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Selective Imaging of Malignant Ascites in a Mouse Model of Peritoneal Metastasis Using in Vivo Dynamic Nuclear PolarizationMagnetic Resonance Imaging Hinako Eto, Fuminori Hyodo,* Kenji Nakano, and Hideo Utsumi Innovation Center for Medical Redox Navigation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan S Supporting Information *
ABSTRACT: The presence of malignant ascites in advanced cancer patients is associated with both a poor prognosis and quality of life with a risk of abdominal infection and sepsis. Contemporary noninvasive visualization methods such as ultrasound, computed tomography, and magnetic resonance imaging (MRI) often struggle to differentiate malignant ascites from surrounding tissues. This study aimed to determine the utility of selective H2O imaging in the abdominal cavity with a free radical probe and deuterium oxide (D2O) contrast agent using in vivo dynamic nuclear polarization-MRI (DNP-MRI). Phantom imaging experiments established a linear relationship between H2O volume and image intensity using in vivo DNPMRI. Similar results were obtained when the radical-D2O probe was used to determine selective and spatial information on H2O in vivo, modeled by the injection of saline into the abdominal cavity of mice. To demonstrate the utility of this method for disease, malignant ascites in peritoneal metastasis animal model was selected as one of the typical examples. In vivo DNP-MRI of peritoneal metastasis animal model was performed 7−21 days after intraperitoneal injection of luciferase, stably expressing the human pancreatic carcinoma (SUIT-2). The image intensity with increasing malignant ascites was significantly increased at days 7, 16, and 21. This increase corresponded to in vivo tumor progression, as measured by bioluminescent imaging. These results suggest that H2O signal enhancement in DNP-MRI using radical-D2O contrast is positively associated with the progression of dissemination and could be a useful biomarker for malignant ascites with cancer metastasis.
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accumulation, the interaction of contrast agents including metals and free radicals such as Gd-DTPA. For example, during cerebral stroke, H2O leaks into the damaged parenchyma, resulting in brain edema that can be evaluated by a contrast change in diffusion-weighted and T2-weighted MRI.1,8 Because both the H2O of tissue and that of transudate result in an image contrast in MRI, it can be difficult to discriminate pathological transudate from MRI images. Particularly in the case of low transudate infusion, there is a risk for them to be overlooked because of very low MRI contrast changes. In vivo dynamic nuclear polarization (DNP)-MRI is a powerful method to obtain the spatiotemporal information on free radicals and could be used to visualize the functional status of tissue, such as redox state, oxygen partial pressure, or pH.9−16 For example, the proton signal of tissue, including free radicals, can be dramatically enhanced by electron spin resonance (ESR) irradiation at the resonance frequency of the free radical prior to applying the MRI pulse sequence. Nitroxyl radicals are stable free radical molecules that could modulate redox reactions, and
ater occupies 60−70% of our body weight and plays a critical role in homeostasis. Water responds sensitively and dynamically to tissue patho/physiological alterations such as organ dysfunction, an increase in internal vascular pressure, inflammation, abnormal osmolality, and peritoneal metastasis. It is known that H2O infiltrates into the intercellular space of the tissue and peritoneal cavity, typically resulting in edema and ascites.1,2 Peritoneal metastasis implies an advanced stage and poor prognosis with associated malignant ascites in the peritoneal cavity.3,4 The mean survival period of cancer patients with malignant ascites is estimated to be less than several months. This poor prognosis is principally due to a lack of noninvasive methods to detect these tiny and disseminated tumors and/or the accumulation of malignant ascites at the early stage. With this in mind, noninvasive imaging may be a useful approach to detect this early accumulation of ascites. Transudate has been previously investigated by imaging methods such as ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI).1,5−7 MRI is an imaging method that can exploit the hydrogen proton of H2O to visualize internal anatomical structures and reveal tissue abnormalities. Further MR image contrast could be employed to complement typical imaging with the aim of identifying abnormal fluid © 2016 American Chemical Society
Received: December 20, 2015 Accepted: January 21, 2016 Published: January 21, 2016 2021
DOI: 10.1021/acs.analchem.5b04821 Anal. Chem. 2016, 88, 2021−2027
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Analytical Chemistry
Figure 1. Phantom imaging of H2O with in vivo DNP-MRI: (A) malignant ascites was collected from SUIT-2 tumor-bearing mice (18 days after implantation of SUIT-2 fluorescent cells). (B) Phantom images of H2O-dependent signal change with in vivo DNP-MRI. The volume of malignant ascites was set from 0.1% to 100% in 2.5 mM CmP-D2O solution (final concn). (C) Ascites-dependent DNP image intensity change with in vivo DNPMRI. The image intensity of the regions of interest for each phantom was plotted with or without DNP. Error bars represent standard deviation. Arbitrary unit (a.u.) is a relative unit of image intensity.
the reduction rate is sensitively shifted depending on the tissue redox environment such as the overgeneration of reactive oxygen species (ROS) or reduced antioxidant defense.17,18 Because of this, nitroxyl radicals have been utilized as a redox-sensitive in vivo contrast agent in in vivo DNP-MRI.19−23 Deuterium oxide (D2O) is a form of water in which both hydrogen atoms are substituted by deuterium. D2O has been utilized as an exogenous diffusible tracer for in vivo MRI perfusion experiments.24,25 Although the chemical and physical properties of D2O are very similar to H2O, D2O is undetectable in nuclear magnetic resonance (NMR) at a resonance frequency of proton (42.57 MHz/T at 1 T) because of its gyromagnetic ratio of 6.5 MHz/T, which is one-seventh that of the water
proton. D2O is nontoxic and has been safely administered for NMR analysis in humans.26,27 In this study, we describe a method for selective imaging H2O with a free radical and D2O and in vivo DNP-MRI. When the free radical probe is dissolved in D2O (radical-D2O solution), there is no signal and no enhancement by in vivo DNP-MRI. However, when the intrinsic water proton comes into contact with the radical-D2O probe, the MRI signal is strongly enhanced by DNP and becomes visible. This feature allows the spatiotemporal visualization of H2O in tissues in vivo. Here, we first demonstrate the high sensitivity and distinct H2O imaging with a radical-D2O probe using DNP-MRI in vitro and show the in vivo selective imaging of malignant ascites in mice with peritoneal metastasis. 2022
DOI: 10.1021/acs.analchem.5b04821 Anal. Chem. 2016, 88, 2021−2027
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Figure 2. In vivo evaluation of H2O imaging with in vivo DNP-MRI: (A) mice with administration of different volumes of saline were measured after injection of 30 mM CmP-D2O isotonic solution. (B) Sum of image intensity was calculated by DNP images from the ROI of the abdomen of mice (n = 4). Error bars represent standard deviation.
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Bio LLC, Tokyo, Japan) and 100 U/mL penicillin and 100 μg/ mL streptomycin (Gibco Life Technologies Tokyo, Japan) at 37 °C in a humidified incubator containing 5% CO2. SUIT-2 cells (1.3 × 106/mouse) were intraperitoneally administered to nude mice. Peritoneal fluid for the phantom study was collected from day 18 mice after the intraperitoneal administration of SUIT-2 cells. Phantom Imaging with in Vivo DNP-MRI. To demonstrate the capabilities of H2O imaging with in vivo DNP-MRI, a seven-tube phantom (3 mm-diameter and 30 mm-length) was prepared as shown in Figure 1, where each tube was filled with 2.5 mM CmP (final concentration) dissolved in D2O and various volumes of malignant ascites (0−100%) derived from SUIT-2 metastasis mice (Figure 1A). These phantoms were measured by in vivo DNP-MRI, with or without ESR irradiation at 527.5 MHz using a surface coil.28 The scanning conditions for the in vivo DNP-MRI experiment were as follows: the power of ESR irradiation = 4 W, flip angle = 90 deg, repetition time (TR) × Echo time (TE) × TESR = 1000 ms × 40 ms × 500 ms, number of acquisitions = 4, and number of phase-encoding steps = 32. The image field of view (FOV, 32 mm × 32 mm) was represented by a 64 × 64 matrix after image reconstruction. In Vivo Imaging of Peritoneal H2O with in Vivo DNPMRI. BALB/cA nu/nu mice were anesthetized by 2% isoflurane and secured on a special holder by adhesive skin tape, stomach side down. During the procedure, the body temperature on the
MATERIALS AND METHODS Chemicals. 3-Carbamoyl-2,2,5,5-tetramethyl-1-pyrrolidine1-oxyl (Carbamoyl-PROXYL; CmP) and D2O were purchased from Sigma-Aldrich Chemical Co. (Milwaukee, WI). Trityl radical (Oxo 63) for in vitro experiments was purchased from Oxford Instruments (Oxford, U.K.). All other chemicals were commercially available and of reagent grade quality. Animals. BALB/cA nu/nu mice (4-week-old females) were purchased from Kyudo Co. (Saga, Japan) and housed in a climate-controlled, circadian rhythm-adjusted room, with free access to water and food (MF, Oriental Yeast Co., Tokyo, Japan) for 1 week prior to experiments. At the time of the experiments, mice were 5−9 weeks old and weighed 16−20 g. All procedures and animal care were approved by the Animal Ethics Committee, Kyushu University, and were conducted in accordance with the Guidelines for Animal Experiments of Kyushu University. Animal Model of Peritoneal Metastasis and Collection of Malignant Ascites. Human pancreatic carcinoma (SUIT-2) cells for the peritoneal metastasis animal model were obtained from the American Type Culture Collection (Manassas, VA). SUIT-2 cells stably expressing luciferase (SUIT-2/Luc) were established as previously reported using an attenuated lentiviral vector with a luciferase reporter gene (donated by Dr. Takafumi Nakamura, University of Tokyo). SUIT-2 cells were grown in RPMI1640 (Wako Pure Chemical Industries, Ltd., Osaka Japan) supplemented with 10% heat-inactivated fetal bovine serum (MP 2023
DOI: 10.1021/acs.analchem.5b04821 Anal. Chem. 2016, 88, 2021−2027
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Analytical Chemistry holder was kept at 37 ± 1 °C with a water pad. To demonstrate the possibility of H2O imaging in vivo, in vivo DNP-MRI experiments were performed after intraperitoneal injection of 30 mM CmP-D2O isotonic solution as a tracer of H2O and different volumes of saline (200, 400, 1000, 3000 μL; n = 4) as shown in Figure 2A. The normal MRI images were obtained without ESR irradiation. Image intensity of whole abdominal region in region of interest (ROI) was calculated by ImageJ software. The scanning conditions for the in vivo DNP-MRI experiment (Coronal plane) were as follows: power of EPR irradiation = 8W, flip angle = 90 deg, TR × TE × TEPR = 1000 ms × 40 ms × 700 ms; number of acquisitions = 2, phase-encoding steps = 32, total time for 1 image = 70 s. FOV = 48 mm × 48 mm, the image field was constructed by a 64 × 64 matrix. In Vivo Imaging of Malignant Ascites in the SUIT-2 Peritoneal Metastasis Model. SUIT-2 peritoneal metastasis BALB/cA nu/nu mice were utilized in this experiment at day 7, 14, and 21 following tumor cell injection. The in vivo DNP-MRI measurements, with ESR irradiation (ESR-ON), began 4 min after intraperitoneal injection of CmP-D2O isotonic solution (30 mM, 400 μL), as the DNP-MRI silent probe. Malignant ascites were collected from peritoneal metastasis mice after in vivo DNPMRI experiments. In Vivo Bioluminescent Imaging of SUIT-2 Peritoneal Metastasis Mice. In vivo bioluminescent imaging (BLI) was performed with an IVIS Imaging System (Xenogen Biosciences, Alameda, CA) at 7, 14, 21, and 28 days after SUIT-2 tumor cell injection. The bioluminescent signals of tumor were analyzed using Living Image software (Xenogen Biosciences). At 20 min prior to in vivo imaging, animals received the substrate DLuciferin (Summit Pharmaceuticals International Corporation, Tokyo, Japan) at 75 mg/kg in 10 mM phosphate buffered solution (pH = 7.4) by intraperitoneal injection under anesthesia (3% isoflurane). Data Analysis. The DNP-MRI data were analyzed using the ImageJ software package, available in the public domain.29,30 Data are presented as means ± standard deviations and differences in means were evaluated by t tests. Differences were considered statistically significant if p < 0.05.
malignant ascites collected from SUIT-2 peritoneal metastasis mice for a phantom study to confirm whether these factors influence the DNP-MRI signal. First, we confirmed the dynamics of peritoneal metastasis and ascites after tumor implantation (Figure S2). Next, approximately 4.0 mL of hemorrhagic malignant ascites was collected from SUIT-2 peritoneal metastasis mice at 18 day (Figure 1A). Various volumes of malignant ascites (0.1−100%) were prepared with the 2.5 mM CmP-D2O probe (final concn) and the solutions were measured by in vivo DNP-MRI (Figure 1B). With ESR-ON, images of the phantom with malignant ascites clearly showed a significant intensity enhancement (up to 20%), which linearly increased as a concentration of ascites (R2 = 0.999) (Figure 1C). By contrast, ESR-OFF images showed comparatively low intensities. The image intensity in 10% and 20% ascites tubes were 31 and 82 times higher than that of the control tube (ascites without probe), respectively. The enhancement appeared to plateau over 50% ascites and enhancement was stable over 30 min, suggesting the reactivity between CmP and ascites has a negligible effect under these conditions. In the phantom study, we also confirmed that contamination of the blood cells (10% RBC) and protein (4 g/dL BSA) did not affect the enhancement of the image intensity by DNP-MRI (Figure S3). These data suggested that the CmPD2O probe method is capable of imaging H2O with a high sensitivity using DNP, even at a very low magnetic field strength (15 mT). We next sought to apply this method to selective imaging of H2O in living mice. The microwave power for EPR irradiation is very important factor for in vivo experiments because excess irradiation courses the heating of the tissue. Therefore, we carefully determined the microwave power before starting experiments. In fact, we did not observe the change of mice body temperature in our experiments. Here, endogenous peritoneal fluid in the peritoneal cavity of the normal mouse was visualized by 2.5 mM CmP-D2O isotonic solution, likely due to the high sensitivity of this method. Therefore, the higher concentration of CmP was used to dampen the DNP-MRI signal by the spin−spin interactions of the radical and to model the dilution factor of the probe solution in the malignant ascites model (data not shown). No enhancement was observed until 6 min after peritoneal injection of 30 mM CmP-D2O probe in normal mice. The small increase in image intensity after 6 min may have been due to absorption of the probe into neighboring tissue and subsequent image enhancement by the interaction between the probe and the tissue protons. Therefore, all in vivo experiments were performed until 4 min after probe injection to minimize this probe absorption artifact. After intraperitoneal injection of the probe in normal BALB/ cA mice, several volumes of saline were also injected into the abdominal cavity, and image enhancement was monitored in real-time using in vivo DNP-MRI (Figure 2A). Although the DNP-MR image after the only injection of probe into mice showed no enhancement, the image intensity was clearly increased as a function of the volume of saline and the intensity appeared wholly distributed in the peritoneal cavity. The enhancement of signal intensity was observed even following a 200 μL dose of saline. Moreover, the sum of image intensities with ESR-ON linearly increased as a volume of saline (Figure 2B). These data suggest that the radical-D2O and DNP-MRI method could be used to selectively visualize H2O in the peritoneal cavity of mice. We next sought to apply the radical-D2O and DNP-MRI protocol to investigate transudate as a marker in a disease model.
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RESULTS AND DISCUSSION Initial experiments tested the dose dependent properties of DNP image contrast with the radical-D2O probe to detect H2O in the phantom study. First, we chose Oxo 63 as a radical probe to confirm the principle and the sensitivity of this method because Oxo 63 exhibits one of the highest sensitivities for DNP-MRI due to its narrow line width. Oxo 63-D2O solution (final concn = 2.5 mM) was prepared and mixed with various volume of H2O (0.1− 100%) in the phantom tubes and imaged by in vivo DNP-MRI (Figure S1A). The image intensity of the phantom in ESR-ON mode linearly increased as a concentration of H2O. Phantom imaging with ESR-ON of a H2O concentration of 0.1% showed significant enhancement compared to the ESR-OFF image (ESR-ON/ESR-OFF = 1.59). The DNP-MR image intensity of the 100% H2O phantom was 113 times higher than that of 0.1% H2O. This indicates that the dynamic range of image intensity affected by H2O is very large and that DNP-MRI could be used to sensitively detect changes in H2O volume. In addition, we confirmed that the shape and intensity of the ESR spectrum of CmP is not affected by replacement of H2O by D2O (Figure S1B). Malignant ascites often includes albumin, blood corpuscles, cancerous and inflammatory cells, in addition to water. We used 2024
DOI: 10.1021/acs.analchem.5b04821 Anal. Chem. 2016, 88, 2021−2027
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Figure 3. In vivo imaging of malignant ascites on SUIT-2 metastasis mice with in vivo DNP-MRI: (A) in vivo DNP images of peritoneal fluid in SUIT-2 metastasis mice. DNP images were obtained 7, 16, and 21 days after administration of SUIT-2 cells. (B) Correlation between volume of peritoneal fluid and DNP image intensity. Image intensity of the abdominal area of mice was plotted against the volume of malignant ascites collected from SUIT-2 metastasis mice after in vivo DNP-MRI. (C) In vivo bioluminescent imaging of SUIT-2 metastasis mice. Photon flux was measured at 0, 7, 14, 21 days after tumor cell administration. Error bars represent the standard deviation.
In particular, we targeted the in vivo spatiotemporal alteration of peritoneal fluid with tumor dissemination. To begin with, we imaged a day 18 SUIT-2 tumor cell-injected peritoneal
metastasis mouse with 4.0 mL of malignant ascites using a 1.5 T animal MRI, and as expected, MRI could not delineate the ascites only component by T1- or T2-weighted MRI (Figure S4). 2025
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demonstrated the visualization of malignant ascites in this report, our method should also be applicable to other diseases accompanied by transudate such as hepatitis, hypoalbuminemia, and stroke, because enhancement by DNP-MRI is not affected by the contamination of blood corpuscles and protein. Therefore, our method for the noninvasive monitoring of H2O enables the early detection of disease progression and provides useful information for planning therapeutic strategies and evaluating drug efficacy.
We then moved on to explore imaging according to our proposed method. In vivo DNP-MRI of SUIT-2 disseminated mice was performed between 7 and 21 days after intraperitoneal injection of tumor cells and the volume of malignant ascites was measured. Despite the absence of obvious signs (Figure S2A) and no alteration of luciferase activity (Figure 3C) in mice at day 7, the image intensity by DNP-MRI increased, and became significantly amplified at day 16 and 21 (Figure 3A). The image intensity derived from fluids was spread across the whole abdomen. In contrast, there were no enhancements after injection of the probe using ESR-OFF (regular MRI mode) due to the low magnetic field, as expected. The correlation between the sums of the image intensity and the malignant ascites was plotted in Figure 3B (n = 26). The sum of the image intensity increased with increasing malignant ascites and appeared to reach a plateau. These data corresponded well to in vivo tumor progression, as measured by bioluminescent imaging (Figure 3C). Ultimately, these results suggest that enhancement in DNP-MRI is tightly associated with the degree of peritoneal metastasis and could be of use in detecting alterations in metastatic fluid. The abdominal fluid accumulation in advanced oncological patients is a risk factor for abdominal infection and sepsis. The pathophysiology of malignant ascites is multifactorial, yet the permeability of tumor vessels is a significant contributing factor. Vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP) play a major role in altering vascular permeability and disruption of tissue matrixes resulting in peritumoral inflammation.4,31 Some reports suggest that malignant ascites is frequently accompanied by a high level of VEGF in cancer patients.2,32 While this may be the case, the direct detection of ascites during progression of metastasis remains troublesome. Asanuma et al. have demonstrated fluorescence-guided diagnostic approaches for the detection of cancer in situ and succeeded in visualizing small peritoneal metastatic tumors using β-galactosidase-targeting fluorescence probes in vivo.33,34 Although this technique has a high sensitivity and promises to improve surgical quality and outcomes, it requires the peritoneum to be opened and the probes splayed.33,34 Unfortunately, it is difficult to visualize peritoneal metastasis with very small tumors and to diagnose peritoneal fluids based on image contrast using current MRI methods. On the other hand, we demonstrated the use of the DNP-MRI method for selective visualization of malignant ascites in peritoneal metastasis model mice and the possibility of early detection based on changes to malignant ascites in preclinical study (Figure 3), although further improvement of probe concentration and injection dose are needed for human study to reduce the injection dose and volume. The idea of our proposed approach is detection of the early alteration after starting small accumulation of ascites (not detectable or distinguishable by current methods). Of course, it is a simple task to remove a small amount of fluid for diagnosis, if a patient has enough amounts of malignant ascites detected by current imaging methods. Our method using nitroxyl radical-D2O also detected the intrinsic peritoneal fluid of the mouse. Because enhancement and sensitivity is directly dependent on the probe, the use of a highly sensitive probe with a narrow radical spectrum such as Oxo63 could increase the detection limit to allow measurements of imperceptible changes in tissue fluid. Our original idea is development of water imaging and one of the typical examples is malignant ascites in peritoneal metastasis animal model. Therefore, this method is for diagnosis of ascites (H2O) with disease and is not for diagnosis of disease. Although we
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CONCLUSION In this study, we demonstrated a method for noninvasive monitoring of ascites using in vivo DNP-MRI, where other contemporary methods have significant limitations. Therefore, the selective imaging of ascites demonstrated by the present technique could be a powerful technique to detect ascites during the early stage of cancer metastasis, which may also lead to applications involving the evaluation of therapeutic drug efficacy.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b04821. Additional methods and results including a complete description of chemicals, materials, instrumentation, and imaging (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Phone: +8192-642-6277. Fax: +81-92-642-6024. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by Creation of Innovation Centers for Advanced Interdisciplinary Research Areas Program from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and by a Development of Advanced Measurement and Analysis Systems grant from the Japan Science and Technology Agency (H.U.). This work was also supported by KAKENHI Grant Numbers 22249003 (H.U.), 25253005 (H.U.), 25713004 (F.H.), and 26670016 (K.N.) from the Japan Society for the Promotion of Science.
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