be used to monitor CSCs by radioimmunoimaging noninvasively. To the best of our knowledge, this is the first study that has successfully monitored colorectal CSCs with radioimmunoimaging in vivo. CSCs are a subpopulation within the tumor, which possesses the characteristics of self-renewal and differentiation, and can form tumors and resist drug therapies [13,19,20]. These characteristics may be the reasons for tumor recurrence and metastasis after therapy. Extensive experimental and clinical research of colorectal cancer has been undertaken [11–14,16,17,19,34,38,45–49] to isolate and identify CSCs. Recent studies [13,19,20] of CSC properties have been primarily in vitro, which have applied techniques such as flow cytometry, MACS, PCR, and western blotting. However, in vitro identification of CSCs is usually traumatic, and it cannot monitor CSCs dynamically during traditional chemotherapy or radiotherapy. Therefore, methods are needed to dynamically monitor CSCs in vivo. Thus far, there are limited studies [39,40] that have attempted to observe CSCs in vivo with molecular imaging methods. Tsurumi et al. [39] selected two sets of paired CD133(+/−) cell lines, lentivirally transduced CD133-overexpressing U251 cells/wild-type U251 cells and HCT116 colon carcinoma cells/ CD133(−) HCT116-derived cells. The CD133-specific monoclonal antibody AC133 mAb was labeled with Alexa Fluor as a tracer that was used for optical imaging of xenografts. Jin et al. [40] labeled CD133 antibodies with 125I and examined the ex vivo biodistribution of CD133 in HCT116 xenografts. However, no comprehensive investigations have been performed to monitor colorectal cancer CSCs in vivo with a radioimmunoimaging method. An appropriate target is the basis for targeting CSCs. The pentaspan membrane glycoprotein CD133 is considered as a specific target for colorectal CSCs. O'Brien et al. [13] and Ricci-Vitiani et al. [19] verified that sorted CD133(+) colorectal cancer cells show the potentials to selfrenew, differentiate, proliferate, and form tumors in vivo. Moreover, CD133(+) cells play an important role in the occurrence and development of tumors [7,47,50]. All these data suggest that CD133 is an important stemness biomarker for colorectal CSCs. Therefore, we selected CD133 as a biomarker and successfully sorted CD133(+) and (−) cells from the colorectal adenocarcinoma cell line LoVo with moderate CD133 expression. Some studies have shown that AC133, a glycosylated form of CD133, is specifically expressed in CSCs (29) and subsequently lost after CSC differentiation (34). Furthermore, its monoclonal antibody, AC133 mAb, shows

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Table 1 The biodistribution of 131I-AC133 mAb in LoVo cell line tumors, sorted CD133(+) tumors, sorted CD133(−) tumors, and LoVo cell line tumors blocked by unlabeled AC133 mAb (Data were shown as mean ± SD, % ID/g, n = 4). Tissue

Tumor Blood Thyroid Brain Heart Lung Liver Spleen Kidney Stomach Small intestine Large intestine Muscle Bone Tumor/Blood Tumor/Muscle

1 day

4.71 24.00 7.28 0.54 5.67 10.59 6.78 6.19 6.16 2.63 2.31 2.79 1.73 2.43 0.20 2.78

3 day

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.25 1.61 0.47 0.19 0.92 3.56 1.08 1.45 0.65 0.38 0.69 0.78 0.28 0.58 0.01 0.46

7.34 15.09 2.69 0.33 3.69 7.15 8.39 7.38 4.97 2.25 3.01 1.65 1.17 2.07 0.49 7.56

5 day

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.35 0.95 0.45 0.12 1.57 2.05 2.27 1.68 0.78 0.55 0.80 0.42 0.60 0.44 0.11 3.77

6.59 7.95 2.33 0.31 1.97 4.92 3.73 2.89 2.15 1.25 1.15 1.13 0.75 1.74 0.85 10.93

7 day

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.59 1.19 1.46 0.41 1.14 2.06 4.19 1.59 1.98 0.74 1.04 1.29 0.42 1.76 0.28 5.99

high affinity for CD133 on CSCs. These findings suggest that AC133 mAb can be used as a probe for radiolabeling and radioimmunoimaging. In our study, AC133 mAb was labeled with 131I by the iodogen method, and the labeled antibody exhibited high radiochemical purity (84.358 ± 0.452%) and relatively good stability. In the cell binding assay, sorted CD133(+)cells showed much higher uptake of the labeled antibody (around 70%) than that of sorted CD133(−) cells (about 2.3%). For unsorted LoVo cells, the cell binding ratio was nearly half of that for sorted positive cells (30%). After blocking with a 100-fold excess of unlabeled antibody, the binding ratio of LoVo cells was similar to that of sorted CD133(−)cells

Unsorted

Positive

6.12 6.38 1.42 0.11 1.15 2.85 2.06 3.06 1.81 0.48 0.93 0.94 0.38 1.30 0.95 16.05

6.97 6.93 1.86 0.20 1.27 2.62 2.06 2.95 1.94 0.50 0.70 0.69 0.40 1.44 1.06 17.89

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.91 0.77 0.23 0.05 0.22 1.30 0.55 0.43 0.19 0.09 0.21 0.25 0.07 0.48 0.21 3.74

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

1.40 1.59 0.31 0.06 0.07 0.39 0.70 0.59 0.10 0.18 0.14 0.13 0.06 0.15 0.34 4.08

Negative

Blocked

1.35 5.98 1.77 0.17 1.43 3.59 2.27 2.62 1.78 0.62 0.82 0.87 0.47 1.23 0.20 2.92

1.61 6.92 1.55 0.16 1.25 2.73 1.86 2.90 1.21 0.54 0.65 0.60 0.43 1.33 0.23 3.69

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.48 0.69 0.14 0.10 0.55 1.93 0.47 0.27 0.52 0.23 0.29 0.26 0.06 0.22 0.06 1.18

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.44 1.33 0.29 0.06 0.14 0.86 0.43 1.06 0.18 0.22 0.11 0.24 0.07 0.64 0.05 0.50

(about 2.7%). The main reason for the difference is the different expression levels of CD133 in cell populations, suggesting that the probe binds specifically to CD133. Moreover, our results indicate that the iodogen method for iodine labeling shows high efficiency and does not impair the specificity and immunoactivity of AC133 mAb. These data provided the feasibility for in vivo SPECT imaging with 131I-AC133 mAb. For tracing CSCs in vivo, we used four types of tumor xenografts for SPECT/CT imaging and follow-up ex vivo studies, unsorted LoVo, CD133(+), CD133(−) tumors and unsorted LoVo tumors that were blocked by unlabeled AC133 mAb. Using SPECT/CT, clear images could be

Fig. 5. Immunofluorescence of tumor frozen slicer after 7 day imaging with antibody against AC133 mAb and nuclear counterstaining (DAPI). There was abundant CD133 expression on apical membranous of gland cavity in LoVo tumors (A), CD133(+) tumors (B), and specific blocking tumors (D), but rare expression in CD133(−) tumor (C), liver (F), kidney (G) and muscle tissue (I). E showed the CD133 expression in Lovo tumor with no labeled mAb injection (×400).

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Fig. 6. ARG images of tumor frozen slicer after 7 day imaging. A, ARG of unsorted tumor model showed obvious radioactivity accumulation in unsorted Lovo tumor, but there was rare uptake in liver, kidney and muscle. B, ARG images showed obvious radioactivity accumulation in CD133(+) tumor, but there was rare expression in CD133(−) tumor and blocking tumor. Frozen slicer of unsorted Lovo tumor with no tracer injection was set as blank control. C, CD133 expression in different tumors and muscle at the protein level. There was abundant CD133 expression in LoVo tumors, CD133(+) tumors, and specific blocking.

obtained for unsorted LoVo and CD133(+) tumors. However, very little or no uptake of 131I-AC133 mAb was found in the other two types of tumor xenografts. In biodistribution experiments, % ID/g of unsorted LoVo and CD133(+) tumors was significantly higher than that of CD133(−) tumors, and the high accumulation could be blocked by excess unlabeled AC133 mAb. At day 7 after injection, there was rare radioactivity accumulation in muscle, resulting in a high tumor/muscle ratio of 16.05 ± 3.74 and 17.89 ± 4.08 for unsorted LoVo and CD133(+) tumors, respectively. To further confirm the association of radioactivity uptake and CD133 expression, we performed immunofluorescence and western blot analyses to check CD133 expression on the surface of tumor cells and the total protein level. These ex vivo experiments showed that there was abundant CD133 expression on the surface of unsorted LoVo and CD133(+) tumors, which accounted for the high uptake of 131I-AC133 mAb in these tumors. In CD133(−) tumors and muscle, there was rare CD133 expression. Moreover, in blocked LoVo tumors, there was abundant CD133 expression but little radioactivity, which further verified the specific binding of 131I-AC133. Therefore, the specific radioactivity accumulation in tumors and low uptake in normal tissues resulted in clear images of the tumors by SPECT/CT. Our study indicated that the 131 I-AC133 mAb could bind specifically to the CD133 molecule, and we successfully traced CD133(+) CSCs in vivo. A related study [39] using dye-labeled AC133.1 mAb also showed the feasibility of tracing CSCs by antibody-based targeting of CD133, which supports our study. In addition, we found that the differences of tracer uptake were not significant (P = 0.504) in unsorted LoVo and CD133(+) tumors. A possible reason for this observation is differentiation of CSCs in vivo. In vitro experiments have indicated that a large proportion of CSCs can maintain CD133 expression for a long period in the absence of serum [19]. However, CSCs

rapidly differentiate, and the CD133(+) cell population significantly decreases after addition of serum [20] or transplantation [13]. 131 I is widely used for radioimmunotherapy because of its appropriate half-life and β-particle emission. This study showed that the iodogen method for coupling 131I to mAbs is appropriate, and the radiolabeled compound showed high specificity and relative stability. Although this is only a preliminary study, 131I-AC133 mAb could be used as a new theranostics agent and may have broad clinical prospects in radioimmunoimaging and radioimmunotherapy. 5. Conclusions The results from in vitro, ex vivo, and in vivo experiments in this study demonstrate the possibility of targeting CSCs with 131I-AC133 mAb in colorectal tumor xenografts. The characteristics of specificity and stability of 131 I-AC133 mAb make it a promising agent to identify and trace CD133(+) CSCs in vivo. Furthermore, our research provides a basis for further study of radioimmunoimaging and radioimmunotherapy for CSCs. Acknowledgment This study was supported by the National Natural Science Foundation of China (81071178). References [1] Wang XL, Yuan Y, Zhang SZ, Cai SR, Huang YQ, Jiang Q, et al. Clinical and genetic characteristics of Chinese hereditary nonpolyposis colorectal cancer families. World J Gastroenterol 2006;12:4074–7. [2] Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer J Clin 2012;62:10–29.

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