CANCER-ONCOGENES
In Vivo Fluorescence Imaging and Urinary Monoamines as Surrogate Biomarkers of Disease Progression in a Mouse Model of Pheochromocytoma Martin Ullrich, Ralf Bergmann, Mirko Peitzsch, Marc Cartellieri, Nan Qin, Monika Ehrhart-Bornstein, Norman L. Block, Andrew V. Schally, Jens Pietzsch, Graeme Eisenhofer, Stefan R. Bornstein, and Christian G. Ziegler Department of Radiopharmaceutical and Chemical Biology (M.U., R.B., J.P.), Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany; Department of Medicine III (M.U., N.Q., M.E.-B., G.E., S.R.B., C.G.Z.), University Hospital Carl Gustav Carus, 01307 Dresden, Germany; Institute for Clinical Chemistry and Laboratory Medicine (M.P., N.Q., G.E.), Technische Universität Dresden, Germany; Department of Radioimmunology (M.C.), Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, 01328 Dresden, Germany; Department of Chemistry and Food Chemistry (J.P.), Technische Universität Dresden, Dresden, Germany; and VA Medical Center Miami FL and Department of Pathology and Medicine (N.L.B., A.V.S.), Division of Endocrinology and Hematology-Oncology, University of Miami Miller School of Medicine, Miami, Florida 33136
Pheochromocytoma (PHEO) is a rare but potentially lethal neuroendocrine tumor arising from catecholamine-producing chromaffin cells. Especially for metastatic PHEO, the availability of animal models is essential for developing novel therapies. For evaluating therapeutic outcome in rodent PHEO models, reliable quantification of multiple organ lesions depends on dedicated smallanimal in vivo imaging, which is still challenging and only available at specialized research facilities. Here, we investigated whether whole-body fluorescence imaging and monitoring of urinary free monoamines provide suitable parameters for measuring tumor progression in a murine allograft model of PHEO. We generated an mCherry-expressing mouse PHEO cell line by lentiviral gene transfer. These cells were injected subcutaneously into nude mice to perform whole-body fluorescence imaging of tumor development. Urinary free monoamines were measured by liquid chromatography with tandem mass spectrometry. Tumor fluorescence intensity and urinary outputs of monoamines showed tumor growth– dependent increases (P ⬍ .001) over the 30 days of monitoring post-tumor engraftment. Concomitantly, systolic blood pressure was increased significantly during tumor growth. Tumor volume correlated significantly (P ⬍ .001) and strongly with tumor fluorescence intensity (rs ⫽ 0.946), and urinary outputs of dopamine (rs ⫽ 0.952), methoxytyramine (rs ⫽ 0.947), norepinephrine (rs ⫽ 0.756), and normetanephrine (rs ⫽ 0.949). Dopamine and methoxytyramine outputs allowed for detection of lesions at diameters below 2.3 mm. Our results demonstrate that mouse pheochromocytoma (MPC)-mCherry cell tumors are functionally similar to human PHEO. Both tumor fluorescence intensity and urinary outputs of free monoamines provide precise parameters of tumor progression in this sc mouse model of PHEO. This animal model will allow for testing new treatment strategies for chromaffin cell tumors. (Endocrinology 155: 4149 – 4156, 2014)
heochromocytomas (PHEOs) are rare catecholamineproducing tumors mainly deriving from sympathetic chromaffin tissue of the adrenal medulla. Tumors from
P
extra-adrenal sympathetic chromaffin tissue are referred to as extra-adrenal PHEOs or paragangliomas. Other paragangliomas deriving from parasympathetic tissue do
ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received May 27, 2014. Accepted August 6, 2014. First Published Online August 19, 2014
Abbreviations: BP, blood pressure; DA, dopamine; EGFP, enhanced green fluorescent protein; EPI, epinephrine; FLI, fluorescence imaging; LC-MS/MS MC, liquid chromatography tandem mass spectrometry; MN, metanephrine; MTY, metabolite 3-methoxytyramine; NE, norepinephrine; NK, natural killer; NMN, normetanephrine; NMRI nu/nu, nude mice with rudimentary thymus; PBS, phosphate-buffered saline; PHEO, pheochromocytomas.
doi: 10.1210/en.2014-1431
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not produce catecholamines (1–3). PHEOs are rare with an incidence of approximately eight or fewer cases per million (4, 5). Approximately 10% of PHEOs are metastatic (2). To date, a few markers, such as mutant succinate dehydrogenase subunit B and the Ki67 proliferation index (6), as well as splice isoforms of carboxypeptidase E (7), have been identified to be related to the metastatic phenotype. However, malignant PHEO is mainly diagnosed by identification of distant metastases (1, 2, 8). Generally, treatment options for metastatic PHEO include radionuclide therapy, radiofrequency ablation, or chemotherapy, which sometimes show suboptimal results, with shortterm remission in some patients (2, 9). Thus, it is necessary to develop new therapeutic strategies for the treatment of metastatic PHEO. Over the years, there has been great interest in developing PHEO cell-line models. To date, with few exceptions (10), most efforts focused on animal cell lines. Thereafter, animal models have limited utilities for evaluating therapeutic strategies. Models available with spontaneously or genetically emerging tumors (11–14) or allograft models (15–18) are the most straightforward models for evaluating therapies. The mouse pheochromocytoma (MPC) cell line was developed from PHEOs arising in neurofibromatosis type 1 (Nf1) knockout mice (14, 19) showing high-level expression of receptor tyrosine kinase RET (20), as such, involved in activation of ras-signaling and the mTOR pathway (21). Furthermore, MPC cells, instead of the frequently used PC12 cells (22, 23), resemble human chromaffin cells as they express the enzyme phenylethanolamine-N-methyltransferase converting norepinephrine to epinephrine (14, 16). To date, monitoring of tumor progression in rodent PHEO models is solely based on dedicated small-animal imaging strategies only available in specialized research centers. However, conventional anatomic imaging as well as functional imaging in small animals, especially for quantifying the total burden of metastatic lesions, remains challenging and time consuming (15, 24, 25). PHEO is characterized by an overproduction of catecholamines, which is most readily assessed from increases in their plasma or urinary O-methylated metabolites (2, 26). In our current study, we investigated whether whole-body fluorescence imaging (FLI) and monitoring of urinary monoamines (catecholamines and metanephrines) provide suitable parameters for quantification of tumor progression in a murine allograft model of PHEO based on our far-red-fluorescence-tagged MPC cell line, named MPC-mCherry. We demonstrate that whole-body FLI is a rapid, quantitative, and powerful tool to monitor tumor progression in an sc mouse model of PHEO. We also found that, to
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some extent, MPC-mCherry cell tumors in mice functionally recapitulate the endocrine situation in human PHEO by producing both catecholamines and O-methylated metabolites of catecholamines. Our findings, that urinary monoamines quantitatively reflect total tumor burden, may offer a perspective to substantially facilitate comprehensive preclinical diagnostic and therapeutic evaluations of multiple organ lesions of PHEO.
Materials and Methods Cell culture Mouse PHEO cells [MPC; clone 4/30PRR (14), passage 32] were cultured in collagen-coated flasks and maintained in RPMI 1640 including HEPES (GIBCO) in a humidified 5% CO2/95% (v/v) O2 atmosphere at 37°C. Culture medium was supplemented with 10% (v/v) heat-inactivated horse serum (GIBCO), 5% (v/v) fetal bovine serum superior (BIOCHROM) and 0.1% (v/v) gentamicin (GIBCO) and was replaced every 48 –72 hours. Cells were routinely passaged every 7–10 days. Before injection into animals, cells at 70 – 80% confluence were detached using 0.05% (w/v) Trypsin-EDTA in magnesium- and calcium-free phosphate-buffered saline (PBS) and were adjusted to a concentration of 2 ⫻ 106 cells per 60 L in 50% (v/v) Matrigel (BD Biosciences)/PBS.
Lentiviral vector construction and genetic modification of cancer cells The lentiviral transfer vector p6NST70-MC is a derivative of the recently described p6NST90, harboring a multiple-cloning site with unique XbaI and HpaI sites downstream of an internal elongation factor 1 alpha promoter instead of the human ubiquitin C promoter– driven enhanced green fluorescent protein (EGFP) expression cassette (27). The open reading frame (orf) of the mCherry expression cassette was cloned into the discussed restriction sites of the multiple cloning site using NheI and PmeI restriction sites at the 5⬘ and 3⬘ end of both orf, respectively. For selection of successfully gene-modified MPC cells (passage 32; ⫽ MPC-mCherry cells; passage 0), the p6NST70-MC harbors a zeocin resistance expression cassette. Bicistronic expression for the selection marker downstream of the transgene stop codon is mediated by an internal ribosomal entry site derived from the encephalomyocarditis virus. To generate vesicular stomatitis virus G glycoprotein pseudotyped lentivirus, the lentiviral vector DNA was cotransfected into HEK293T cells by linear polyethylenimine (Polysciences Europe) with lentiviral packaging plasmid pCD/NL-BH and vesicular stomatitis virus G glycoprotein– encoding pMD-GM plasmid. Viral supernatants were collected, concentrated by ultracentrifugation, and stored at ⫺80°C, as described elsewhere (28, 29). Viral titers were determined by limiting dilution transduction on HT1080 cells, as described elsewhere (27, 29).
Animal experimentation All animal procedures and experiments were carried out according to the guidelines of the German Regulations for Animal Welfare. Nude mice with rudimentary thymus (NMRI nu/nu)
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were purchased from the pathogen-free breeding facility of the Medical Faculty Carl Gustav-Carus Experimental Center (Technische Universität). Nude mice were preferred to ensure maximal optical tissue penetration during whole-body FLI. The protocols were approved by the local Ethical Committee for Animal Experiments. A number of 2 ⫻ 106 MPC-mCherry cells (passage 12) were transplanted subcutaneously into the right shoulder of 10-week-old male and female animals. Experimental groups consisted of 10 animals housed in a pathogen-free facility. General anesthesia was induced and maintained with inhalation of 10% (v/v) desflurane in 30/10% (v/v) oxygen/air. Imaging studies were performed every 3– 4 days. Noninvasive blood pressure (BP) data and single-voided urine samples were collected weekly on different days, respectively, and in advance of any imaging study. Tumor diameters were measured using a caliper. Tumor volume V was determined according to Tomayko and Reynolds (30), assuming a triaxial ellipsoid with distinct axes a, b, and c (V ⫽ (/6)⫻abc. When tumor size reached a diameter of 1.5 cm, animals were killed using CO2 inhalation and cervical dislocation.
Fluorescence imaging All imaging studies were performed using the In-Vivo Xtreme System (BRUKER) equipped with a 400W xenon illuminator and a back-illuminated 4 MP CCD detector. For quantification of mCherry in vitro, cells were serially diluted from 2 ⫻ 106 to 6.25 ⫻ 104 cells in PBS in a 96-well plate and imaged for 1–5 seconds with a 550-nm excitation band-pass filter and a 600-nm emission long-pass filter. Fluorescence microscopy of paraformaldehyde-fixed cells was performed using a laser-scanning microscope IX83 (OLYMPUS). Nuclei were stained with Hoechst 33258 (1:10 000; stock solution: 1 mg mL⫺1 in dimethylsulfoxide). For detection of mCherry in vivo, continuously anesthetized animals were imaged for 1–5 seconds with a 600-nm excitation band-pass filter and a 700-nm emission long pass filter. All images were analyzed using MI 7.1.1 software (Bruker). Fluorescence intensity (total photon counts) was determined as the sum of the background-subtracted pixel values within a region of interest of constant size and shape for each animal. Local background was defined as the median of each region of interest perimeter intensity.
Determination of urinary free monoamines Each animal was allowed to roam in an empty, clean, conventional cage. After spontaneous urination, the animal was removed and the voided urine was aspirated with a pipet tip, avoiding contamination with feces. Samples were aliquoted and stored at ⫺66°C. Urinary free metanephrines and catecholamines were determined simultaneously by liquid chromatography tandem mass spectrometry (LC-MS/MS), as described elsewhere (26). Urinary creatinine excretion was determined using the picric acid method of Jaffe in a DxC 800 analytical system (Beckman Coulter) and was used for volume corrections.
Blood pressure measurement Blood pressure measurements were performed using the noninvasive CODA 2 system for mice and rats (Emka Technologies). Awake animals were allowed to acclimatize to the holder for 10
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minutes. Blood pressure was determined by averaging at least five consecutive measurements in a 20-minute session.
Statistical analysis Statistical analysis was performed using GraphPad Prism version 5.02 for Windows (Graphpad Software, www.graphpad.com). Experimental groups consisted of randomly selected animals; n represents the number of animals or cell culture dishes investigated. Data are presented as arithmetic means ⫾ SEM (for n ⬍ 10) or mean ⫾ SD (for n ⱖ 10). In vitro growth data were fitted with the logistic equation:
y共t兲 ⫽ ymax ⫻ y0共共 ymax ⫺ y0兲e ⫺ kx ⫹ y0兲 ⫺ 1 In vivo tumor growth data were fitted with the exponential equation:
y共t兲 ⫽ y0 ⫹ ekx. Comparison of fits was done using the Extra sum-of-squares F-test. Significance of differences was tested by analysis of variance using Sidak’s post hoc test. Differences were considered significant at values of P ⬍ .05. Significance of relationships was analyzed using Spearman’s linear correlation test and displayed as Spearman’s correlation coefficient, rs.
Results Generation of a stably transduced mCherryexpressing MPC cell line In this study, consecutive expression of mCherry in MPC cells was used for observing tumor progression in a murine sc allograft model of PHEO. For stable genetic modification of MPC cells, we generated the lentiviral transfer vector p6NST70-MC harboring an mCherry expression cassette driven by the elongation factor 1 alpha promoter. We named this new cell line MPC-mCherry. As demonstrated by fluorescence microscopy, mCherry was located throughout the cytoplasm and around the nucleus of MPC-mCherry cells (Figure 1B). To examine other effects of the genetic modification, we compared the biological properties of MPC-mCherry cells with the nontransfected parental MPC cells in vitro. Both cell lines were found to exhibit identical cell morphology (Figure 1, A and B). Logistic growth characteristics (Figure 1 C) did not differ significantly (P ⫽ .551, F ⫽ 0.705 [3;90]) between MPC cells (ymax ⫽ 71.3, y0 ⫽ 1.65, k ⫽ 0.457) and MPCmCherry cells (ymax ⫽ 65.0, y0 ⫽ 1.21, k ⫽ 0.520). mCherry expression was quantified by measuring the fluorescence intensity of serially diluted MPC-mCherry cells at excitation/emission wavelengths of 550/600 nm (Figure 1E). We observed a proportional relation (y ⫽ 9.93 ⫻ 10⫺4x; R2 ⫽ 0.999) of cell number and fluorescence intensity (rs ⫽ 1; P ⫽ .003) confirming a stable genomic integration of the mCherry expression cassette.
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Figure 1. Biological properties of MPC-mCherry cells compared with nontransduced parent MPC cells in vitro; A and B, Morphological appearance and fluorescence under microscopy; nuclei stained with Hoechst 33258; scale bars, 30 m. C, Growth of 2 ⫻ 105 MPC and MPCmCherry cells during 16 days post subculture quantified as protein of adherent cells per cm2, presented as means ⫾ SEM, n ⫽ 4. D, Positive proportional relation of MPC-mCherry cell number and corresponding fluorescence intensities at Ex/Em ⫽ 550/600 nm.
Fluorescence imaging and progression of MPCmCherry-derived tumors To study tumor progression in vivo, we injected 2 ⫻ 106 MPC-mCherry cells subcutaneously into the shoulders of nude mice with rudimentary thymus (NMRI nu/nu). To support the engraftment, we used Matrigel in PBS as the injection vehicle. Tumor progression was monitored at regular intervals over 4 weeks. Tumor fluorescence intensity was measured by performing whole-body FLI at excitation/emission wavelengths of 600/700 nm allowing for deep tissue penetration at the optimal signal-to-background ratio, as determined by multispectral analysis (data not shown). As a reference parameter, tumor volume was determined with calipers (range from 2– 463 mm3). To exclude any possible sex effects on tumor progression, male and female animals were monitored specifically using the identical experimental setup. Progression of the sc tumors is shown in Figure 2, A–C. During the first 7 days after cell injection, the initial fluorescence intensity at the injection site decreased, probably due to cell death or dispersal. The remaining MPCmCherry cells gave rise to a solid sc tumor, which did not metastasize during the period of observation. Monitoring tumor progression by FLI (Figure 2B) revealed that the exponential tumor growth characteristics between male animals (y0 ⫽ 365083, k ⫽ 0.132, R2 ⫽ 0.604) and female
animals (y0 ⫽ 756 451, k ⫽ 0.110, R2 ⫽ 0.544) did not differ significantly (P ⫽ .165, F ⫽ 1.825 [2;133]). Monitoring tumor progression using volume measurement by caliper also showed that the exponential tumor growth characteristics between male animals (y0 ⫽ 2.66, k ⫽ 0.169, R2 ⫽ 0.728) and female animals (y0 ⫽ 6.41; k ⫽ 0.137; R2 ⫽ 0.795) did not differ significantly (P ⫽ .249, F ⫽ 1.405 [2;132]). Analysis of covariance between tumor volume (mm3) and tumor fluorescence intensity (total photon counts) revealed a significant positive correlation (rs ⫽ 0.946, P ⬍ .001). Taken together, whole-body FLI enabled us to noninvasively monitor the tumor cells in nonmetastatic MPC-mCherry cellderived sc tumors in vivo.
Physiologic parameters during progression of MPC-mCherry cell-derived tumors To investigate whether urinary free monoamines reflect the total tumor burden in mice bearing MPC-mCherry definable tumors, we regularly measured concentrations of urinary free catecholamines and their corresponding metanephrines by LC-MS/MS. In addition, BP was monitored at regular intervals. To examine any possible sex effects, we systematically compared male and female animals separately. In all animals, we found steadily increasing urinary concentrations of the several monoamines during 27 days of tumor progression compared with basal concentrations (⫺8 and ⫺2 days) before cell injection (Figure 3, A–E). In this effect, we did not observe any significant differences between male and female animals. We observed the earliest alterations in urinary monoamine concentrations at 6 days after cell injections. The animals showed a 1.7-fold increase in dopamine (DA) from 0.68 –1.18 mol/mmol creatinine and a 2.1-fold increase in its corresponding metabolite 3-methoxytyramine (MTY) from 0.35– 0.76 mol/mmol creatinine (Figure 4). In contrast, during this time period, norepinephrine (NE) decreased from 0.60 – 0.30 mol/mmol creatinine and its corresponding metabolite normetanephrine (NMN) decreased from 1.20 – 0.70 mol/mmol creatinine. At this time the average tumor volume was approximately 12.3 mm3 (tumor diameter of approximately 2.3 mm) and the fluorescence intensity at the cell injection site reached the minimum level for detection (Figure 2, A and B).
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0.68 –35.18 mol/mmol creatinine, a significant 70-fold increase in MTY from 0.35–24.26 mol/mmol creatinine, a nonsignificant 3-fold increase in NE from 0.55–1.56 mol/mmol creatinine, and a significant 11-fold increase in NMN from 1.21–13.03 mol/mmol creatinine. Throughout the experiment, epinephrine (EPI) and metanephrine (MN) remained at basal levels of 0.15– 0.30 mol/mmol creatinine, respectively (Figure 3E). Tumor volume (mm3) showed significant positive correlations with urinary outputs of DA (rs ⫽ 0.952, P ⬍ .001), MTY (rs ⫽ 0.947, P ⬍ .001), NE (rs ⫽ 0.756, P ⬍ .001), and NMN Figure 2. Progression of sc tumors in male and female nude mice after sc injection of 2 ⫻ 106 (rs ⫽ 0.949, P ⬍ .001). MPC-mCherry cells. A, Overlays of x-ray and fluorescence images (Ex/Em ⫽ 600/700 nm) of a representative animal at 1 h to 29 d post injection. B, Tumor fluorescence intensities in vivo over time. In addition to the altered cateC, Tumor volumes over time; data are presented as mean ⫾ SD, (male animals, n ⫽ 10; female cholamine pattern, we observed inanimals, n ⫽ 10). D, Correlation analysis between tumor volume and tumor fluorescence intensity; creasing systolic BP (Figure 3F). n ⫽ 20; confidence interval, 95%; number of XY pairs, 136; p.i., post injection; px, pixel. With this effect, we did not observe Within 27 days, we observed dramatic increases in uri- any significant differences between male and female aninary monoamine outputs of DA, MTY, and NMN. The mals. Changes in systolic BP were found to be significant animals showed a significant 50-fold increase in DA from (P ⬍ 0.01) after 24 days’ post-cell injections among a mixed population of both male (n ⫽ 10) and female (n ⫽ 10) animals. In conclusion, MPC-mCherry cell-derived sc tumors altered the physiologic catecholamine pattern and BP in nude mice. Among all monoamines investigated, urinary concentrations of free DA, MTY, NE, and NMN allowed for biochemical monitoring of tumor progression in vivo.
Discussion
Figure 3. Physiologic parameters during tumor progression in male and female nude mice after sc injection of 2 ⫻ 106 MPC-mCherry cells. A–D, Urinary concentration of free monoamines ⫺10 and ⫺2 d before, and 6, 13, 20, and 27 d after cell injection; presented as mean ⫾ SD (male animals, n ⫽ 10, female animals, n ⫽ 10). E, Changes in the urinary concentration pattern of free monoamines (net chart) presented as mean (male ⫹ female animals, n ⫽ 20). F, Systolic BP ⫺10 and ⫺4 d before and 3, 10, 17, 24, 31 d after cell injection presented as mean (male animals, n ⫽ 10; female animals, n ⫽ 10) and mean ⫾ SD (male ⫹ female animals, n ⫽ 20); DA, dopamine; MTY, 3-methoxytyramine; NE, norepinephrine; NMN, normetanephrine; EPI, epinephrine; MN, metanephrine; p.i., post injection. **, P ⬍ 0.01; ***, P ⬍ 0.001.
Our study characterized a sophisticated animal model developed for evaluating novel therapeutic strategies for the treatment of PHEO. We employed the murine MPC cell line for developing an allograft mouse model of PHEO. MPC cells are biochemically and molecularly similar to human PHEOs, and are a suitable tool for testing new therapies for PHEO (18). However, this model
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To monitor tumor progression using FLI, we generated an mCherry-expressing MPC cell line by lentiviral transduction, named MPC-mCherry. Genetic manipulation neither altered the morphology nor the growth characteristics compared with the parental MPC cell line. Genetic properties of this particular MPC-mCherry clone remain to be investigated. MPCmCherry cells consecutively express red fluorescent mCherry protein driven by the elongation factor alpha promoter, enabling easy optical detection of the cells both in vitro and in vivo. To investigate whether the intensity of tumor fluorescence is a suitable parameter to monitor disease progression in nude mice, we performed whole-body FLI in the far-red portion of the spectrum, detecting only the tumor cells in MPC-mCherry cell-derived sc tumors in vivo. FLI allows for semiquantitative measurements of tumor progression, metastasis, and treatment response (32), and unlike other in vivo imaging strategies, it does Figure 4. Analysis of covariance between urinary free monoamines and tumor volume. DA, not require the addition of a substrate, dopamine; MTY, 3-methoxytyramine; NE, norepinephrine; EPI, epinephrine; NMN, normetanephrine. n ⫽ 20, confidence interval, 95%, number of XY pairs, 78. an imaging probe, or a radiotracer. Thus, FLI is preferred for multiple examinations at a minimum stress level may not be suitable for investigating metastatic spread, for animals. Fluorescent proteins emitting in the far-red porgenerally arising from a primary tumor. However, in nude tion of the spectrum allow for biological interrogations at mice, iv and ip, but potentially not sc injections of MPC deep optical tissue penetration (33, 34). Among these procells, can be used to generate multiple organ lesions (31). teins, mCherry, owing to its spectral class (excitation maxThe difficulty thereafter is to monitor disease progression. imum, 587 nm; emission maximum, 610 nm), brightness, We have therefore first employed MPC cells to develop an and superior photostability, is the best general purpose agent sc nonmetastatic mouse model of PHEO in which the lofor whole-body optical imaging (35, 36). These optical propcation of one single lesion is known and its size and morerties allow for detection of especially small changes in numphology can easily be determined and matched to other ber of tumor cells observed during the first 7 days after cell parameters such as fluorescence intensity and urinary injection. However, these effects on the number of tumor monoamine concentrations to validate their use for mon- cells may be due to cell death, diffusion, invasion of neutroitoring disease progression. phils, or newly built blood vessels and connective tissue. The In our study, the use of Matrigel increased the repro- strongly positive correlation between tumor size and fluoducibility of tumor cell engraftment as well as the homo- rescence intensity suggests a minor influence of light-scattergeneity in tumor shapes among the animals and, thereby ing artifacts when the total fluorescence intensity of the tuimproved tumor volume measurements by caliper. Precise mor is used as a quantitative parameter. and complete determination of tumor volume was a preUnlike caliper measurements, FLI eliminates most conrequisite for studying intensities of tumor fluorescence and tributions of the nontumor cell-derived volume, such as concentrations of urinary free monoamines as surrogate capsule, edema, necrotic areas, blood vessels, or immune markers of disease progression in this murine model of cell infiltration, inside the heterogeneous structure of a PHEO. solid tumor. Taken together, FLI of MPC-mCherry cell-
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derived tumors is considered to provide a powerful and rapid method for evaluating tumor progression during preclinical therapeutic investigations in nude mice. In our study, we used nude mice to ensure maximal optical tissue penetration during whole-body FLI. In addition, the immunodeficiency of athymic mice will allow for extending the animal model to the engraftment of primary human PHEO cells or newly developed human PHEO cell lines in the future. Referring to the latter, a recent study reported about a human PHEO precursor cell line established form a primary human tumor (10). Our findings that sc MPC-mCherry cell-derived tumors did not metastasize in nude mice verify earlier studies (31). This outcome may be due to the fact that MPC cells were generated from adrenal tumors of BL-6 heterozygous neurofibromin-1 knockout mice, which generally exhibit a low metastatic potential (14). Despite their immunodeficiency, nude mice are capable of generating a nonspecific immune response against cells from different strains, for example by natural killer (NK) cell activation (37). This effect may also contribute to the initial tumor cell death at the injection site observed during the first 7 days after injection. We also investigated whether tumor-induced alterations of physiologic parameters reflect the total tumor burden of PHEO in mice. For this, we monitored the concentrations of urinary free monoamines as well as BP at regular intervals during 4 weeks of tumor progression. Among all monoamines investigated, our analyses revealed that the most dramatic increases involved urinary levels of DA and its corresponding metabolite MTY. These results suggest that MPC-mCherry cell-derived sc tumors mainly produce DA. Most importantly, measuring urinary free DA and/or MTY allowed for the most sensitive biochemical detection of tumors at diameters below 2.3 mm in vivo, although possessing similar tumor detection limits compared with anatomic and functional smallanimal imaging (magnetic resonance imaging, computed tomography, positron emission tomography, single-photon emission computed tomography) in vivo. Therefore, our investigations suggest that measuring urinary free DA and/or MTY allows for easy, noninvasive quantification of catecholamine-producing lesions. In contrast with the increase in urinary DA, we found relatively small increases in urinary NE but larger increases in its corresponding metabolite, NMN. These findings suggest that much of the NE produced within tumors is metabolized to NMN. We did not observe tumor-induced increases in urinary free EPI or its corresponding metabolite, MN. Although MPC cells are known to express phenyl-N-methyltransferase, they also only produce small amounts of EPI from NE (14), explaining why we
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did not observe significant increases in urinary EPI and MN. However, adaptation of peripheral catecholamine metabolism, such as in the liver, cannot be excluded as a factor influencing the observed metabolite pattern. Systolic BP was found to be significantly increased during tumor growth, demonstrating the physiologic influence of tumor-related NE release. Nevertheless, increases in BP were not severe, although reflecting the pattern of increases in urinary outputs of catecholamines (38). In summary, we demonstrated that the MPC-mCherry cell-derived sc allograft mouse model exhibits functional similarities to the endocrine situation in human PHEO, characterized by increased urinary outputs of catecholamines and BP. In particular, measurement of tumor progression by whole-body FLI and urinary free DA and/or MTY by LC-MS/MS allows for noninvasive quantification of tumor progression. The introduced sc PHEO model provides a useful tool for comprehensively evaluating therapeutic strategies for PHEO, but may not be a predictor of organ metastatic lesion responses to treatment. However, FLI and measurements of urinary free monoamines may offer perspectives to substantially facilitate comprehensive preclinical diagnostic and therapeutic evaluations also in other mouse models, which present tumors in the liver, lung, kidneys, bones, and muscles. Diagnosis and monitoring of tumor progression and therapy response in human patients with metastatic PHEO may also benefit from this strategy.
Acknowledgments Address all correspondence and requests for reprints to: Christian G. Ziegler, PhD, Molecular Endocrinology, Medical Clinic III, University Hospital Carl-Gustav Carus, Fiedlerstrasse 42, 01307 Dresden, Germany. E-mail: [email protected]. This work was supported by The Deutsche Forschungsgemeinschaft (Grants ZI-1362/2–1 [to C.G.Z. and G.E.] and BE2607/1–1 [to R.B. and J.P.]). MPC 4/30PRR cells were kindly provided by Professor Arthur Tischler, Dr James Powers, and Professor Karel Pacak. Disclosure Summary: The authors have nothing to disclose.
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paraganglioma syndrome: No longer the 10% tumor. J Surg Oncol. 2005;89(3):193–201. Harding JL, Yeh MW, Robinson BG, Delbridge LW, Sidhu SB. Potential pitfalls in the diagnosis of phaeochromocytoma. Med J Aust. 2005;182(12):637– 640. Kimura N, Takayanagi R, Takizawa N, et al. Pathological grading for predicting metastasis in phaeochromocytoma and paraganglioma. Endocr Relat Cancer. 2014;21(3):405– 414. Lee TK, Murthy SR, Cawley NX, et al. An N-terminal truncated carboxypeptidase E splice isoform induces tumor growth and is a biomarker for predicting future metastasis in human cancers. J Clin Invest. 2011;121(3):880 – 892. Zinnamosca L, Petramala L, Cotesta D, et al. Neurofibromatosis type 1 (NF1) and pheochromocytoma: Prevalence, clinical and cardiovascular aspects. Arch Dermatol Res. 2011;303(5):317–325. Pacak K, Fojo T, Goldstein DS, et al. Radiofrequency ablation: A novel approach for treatment of metastatic pheochromocytoma. J Natl Cancer Inst. 2001;93(8):648 – 649. Ghayee HK, Bhagwandin VJ, Stastny V, et al. Progenitor cell line (hPheo1) derived from a human pheochromocytoma tumor. PLoS One. 2013;8(6):e65624. Fritz A, Walch A, Piotrowska K, et al. Recessive transmission of a multiple endocrine neoplasia syndrome in the rat. Cancer Res. 2002; 62(11):3048 –3051. Molatore S, Liyanarachchi S, Irmler M, et al. Pheochromocytoma in rats with multiple endocrine neoplasia (MENX) shares gene expression patterns with human pheochromocytoma. Proc Natl Acad Sci USA. 2010;107(43):18493–18498. Jacks T, Shih TS, Schmitt EM, Bronson RT, Bernards A, Weinberg RA. Tumour predisposition in mice heterozygous for a targeted mutation in Nf1. Nat Genet. 1994;7(3):353–361. Powers JF, Evinger MJ, Tsokas P, Bedri S, Alroy J, Shahsavari M, Tischler AS. Pheochromocytoma cell lines from heterozygous neurofibromatosis knockout mice. Cell Tissue Res. 2000;302(3):309 – 320. Giubellino A, Woldemichael GM, Sourbier C, et al. Characterization of two mouse models of metastatic pheochromocytoma using bioluminescence imaging. Cancer Lett. 2012;316(1):46 –52. Martiniova L, Lai EW, Elkahloun AG, et al. Characterization of an animal model of aggressive metastatic pheochromocytoma linked to a specific gene signature. Clin Exp Metastasis. 2009;26(3):239 – 250. Ohta S, Lai EW, Morris JC, et al. Metastasis-associated gene expression profile of liver and subcutaneous lesions derived from mouse pheochromocytoma cells. Mol Carcinog. 2008;47(4):245– 251. Korpershoek E, Pacak K, Martiniova L. Murine models and cell lines for the investigation of pheochromocytoma: Applications for future therapies? Endocr Pathol. 2012;23(1):43–54. Ziegler CG, Ullrich M, Schally AV, et al. Anti-tumor effects of peptide analogs targeting neuropeptide hormone receptors on mouse pheochromocytoma cells. Mol Cell Endocrinol. 2013;371(1–2): 189 –194. Powers JF, Schelling K, Brachold JM, et al. High-level expression of receptor tyrosine kinase Ret and responsiveness to Ret-activating
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In Vivo Fluorescence Imaging and Urinary Monoamines as Surrogate Biomarkers of Disease Progression in a Mouse Model of Pheochromocytoma Martin Ullrich, Ralf Bergmann, Mirko Peitzsch, Marc Cartellieri, Nan Qin, Monika Ehrhart-Bornstein, Norman L. Block, Andrew V. Schally, Jens Pietzsch, Graeme Eisenhofer, Stefan R. Bornstein, and Christian G. Ziegler Department of Radiopharmaceutical and Chemical Biology (M.U., R.B., J.P.), Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, Dresden, Germany; Department of Medicine III (M.U., N.Q., M.E.-B., G.E., S.R.B., C.G.Z.), University Hospital Carl Gustav Carus, 01307 Dresden, Germany; Institute for Clinical Chemistry and Laboratory Medicine (M.P., N.Q., G.E.), Technische Universität Dresden, Germany; Department of Radioimmunology (M.C.), Institute of Radiopharmaceutical Cancer Research, Helmholtz-Zentrum Dresden-Rossendorf, 01328 Dresden, Germany; Department of Chemistry and Food Chemistry (J.P.), Technische Universität Dresden, Dresden, Germany; and VA Medical Center Miami FL and Department of Pathology and Medicine (N.L.B., A.V.S.), Division of Endocrinology and Hematology-Oncology, University of Miami Miller School of Medicine, Miami, Florida 33136
Pheochromocytoma (PHEO) is a rare but potentially lethal neuroendocrine tumor arising from catecholamine-producing chromaffin cells. Especially for metastatic PHEO, the availability of animal models is essential for developing novel therapies. For evaluating therapeutic outcome in rodent PHEO models, reliable quantification of multiple organ lesions depends on dedicated smallanimal in vivo imaging, which is still challenging and only available at specialized research facilities. Here, we investigated whether whole-body fluorescence imaging and monitoring of urinary free monoamines provide suitable parameters for measuring tumor progression in a murine allograft model of PHEO. We generated an mCherry-expressing mouse PHEO cell line by lentiviral gene transfer. These cells were injected subcutaneously into nude mice to perform whole-body fluorescence imaging of tumor development. Urinary free monoamines were measured by liquid chromatography with tandem mass spectrometry. Tumor fluorescence intensity and urinary outputs of monoamines showed tumor growth– dependent increases (P ⬍ .001) over the 30 days of monitoring post-tumor engraftment. Concomitantly, systolic blood pressure was increased significantly during tumor growth. Tumor volume correlated significantly (P ⬍ .001) and strongly with tumor fluorescence intensity (rs ⫽ 0.946), and urinary outputs of dopamine (rs ⫽ 0.952), methoxytyramine (rs ⫽ 0.947), norepinephrine (rs ⫽ 0.756), and normetanephrine (rs ⫽ 0.949). Dopamine and methoxytyramine outputs allowed for detection of lesions at diameters below 2.3 mm. Our results demonstrate that mouse pheochromocytoma (MPC)-mCherry cell tumors are functionally similar to human PHEO. Both tumor fluorescence intensity and urinary outputs of free monoamines provide precise parameters of tumor progression in this sc mouse model of PHEO. This animal model will allow for testing new treatment strategies for chromaffin cell tumors. (Endocrinology 155: 4149 – 4156, 2014)
heochromocytomas (PHEOs) are rare catecholamineproducing tumors mainly deriving from sympathetic chromaffin tissue of the adrenal medulla. Tumors from
P
extra-adrenal sympathetic chromaffin tissue are referred to as extra-adrenal PHEOs or paragangliomas. Other paragangliomas deriving from parasympathetic tissue do
ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2014 by the Endocrine Society Received May 27, 2014. Accepted August 6, 2014. First Published Online August 19, 2014
Abbreviations: BP, blood pressure; DA, dopamine; EGFP, enhanced green fluorescent protein; EPI, epinephrine; FLI, fluorescence imaging; LC-MS/MS MC, liquid chromatography tandem mass spectrometry; MN, metanephrine; MTY, metabolite 3-methoxytyramine; NE, norepinephrine; NK, natural killer; NMN, normetanephrine; NMRI nu/nu, nude mice with rudimentary thymus; PBS, phosphate-buffered saline; PHEO, pheochromocytomas.
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not produce catecholamines (1–3). PHEOs are rare with an incidence of approximately eight or fewer cases per million (4, 5). Approximately 10% of PHEOs are metastatic (2). To date, a few markers, such as mutant succinate dehydrogenase subunit B and the Ki67 proliferation index (6), as well as splice isoforms of carboxypeptidase E (7), have been identified to be related to the metastatic phenotype. However, malignant PHEO is mainly diagnosed by identification of distant metastases (1, 2, 8). Generally, treatment options for metastatic PHEO include radionuclide therapy, radiofrequency ablation, or chemotherapy, which sometimes show suboptimal results, with shortterm remission in some patients (2, 9). Thus, it is necessary to develop new therapeutic strategies for the treatment of metastatic PHEO. Over the years, there has been great interest in developing PHEO cell-line models. To date, with few exceptions (10), most efforts focused on animal cell lines. Thereafter, animal models have limited utilities for evaluating therapeutic strategies. Models available with spontaneously or genetically emerging tumors (11–14) or allograft models (15–18) are the most straightforward models for evaluating therapies. The mouse pheochromocytoma (MPC) cell line was developed from PHEOs arising in neurofibromatosis type 1 (Nf1) knockout mice (14, 19) showing high-level expression of receptor tyrosine kinase RET (20), as such, involved in activation of ras-signaling and the mTOR pathway (21). Furthermore, MPC cells, instead of the frequently used PC12 cells (22, 23), resemble human chromaffin cells as they express the enzyme phenylethanolamine-N-methyltransferase converting norepinephrine to epinephrine (14, 16). To date, monitoring of tumor progression in rodent PHEO models is solely based on dedicated small-animal imaging strategies only available in specialized research centers. However, conventional anatomic imaging as well as functional imaging in small animals, especially for quantifying the total burden of metastatic lesions, remains challenging and time consuming (15, 24, 25). PHEO is characterized by an overproduction of catecholamines, which is most readily assessed from increases in their plasma or urinary O-methylated metabolites (2, 26). In our current study, we investigated whether whole-body fluorescence imaging (FLI) and monitoring of urinary monoamines (catecholamines and metanephrines) provide suitable parameters for quantification of tumor progression in a murine allograft model of PHEO based on our far-red-fluorescence-tagged MPC cell line, named MPC-mCherry. We demonstrate that whole-body FLI is a rapid, quantitative, and powerful tool to monitor tumor progression in an sc mouse model of PHEO. We also found that, to
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some extent, MPC-mCherry cell tumors in mice functionally recapitulate the endocrine situation in human PHEO by producing both catecholamines and O-methylated metabolites of catecholamines. Our findings, that urinary monoamines quantitatively reflect total tumor burden, may offer a perspective to substantially facilitate comprehensive preclinical diagnostic and therapeutic evaluations of multiple organ lesions of PHEO.
Materials and Methods Cell culture Mouse PHEO cells [MPC; clone 4/30PRR (14), passage 32] were cultured in collagen-coated flasks and maintained in RPMI 1640 including HEPES (GIBCO) in a humidified 5% CO2/95% (v/v) O2 atmosphere at 37°C. Culture medium was supplemented with 10% (v/v) heat-inactivated horse serum (GIBCO), 5% (v/v) fetal bovine serum superior (BIOCHROM) and 0.1% (v/v) gentamicin (GIBCO) and was replaced every 48 –72 hours. Cells were routinely passaged every 7–10 days. Before injection into animals, cells at 70 – 80% confluence were detached using 0.05% (w/v) Trypsin-EDTA in magnesium- and calcium-free phosphate-buffered saline (PBS) and were adjusted to a concentration of 2 ⫻ 106 cells per 60 L in 50% (v/v) Matrigel (BD Biosciences)/PBS.
Lentiviral vector construction and genetic modification of cancer cells The lentiviral transfer vector p6NST70-MC is a derivative of the recently described p6NST90, harboring a multiple-cloning site with unique XbaI and HpaI sites downstream of an internal elongation factor 1 alpha promoter instead of the human ubiquitin C promoter– driven enhanced green fluorescent protein (EGFP) expression cassette (27). The open reading frame (orf) of the mCherry expression cassette was cloned into the discussed restriction sites of the multiple cloning site using NheI and PmeI restriction sites at the 5⬘ and 3⬘ end of both orf, respectively. For selection of successfully gene-modified MPC cells (passage 32; ⫽ MPC-mCherry cells; passage 0), the p6NST70-MC harbors a zeocin resistance expression cassette. Bicistronic expression for the selection marker downstream of the transgene stop codon is mediated by an internal ribosomal entry site derived from the encephalomyocarditis virus. To generate vesicular stomatitis virus G glycoprotein pseudotyped lentivirus, the lentiviral vector DNA was cotransfected into HEK293T cells by linear polyethylenimine (Polysciences Europe) with lentiviral packaging plasmid pCD/NL-BH and vesicular stomatitis virus G glycoprotein– encoding pMD-GM plasmid. Viral supernatants were collected, concentrated by ultracentrifugation, and stored at ⫺80°C, as described elsewhere (28, 29). Viral titers were determined by limiting dilution transduction on HT1080 cells, as described elsewhere (27, 29).
Animal experimentation All animal procedures and experiments were carried out according to the guidelines of the German Regulations for Animal Welfare. Nude mice with rudimentary thymus (NMRI nu/nu)
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were purchased from the pathogen-free breeding facility of the Medical Faculty Carl Gustav-Carus Experimental Center (Technische Universität). Nude mice were preferred to ensure maximal optical tissue penetration during whole-body FLI. The protocols were approved by the local Ethical Committee for Animal Experiments. A number of 2 ⫻ 106 MPC-mCherry cells (passage 12) were transplanted subcutaneously into the right shoulder of 10-week-old male and female animals. Experimental groups consisted of 10 animals housed in a pathogen-free facility. General anesthesia was induced and maintained with inhalation of 10% (v/v) desflurane in 30/10% (v/v) oxygen/air. Imaging studies were performed every 3– 4 days. Noninvasive blood pressure (BP) data and single-voided urine samples were collected weekly on different days, respectively, and in advance of any imaging study. Tumor diameters were measured using a caliper. Tumor volume V was determined according to Tomayko and Reynolds (30), assuming a triaxial ellipsoid with distinct axes a, b, and c (V ⫽ (/6)⫻abc. When tumor size reached a diameter of 1.5 cm, animals were killed using CO2 inhalation and cervical dislocation.
Fluorescence imaging All imaging studies were performed using the In-Vivo Xtreme System (BRUKER) equipped with a 400W xenon illuminator and a back-illuminated 4 MP CCD detector. For quantification of mCherry in vitro, cells were serially diluted from 2 ⫻ 106 to 6.25 ⫻ 104 cells in PBS in a 96-well plate and imaged for 1–5 seconds with a 550-nm excitation band-pass filter and a 600-nm emission long-pass filter. Fluorescence microscopy of paraformaldehyde-fixed cells was performed using a laser-scanning microscope IX83 (OLYMPUS). Nuclei were stained with Hoechst 33258 (1:10 000; stock solution: 1 mg mL⫺1 in dimethylsulfoxide). For detection of mCherry in vivo, continuously anesthetized animals were imaged for 1–5 seconds with a 600-nm excitation band-pass filter and a 700-nm emission long pass filter. All images were analyzed using MI 7.1.1 software (Bruker). Fluorescence intensity (total photon counts) was determined as the sum of the background-subtracted pixel values within a region of interest of constant size and shape for each animal. Local background was defined as the median of each region of interest perimeter intensity.
Determination of urinary free monoamines Each animal was allowed to roam in an empty, clean, conventional cage. After spontaneous urination, the animal was removed and the voided urine was aspirated with a pipet tip, avoiding contamination with feces. Samples were aliquoted and stored at ⫺66°C. Urinary free metanephrines and catecholamines were determined simultaneously by liquid chromatography tandem mass spectrometry (LC-MS/MS), as described elsewhere (26). Urinary creatinine excretion was determined using the picric acid method of Jaffe in a DxC 800 analytical system (Beckman Coulter) and was used for volume corrections.
Blood pressure measurement Blood pressure measurements were performed using the noninvasive CODA 2 system for mice and rats (Emka Technologies). Awake animals were allowed to acclimatize to the holder for 10
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minutes. Blood pressure was determined by averaging at least five consecutive measurements in a 20-minute session.
Statistical analysis Statistical analysis was performed using GraphPad Prism version 5.02 for Windows (Graphpad Software, www.graphpad.com). Experimental groups consisted of randomly selected animals; n represents the number of animals or cell culture dishes investigated. Data are presented as arithmetic means ⫾ SEM (for n ⬍ 10) or mean ⫾ SD (for n ⱖ 10). In vitro growth data were fitted with the logistic equation:
y共t兲 ⫽ ymax ⫻ y0共共 ymax ⫺ y0兲e ⫺ kx ⫹ y0兲 ⫺ 1 In vivo tumor growth data were fitted with the exponential equation:
y共t兲 ⫽ y0 ⫹ ekx. Comparison of fits was done using the Extra sum-of-squares F-test. Significance of differences was tested by analysis of variance using Sidak’s post hoc test. Differences were considered significant at values of P ⬍ .05. Significance of relationships was analyzed using Spearman’s linear correlation test and displayed as Spearman’s correlation coefficient, rs.
Results Generation of a stably transduced mCherryexpressing MPC cell line In this study, consecutive expression of mCherry in MPC cells was used for observing tumor progression in a murine sc allograft model of PHEO. For stable genetic modification of MPC cells, we generated the lentiviral transfer vector p6NST70-MC harboring an mCherry expression cassette driven by the elongation factor 1 alpha promoter. We named this new cell line MPC-mCherry. As demonstrated by fluorescence microscopy, mCherry was located throughout the cytoplasm and around the nucleus of MPC-mCherry cells (Figure 1B). To examine other effects of the genetic modification, we compared the biological properties of MPC-mCherry cells with the nontransfected parental MPC cells in vitro. Both cell lines were found to exhibit identical cell morphology (Figure 1, A and B). Logistic growth characteristics (Figure 1 C) did not differ significantly (P ⫽ .551, F ⫽ 0.705 [3;90]) between MPC cells (ymax ⫽ 71.3, y0 ⫽ 1.65, k ⫽ 0.457) and MPCmCherry cells (ymax ⫽ 65.0, y0 ⫽ 1.21, k ⫽ 0.520). mCherry expression was quantified by measuring the fluorescence intensity of serially diluted MPC-mCherry cells at excitation/emission wavelengths of 550/600 nm (Figure 1E). We observed a proportional relation (y ⫽ 9.93 ⫻ 10⫺4x; R2 ⫽ 0.999) of cell number and fluorescence intensity (rs ⫽ 1; P ⫽ .003) confirming a stable genomic integration of the mCherry expression cassette.
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Figure 1. Biological properties of MPC-mCherry cells compared with nontransduced parent MPC cells in vitro; A and B, Morphological appearance and fluorescence under microscopy; nuclei stained with Hoechst 33258; scale bars, 30 m. C, Growth of 2 ⫻ 105 MPC and MPCmCherry cells during 16 days post subculture quantified as protein of adherent cells per cm2, presented as means ⫾ SEM, n ⫽ 4. D, Positive proportional relation of MPC-mCherry cell number and corresponding fluorescence intensities at Ex/Em ⫽ 550/600 nm.
Fluorescence imaging and progression of MPCmCherry-derived tumors To study tumor progression in vivo, we injected 2 ⫻ 106 MPC-mCherry cells subcutaneously into the shoulders of nude mice with rudimentary thymus (NMRI nu/nu). To support the engraftment, we used Matrigel in PBS as the injection vehicle. Tumor progression was monitored at regular intervals over 4 weeks. Tumor fluorescence intensity was measured by performing whole-body FLI at excitation/emission wavelengths of 600/700 nm allowing for deep tissue penetration at the optimal signal-to-background ratio, as determined by multispectral analysis (data not shown). As a reference parameter, tumor volume was determined with calipers (range from 2– 463 mm3). To exclude any possible sex effects on tumor progression, male and female animals were monitored specifically using the identical experimental setup. Progression of the sc tumors is shown in Figure 2, A–C. During the first 7 days after cell injection, the initial fluorescence intensity at the injection site decreased, probably due to cell death or dispersal. The remaining MPCmCherry cells gave rise to a solid sc tumor, which did not metastasize during the period of observation. Monitoring tumor progression by FLI (Figure 2B) revealed that the exponential tumor growth characteristics between male animals (y0 ⫽ 365083, k ⫽ 0.132, R2 ⫽ 0.604) and female
animals (y0 ⫽ 756 451, k ⫽ 0.110, R2 ⫽ 0.544) did not differ significantly (P ⫽ .165, F ⫽ 1.825 [2;133]). Monitoring tumor progression using volume measurement by caliper also showed that the exponential tumor growth characteristics between male animals (y0 ⫽ 2.66, k ⫽ 0.169, R2 ⫽ 0.728) and female animals (y0 ⫽ 6.41; k ⫽ 0.137; R2 ⫽ 0.795) did not differ significantly (P ⫽ .249, F ⫽ 1.405 [2;132]). Analysis of covariance between tumor volume (mm3) and tumor fluorescence intensity (total photon counts) revealed a significant positive correlation (rs ⫽ 0.946, P ⬍ .001). Taken together, whole-body FLI enabled us to noninvasively monitor the tumor cells in nonmetastatic MPC-mCherry cellderived sc tumors in vivo.
Physiologic parameters during progression of MPC-mCherry cell-derived tumors To investigate whether urinary free monoamines reflect the total tumor burden in mice bearing MPC-mCherry definable tumors, we regularly measured concentrations of urinary free catecholamines and their corresponding metanephrines by LC-MS/MS. In addition, BP was monitored at regular intervals. To examine any possible sex effects, we systematically compared male and female animals separately. In all animals, we found steadily increasing urinary concentrations of the several monoamines during 27 days of tumor progression compared with basal concentrations (⫺8 and ⫺2 days) before cell injection (Figure 3, A–E). In this effect, we did not observe any significant differences between male and female animals. We observed the earliest alterations in urinary monoamine concentrations at 6 days after cell injections. The animals showed a 1.7-fold increase in dopamine (DA) from 0.68 –1.18 mol/mmol creatinine and a 2.1-fold increase in its corresponding metabolite 3-methoxytyramine (MTY) from 0.35– 0.76 mol/mmol creatinine (Figure 4). In contrast, during this time period, norepinephrine (NE) decreased from 0.60 – 0.30 mol/mmol creatinine and its corresponding metabolite normetanephrine (NMN) decreased from 1.20 – 0.70 mol/mmol creatinine. At this time the average tumor volume was approximately 12.3 mm3 (tumor diameter of approximately 2.3 mm) and the fluorescence intensity at the cell injection site reached the minimum level for detection (Figure 2, A and B).
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0.68 –35.18 mol/mmol creatinine, a significant 70-fold increase in MTY from 0.35–24.26 mol/mmol creatinine, a nonsignificant 3-fold increase in NE from 0.55–1.56 mol/mmol creatinine, and a significant 11-fold increase in NMN from 1.21–13.03 mol/mmol creatinine. Throughout the experiment, epinephrine (EPI) and metanephrine (MN) remained at basal levels of 0.15– 0.30 mol/mmol creatinine, respectively (Figure 3E). Tumor volume (mm3) showed significant positive correlations with urinary outputs of DA (rs ⫽ 0.952, P ⬍ .001), MTY (rs ⫽ 0.947, P ⬍ .001), NE (rs ⫽ 0.756, P ⬍ .001), and NMN Figure 2. Progression of sc tumors in male and female nude mice after sc injection of 2 ⫻ 106 (rs ⫽ 0.949, P ⬍ .001). MPC-mCherry cells. A, Overlays of x-ray and fluorescence images (Ex/Em ⫽ 600/700 nm) of a representative animal at 1 h to 29 d post injection. B, Tumor fluorescence intensities in vivo over time. In addition to the altered cateC, Tumor volumes over time; data are presented as mean ⫾ SD, (male animals, n ⫽ 10; female cholamine pattern, we observed inanimals, n ⫽ 10). D, Correlation analysis between tumor volume and tumor fluorescence intensity; creasing systolic BP (Figure 3F). n ⫽ 20; confidence interval, 95%; number of XY pairs, 136; p.i., post injection; px, pixel. With this effect, we did not observe Within 27 days, we observed dramatic increases in uri- any significant differences between male and female aninary monoamine outputs of DA, MTY, and NMN. The mals. Changes in systolic BP were found to be significant animals showed a significant 50-fold increase in DA from (P ⬍ 0.01) after 24 days’ post-cell injections among a mixed population of both male (n ⫽ 10) and female (n ⫽ 10) animals. In conclusion, MPC-mCherry cell-derived sc tumors altered the physiologic catecholamine pattern and BP in nude mice. Among all monoamines investigated, urinary concentrations of free DA, MTY, NE, and NMN allowed for biochemical monitoring of tumor progression in vivo.
Discussion
Figure 3. Physiologic parameters during tumor progression in male and female nude mice after sc injection of 2 ⫻ 106 MPC-mCherry cells. A–D, Urinary concentration of free monoamines ⫺10 and ⫺2 d before, and 6, 13, 20, and 27 d after cell injection; presented as mean ⫾ SD (male animals, n ⫽ 10, female animals, n ⫽ 10). E, Changes in the urinary concentration pattern of free monoamines (net chart) presented as mean (male ⫹ female animals, n ⫽ 20). F, Systolic BP ⫺10 and ⫺4 d before and 3, 10, 17, 24, 31 d after cell injection presented as mean (male animals, n ⫽ 10; female animals, n ⫽ 10) and mean ⫾ SD (male ⫹ female animals, n ⫽ 20); DA, dopamine; MTY, 3-methoxytyramine; NE, norepinephrine; NMN, normetanephrine; EPI, epinephrine; MN, metanephrine; p.i., post injection. **, P ⬍ 0.01; ***, P ⬍ 0.001.
Our study characterized a sophisticated animal model developed for evaluating novel therapeutic strategies for the treatment of PHEO. We employed the murine MPC cell line for developing an allograft mouse model of PHEO. MPC cells are biochemically and molecularly similar to human PHEOs, and are a suitable tool for testing new therapies for PHEO (18). However, this model
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To monitor tumor progression using FLI, we generated an mCherry-expressing MPC cell line by lentiviral transduction, named MPC-mCherry. Genetic manipulation neither altered the morphology nor the growth characteristics compared with the parental MPC cell line. Genetic properties of this particular MPC-mCherry clone remain to be investigated. MPCmCherry cells consecutively express red fluorescent mCherry protein driven by the elongation factor alpha promoter, enabling easy optical detection of the cells both in vitro and in vivo. To investigate whether the intensity of tumor fluorescence is a suitable parameter to monitor disease progression in nude mice, we performed whole-body FLI in the far-red portion of the spectrum, detecting only the tumor cells in MPC-mCherry cell-derived sc tumors in vivo. FLI allows for semiquantitative measurements of tumor progression, metastasis, and treatment response (32), and unlike other in vivo imaging strategies, it does Figure 4. Analysis of covariance between urinary free monoamines and tumor volume. DA, not require the addition of a substrate, dopamine; MTY, 3-methoxytyramine; NE, norepinephrine; EPI, epinephrine; NMN, normetanephrine. n ⫽ 20, confidence interval, 95%, number of XY pairs, 78. an imaging probe, or a radiotracer. Thus, FLI is preferred for multiple examinations at a minimum stress level may not be suitable for investigating metastatic spread, for animals. Fluorescent proteins emitting in the far-red porgenerally arising from a primary tumor. However, in nude tion of the spectrum allow for biological interrogations at mice, iv and ip, but potentially not sc injections of MPC deep optical tissue penetration (33, 34). Among these procells, can be used to generate multiple organ lesions (31). teins, mCherry, owing to its spectral class (excitation maxThe difficulty thereafter is to monitor disease progression. imum, 587 nm; emission maximum, 610 nm), brightness, We have therefore first employed MPC cells to develop an and superior photostability, is the best general purpose agent sc nonmetastatic mouse model of PHEO in which the lofor whole-body optical imaging (35, 36). These optical propcation of one single lesion is known and its size and morerties allow for detection of especially small changes in numphology can easily be determined and matched to other ber of tumor cells observed during the first 7 days after cell parameters such as fluorescence intensity and urinary injection. However, these effects on the number of tumor monoamine concentrations to validate their use for mon- cells may be due to cell death, diffusion, invasion of neutroitoring disease progression. phils, or newly built blood vessels and connective tissue. The In our study, the use of Matrigel increased the repro- strongly positive correlation between tumor size and fluoducibility of tumor cell engraftment as well as the homo- rescence intensity suggests a minor influence of light-scattergeneity in tumor shapes among the animals and, thereby ing artifacts when the total fluorescence intensity of the tuimproved tumor volume measurements by caliper. Precise mor is used as a quantitative parameter. and complete determination of tumor volume was a preUnlike caliper measurements, FLI eliminates most conrequisite for studying intensities of tumor fluorescence and tributions of the nontumor cell-derived volume, such as concentrations of urinary free monoamines as surrogate capsule, edema, necrotic areas, blood vessels, or immune markers of disease progression in this murine model of cell infiltration, inside the heterogeneous structure of a PHEO. solid tumor. Taken together, FLI of MPC-mCherry cell-
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doi: 10.1210/en.2014-1431
derived tumors is considered to provide a powerful and rapid method for evaluating tumor progression during preclinical therapeutic investigations in nude mice. In our study, we used nude mice to ensure maximal optical tissue penetration during whole-body FLI. In addition, the immunodeficiency of athymic mice will allow for extending the animal model to the engraftment of primary human PHEO cells or newly developed human PHEO cell lines in the future. Referring to the latter, a recent study reported about a human PHEO precursor cell line established form a primary human tumor (10). Our findings that sc MPC-mCherry cell-derived tumors did not metastasize in nude mice verify earlier studies (31). This outcome may be due to the fact that MPC cells were generated from adrenal tumors of BL-6 heterozygous neurofibromin-1 knockout mice, which generally exhibit a low metastatic potential (14). Despite their immunodeficiency, nude mice are capable of generating a nonspecific immune response against cells from different strains, for example by natural killer (NK) cell activation (37). This effect may also contribute to the initial tumor cell death at the injection site observed during the first 7 days after injection. We also investigated whether tumor-induced alterations of physiologic parameters reflect the total tumor burden of PHEO in mice. For this, we monitored the concentrations of urinary free monoamines as well as BP at regular intervals during 4 weeks of tumor progression. Among all monoamines investigated, our analyses revealed that the most dramatic increases involved urinary levels of DA and its corresponding metabolite MTY. These results suggest that MPC-mCherry cell-derived sc tumors mainly produce DA. Most importantly, measuring urinary free DA and/or MTY allowed for the most sensitive biochemical detection of tumors at diameters below 2.3 mm in vivo, although possessing similar tumor detection limits compared with anatomic and functional smallanimal imaging (magnetic resonance imaging, computed tomography, positron emission tomography, single-photon emission computed tomography) in vivo. Therefore, our investigations suggest that measuring urinary free DA and/or MTY allows for easy, noninvasive quantification of catecholamine-producing lesions. In contrast with the increase in urinary DA, we found relatively small increases in urinary NE but larger increases in its corresponding metabolite, NMN. These findings suggest that much of the NE produced within tumors is metabolized to NMN. We did not observe tumor-induced increases in urinary free EPI or its corresponding metabolite, MN. Although MPC cells are known to express phenyl-N-methyltransferase, they also only produce small amounts of EPI from NE (14), explaining why we
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did not observe significant increases in urinary EPI and MN. However, adaptation of peripheral catecholamine metabolism, such as in the liver, cannot be excluded as a factor influencing the observed metabolite pattern. Systolic BP was found to be significantly increased during tumor growth, demonstrating the physiologic influence of tumor-related NE release. Nevertheless, increases in BP were not severe, although reflecting the pattern of increases in urinary outputs of catecholamines (38). In summary, we demonstrated that the MPC-mCherry cell-derived sc allograft mouse model exhibits functional similarities to the endocrine situation in human PHEO, characterized by increased urinary outputs of catecholamines and BP. In particular, measurement of tumor progression by whole-body FLI and urinary free DA and/or MTY by LC-MS/MS allows for noninvasive quantification of tumor progression. The introduced sc PHEO model provides a useful tool for comprehensively evaluating therapeutic strategies for PHEO, but may not be a predictor of organ metastatic lesion responses to treatment. However, FLI and measurements of urinary free monoamines may offer perspectives to substantially facilitate comprehensive preclinical diagnostic and therapeutic evaluations also in other mouse models, which present tumors in the liver, lung, kidneys, bones, and muscles. Diagnosis and monitoring of tumor progression and therapy response in human patients with metastatic PHEO may also benefit from this strategy.
Acknowledgments Address all correspondence and requests for reprints to: Christian G. Ziegler, PhD, Molecular Endocrinology, Medical Clinic III, University Hospital Carl-Gustav Carus, Fiedlerstrasse 42, 01307 Dresden, Germany. E-mail: [email protected]. This work was supported by The Deutsche Forschungsgemeinschaft (Grants ZI-1362/2–1 [to C.G.Z. and G.E.] and BE2607/1–1 [to R.B. and J.P.]). MPC 4/30PRR cells were kindly provided by Professor Arthur Tischler, Dr James Powers, and Professor Karel Pacak. Disclosure Summary: The authors have nothing to disclose.
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