Establishment and Characterization of a Human Adrenocortical Carcinoma Xenograft Model*
ARMELLE LOGIÉ, PHILIPPE BOUDOU, LILIANE BOCCON-GIBOD, ERIC BAUDIN, GILLES VASSAL, MARTIN SCHLUMBERGER, YVES LE BOUC, AND CHRISTINE GICQUEL
Laboratoire d’Explorations Fonctionnelles Endocriniennes, INSERM U-515 (A.L., Y.L.B., C.G.), and Laboratoire d’Anatomie et Cytologie Pathologique (L.B.G.), Hôpital d’Enfants Armand Trousseau, 75012 Paris, France; Laboratoire de Biologie Hormonale, Hôpital Saint Louis (P.B.), 75010 Paris, France; and Service de Médecine Nucléaire (E.B., M.S.) and Laboratoire de Pharmacotoxicologie (G.V.), Institut Gustave Roussy, 96800 Villejuif, France
ABSTRACT
Adrenocortical carcinomas are rare malignant tumors. They have a poor prognosis, as they are often diagnosed late and are usually resistant to chemotherapy. The lack of a suitable animal model for these tumors has been a major obstacle to the evaluation of new therapeutic agents. The aim of this study was to establish and char- acterize xenografts of the human adrenocortical carcinoma NCI H295R cell line as a model of adrenocortical carcinoma for future therapeutic trials. This cell line was sc injected (6 × 106 cells) into nude mice (n = 20). Solid tumors were locally measurable after 45 days at 90% of the inoculation sites. The xenografts were similar histologically to the original adrenocortical carcinoma from which the cell line was derived. The xenografts precisely reproduced the dys- regulation of the insulin-like growth factor (IGF) system [overexpres-
sion of the IGF-II and IGF-binding protein-2 (IGFBP-2) genes] typical of adrenocortical carcinoma. Similarly to adrenocortical carcinomas, human IGFBP-2 (but not IGF-II) was secreted in mouse plasma. We analyzed steroid production (cortisol, 17-hydroxypregnenolone, 17- hydroxyprogesterone, dehydroepiandrosterone, 44-androstenedione, 11-deoxycortisol, corticosterone, and testosterone). Xenografts pro- duced all three class of steroids, with the preferential production of androgens of the 44 pathway.
The H295R xenograft model is a good model of human adrenocor- tical carcinoma, as it mimics dysregulation of the IGF system usually found in these tumors. It also produces IGFBP-2 and steroids that can be used as tumor markers. This model may therefore be useful for evaluating therapeutic agents. (Endocrinology 141: 3165-3171, 2000)
A DRENOCORTICAL carcinomas are rare malignant tu- mors with a poor prognosis. Almost 50% are meta- static on diagnosis, and metastatic disease develops in most patients (>80%), with apparently localized disease at diag- nosis (1). Systemic treatments, including chemotherapy, are poorly effective. The rarity of these tumors also complicates both their pathogenetic approach and therapeutic evaluation (2, 3).
We recently showed that the insulin-like growth factor (IGF) system plays a major role in the malignant transfor- mation of adrenocortical tumors. Indeed, most malignant tumors (90%) exhibit strong overexpression of the IGF-II gene and a maternal loss of heterozygosity of the imprinted 11p15 region (where the IGF-II gene maps) (4). Moreover, 11p15 loss of heterozygosity and the overexpression of the IGF-II gene in localized adrenocortical tumors are predictors of tumor relapse (our manuscript in preparation). The ex-
Address all correspondence and requests for reprints to: Dr. Christine Gicquel, Laboratoire d’Explorations Fonctionnelles Endocriniennes, Hô- pital Trousseau, 26 avenue Arnold Netter, 75012 Paris, France. E-mail: christine.gicquel@trs.ap-hop-paris.fr.
* This work was supported by Assistance Publique Hôpitaux de Paris: Contrat de Recherche Clinique 97133, the University Paris VI, Faculté Saint Antoine (UPRES EA 1531), Association de Recherche contre le Cancer (no. 1364), INSERM (U-515), Programme Hospitalier de Recher- che Clinique Grant AOM-95201 for the Comete Network, and grants from La Ligue Nationale contre le Cancer and La Fondation pour la Recherche Médicale (to A.L.).
pression of IGF-binding proteins (IGFBPs) is also im- paired, and malignant tumors contain large amounts of IGFBP-2, suggesting that IGFBP-2 is involved in IGF-II proliferative effects (5).
A human tumor cell line, H295, was recently established (6, 7). It was derived from an invasive, secreting primary adrenocortical carcinoma (ACC) (6). H295R cells retain the ability to produce all of the major steroids (mainly andro- gens). As in malignant adrenocortical tumors, cell prolifer- ation is associated with an overexpression of both the IGF-II and IGFBP-2 genes (8). We have also shown that the prolif- erative effects of IGF-II on H295R cells are mediated by the type 1 IGF receptor (8).
Human tumor xenografts are well established tools for the preclinical screening of anticancer drugs, which is particu- larly interesting for rare tumors. The aim of this study was to establish and to characterize the first sc xenograft model of adrenal carcinoma for use in therapeutic trials. We found that the xenografts exhibited features typical of human ACC, dysregulation of the IGF system in particular. The xenografts also produced and secreted steroids and human IGFBP-2, and their plasma concentrations may be useful markers for the evaluation of anticancer drugs in mice.
Materials and Methods
Cell cultures
NCI H295R cells (7) were cultured to confluence in a 1:1 mixture of DMEM and Ham’s F-12 medium (Sigma, St. Louis, MO). The medium
was supplemented with transferrin (5 µg/ml; Sigma), sodium selenite (5 ng/ml; Sigma), L-glutamine (2.5 mm; Life Technologies, Inc., Paisley, UK), and antibiotics (50 µg/ml streptomycin, 50 IU/ml penicillin; Life Technologies, Inc.) with or without 2% Ultroser G (Biosepra, Marlbor- ough, MA) at 37 C in a 5% CO2-95% air atmosphere. Confluent cells were treated using 0.05% trypsin and 0.02% EDTA and were used during passages 3-6 for experiments. For injection into mice, cells were treated with trypsin, collected by centrifugation, rinsed, and resuspended in 1 X PBS. They were then split into aliquots of 6 × 106 cells/100 pl.
Animals and biological samples
Female nude mice (nu/nu) were bred at the INSERM U515 (Paris, France) animal house. They were fed on a standard diet and were subjected to a regulated light cycle (12 h of light, 12-h of darkness). Cell suspensions (100 ul) were bilaterally injected sc into the flanks of 6-week-old mice (n = 20). For each passage, the tumor was ground into small pieces in PBS, and the resulting suspension was injected into the flanks of nude mice. Two perpendicular diameters of the tumor were measured twice a week by the same investigator. The volume of each tumor was calculated according to the following equation: V (mm3) = (d2 × D)/2, where d (mm) and D (mm) are the smallest and largest perpendicular tumor diameters, respectively (9). The take rate was de- fined as the number of tumors per number of inoculation sites. Animals were decapitated, and blood was collected into 4 mM EDTA. Plasma was isolated by centrifugation, aliquoted, and stored at -20 C. Tumors were immediately frozen in liquid nitrogen and stored at -80 C for RNA and protein extraction or immersed in 10% formol for pathological analysis. Experiments were carried out in accordance with the guidelines of the Ministry of Agriculture (authorization 7574).
Pathological methods
Each tumor sample was weighed and fixed in formalin, and serial sections were cut and stained with hematoxylin-eosin-safran.
IGF assays
The method used has been described in detail previously (10). Plasma samples (25 pl) were incubated with 2 ml 0.01 N HCI for 30 min at room temperature to dissociate IGFs from IGFBPs and centrifuged on Cen- tricon 30 (Amicon, Epernon, France) to separate IGFs from IGFBPs. The ultrafiltrate containing IGFs was lyophilized, dissolved in 0.1 M phos- phate buffer (pH 7.4) and 1 mg/ml BSA (BioMérieux, Paris, France), and incubated for 2-3 days in a final volume of 400 ul with either 1) a specific polyclonal antihuman IGF-I antibody that cross-reacts with murine IGF-I (gift from Dr. J. Closset, Liege, Belgium) and [125]]hIGF-I (3000 cpm/tube) or 2) IGFBPs extracted from cerebrospinal fluid that have a selective affinity for IGF-II (11) and [125I]human IGF-II (3000 cpm/tube).
Steroid assays
Steroid secretion was assessed by determining the plasma concen- tration of the steroid concerned. 17-Hydroxypregnenolone (17OH- PREG), 17-hydroxyprogesterone (17OH-P), dehydroepiandrosterone (DHEA), 44-androstenedione (A), 11-deoxycortisol (11-DOF), cortico- sterone (B), and testosterone (T) levels were determined by specific RIA methods after chromatographic purification of plasma samples as pre- viously described (12). The lower limit of detection was 0.76 nmol/liter for 17OH-PREG, 0.84 nmol/liter for 17OH-P, 1.08 nmol/liter for DHEA, 0.42 nmol/liter for A, 0.51 nmol/liter for 11-DOF, 3.1 nmol/liter for B, and 0.19 nmol/liter for T (12). Cortisol (F) was determined directly using the Gamma Coat [125]]F kit (DiaSoring, Stillwater, MN) or was evaluated, with the same kit, after a chromatography step (13). The cross-reactivity of the F assay with the main relevant steroids was 6.3% for 11-DOF, 0.4% for B, and 1.2% for 17OH-P. The lower limit of detection for F was 5.9 nmol/liter for the direct assay (manufacturer’s data) and 7.0 nmol/liter after the purification procedure (13).
Protein extraction
Frozen tissues (100 mg) were quickly homogenized on ice in 3 ml ice-cold lysis buffer [50 mM HEPES (pH 7), 250 mm NaCl, 5 mm EDTA,
1 mM sodium orthovanadate, 2 mm sodium pyrophosphate, and 0.1% Nonidet P-40] containing protease inhibitors (1 mm dithiothreitol, 1 ug/ml aprotinin, 1 µg/ml leupeptin, and 50 µg/ml phenylmethylsul- fonylfluoride) using a Polytron homogenizer (Brinkmann Instruments, Inc., Westbury, NY).
Homogenates were incubated for 1 h at 4 C and centrifuged at 15,000 × g for 30 min at 4 C. The supernatant was collected and frozen at -20 C. Aliquots of supernatant were collected for protein determi- nation by the Bradford method (Bio-Rad protein assay, Bio-Rad Labo- ratories, Inc., Richmond, CA).
Western ligand and immunoblotting
Western ligand blotting and immunoblotting were performed as previously described (14, 15). Briefly, plasma samples (3 µl) or tumor proteins (200 µg) were denatured at 100 C for 2 min and subjected to SDS-11% PAGE under nonreducing conditions. The proteins were then electrotransferred onto nitrocellulose (BA 85, Schleicher & Schuell, Inc., Dassel, Germany). The various IGFBP species were detected 1) by in- cubation with a mixture of [125]]IGF-I and [125]]IGF-II (5 × 105 cpm each) at 4 C for 48 h, followed by autoradiography (ligand blotting), and 2) by incubation with an anti-bovine-IGFBP-2 polyclonal antibody that rec- ognizes both human and mouse IGFBP-2 (Upstate Biotechnology, Inc., Lake Placid, NY). Blots were then incubated with an anti-IgG antibody coupled to horseradish peroxidase (Biosource International, Camarillo, CA), and complexes were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech, Uppsala, Sweden).
Isolation and analysis of RNA
Total RNA was extracted from frozen tumors using the standard CsCl/guanidine isothiocyanate method as previously described (16). Total RNA (10 µg) was loaded onto a 1.0% agarose-formaldehyde gel, subjected to electrophoresis, and transferred to a Hybond XL membrane (Amersham Pharmacia Biotech). The RNA was covalently bound to the membrane by baking at 80 C for 2 h. Northern blots were probed with the human IGF-II complementary DNA (cDNA) (17).
Type 1 and type 2 IGF receptor expression was analyzed by RT-PCR. RNA samples (1 µg) were treated with deoxyribonuclease and reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc.), and the resulting cDNA was amplified by PCR. The sense and antisense primers for the type 1 IGF receptor were 5’-AAC CAC GAG GCT GAG AAG CT and 5’-CAG CAT AAT CAC CAA CCC TC, respectively (18). Oligonucleotide primers for the type 2 IGF recep- tor were designed using sequences from the 3’-untranslated region of the type 2 IGF receptor gene (19). The sense and antisense primers were 5’-TTG CCG GCT GGT GAA TTC AA and 5’-GTA TCA TGA GAA CCT GAA GAG, respectively. Deoxyribonuclease-treated RNA was sub- jected to PCR as a control for DNA contamination of the RNA samples. The amplification products were analyzed by electrophoresis in a 1.5% agarose gel.
Statistical and mathematical analysis
Values are medians (minimum-maximum) unless otherwise stated. The Mann-Whitney test was used to evaluate differences between groups. P < 0.05 was considered significant. An exponential function was fitted to the tumor growth curve, and doubling time was calculated for r2 > 0.95.
Plasma sample volumes were small, so for statistical analysis, the first calibration point (loss-corrected) was retained for steroids with concen- trations lower than the detection threshold (A, DHEA, 17OH-P, and 11-DOF).
Results
Tumorigenicity of the NCI H295R cell line
Cell suspensions were injected sc into both flanks of nude mice. Solid tumors were locally measurable 45 days (range, 32-73) after inoculation. The overall tumor take rate was 90%. The tumor growth curves for the first passage were heter- ogeneous (Fig. 1A). Differences were also seen between the
A
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Tumoral volume (mm3)
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10
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doubling time (days)
tumors growing on the left and right sides of individual mice. However, analysis of tumor growth curves for the second and third passages showed that growth rate was stable through the serial passages. Most xenografts had a doubling time of 12 days (range, 6-26; Fig. 1B).
Histological examination of xenografts
The xenografts were histologically similar to the adreno- cortical tumor from which the NCI H295 cell line was derived (6) (Fig. 2). Indeed, the xenografts contained rather small, uniform cells with prominent nucleoli, a high mitotic index, and foci of necrosis. No vascular invasion was observed. A thin capsule surrounded the tumor, interrupted in a few places by small bulging extracapsular nodules.
Plasma steroid concentrations in mice
We have previously demonstrated the production of glu- cocorticoids, androgens, and mineralocorticoids by the NCI H295R cell line in serum-free medium (8). We therefore ex- pected the xenografts to produce steroids. The concentra-
tions of steroids in the plasma of control and xenografted mice are summarized in Fig. 3.
In the control group of mice, we detected no significant amount of 17OH-P, DHEA, A, or 11-DOF. In contrast, this group secreted 17OH-PREG (7.6 ± 2.1 nmol/liter) and, to a lesser extent, T (1 ± 0.4 nmol/liter), with B the major steroid (103 ± 88 nmol/liter). F was detected (51.5 ± 4.4 nmol/liter) in the control group of mice, probably due to a bias in the direct 125I cortisol assay. Indeed, when F levels were evalu- ated, after extraction chromatography, in three control mice with larger available plasma volumes, the concentration of F was below the detection limit (<27 nmol/liter).
Unlike control mice, xenografted mice had high steroid levels. 17OH-PREG (the main precursor of androgens in H295R cells) was metabolized to give both 17OH-P and DHEA. High concentrations of A were derived from 17OH-P and DHEA, but also from 11-DOF. Like NCI H295R cells, the xenografts produced large amounts of 11-DOF, suggesting an alteration in the 11ß-hydroxylase activity. There was a significant difference in F levels (P = 0.006) between the control and xenografted mouse groups (Fig. 3). Indeed, more
than half of the xenografted mice had F levels above those in the control group (Fig. 3). Although there is cross-reaction with other steroids, in the direct [125I]F assay, this difference between controls and xenografted animals is consistent with F production by xenografts. Due to the plasma volumes required for F assay after chromatography extraction, F as- says could not be carried out for this series of xenografted mice. Instead, we used another series of 10 xenografted mice. We found that mice with F levels above 70 nmol/liter in the direct assay had detectable cortisol levels after the purifica- tion procedure (data not shown). Moreover, xenografts pro- ducing large amounts of one steroid also produced large amounts of the other steroids.
Expression of the components of the IGF system in mice
IGF-II expression was expected in xenografts because they were derived from the NCI H295R ACC cell line. Northern blot analysis of RNA extracts from xenografts showed that the species of IGF-II messenger RNA (mRNA) produced (2.2, 4.8, and 6.0 kb) were the same as those in NCI H295R cells, with the preferential expression of the 2.2 kb P3-driven spe- cies (Fig. 4).
We have shown that NCI H295R cells, grown in serum-free medium produce both IGF-II and IGFBP-2 proteins, which are secreted into the medium (8). Plasma IGF-I and IGF-II were determined by RIA (for 8 control and 10 xenografted mice). Plasma IGF-II levels were similar in control and xe- nografted mice (85 ± 10 vs. 96 ± 28 ng/ml; mean ± SD) as were plasma IGF-I levels (577 ± 78 vs. 469 ± 144 ng/ml).
In Western ligand blot analysis of the proteins extracted from xenografts and from xenograft-bearing mouse plasma, we detected a band that migrated to the same position as the 34-kDa band of a control human serum but was absent from control mouse plasma (Fig. 5A). This 34-kDa band was iden- tified as human IGFBP-2 by immunoblotting. On the immu- noblot probed with the anti-IGFBP-2 antibody, a 32-kDa band was also detected in plasma from both xenografted and control mice. This band corresponds to mouse IGFBP-2 (the
IGFBP-2 antibody recognizes both mouse and human IGFBP-2).
In mouse plasma, the human IGFBP-2 appeared as an intact protein, as no proteolytic fragment was detected (Fig. 5B). By contrast, in xenografts, an additional band migrating at 25 kDa was detected on IGFBP-2 immunoblots. This band has also been detected in human ACC (5) and probably corresponds to a proteolytic fragment (Fig. 5B).
Other IGFBPs were detected by Western ligand blotting in tumors, and in control and xenograft-bearing mouse plasma, a 39- to 42-kDa protein corresponding to IGFBP-3, a 32-kDa protein corresponding to IGFBP-1 and IGFBP-2, and a 24- kDa protein corresponding to IGFBP-4 (Fig. 5A).
Finally, we showed that both IGF receptors (types 1 and 2) were expressed similarly in xenografts and H295R cells (Fig. 6).
Discussion
ACC is a rare tumor with an annual incidence of approx- imately two per million people (20). Until recent years, the pathogenesis of adrenocortical tumor was poorly under- stood. The most relevant advance in our understanding of adrenocortical tumorigenesis was the demonstration that the IGF system is dysregulated in malignant adrenocortical tu- mors. The prognosis of ACC is very poor for two major reasons: 1) it is often discovered at an advanced stage, when curative surgery is no longer possible; and 2) no chemother- apy has been shown to be really efficient (1).
Until recently, no experimental model was available. Only a few human ACC cell lines have been established (6, 21), and no ACC xenograft model has previously been described. We recently characterized the IGF system of the human adrenal tumor H295R cell line and demonstrated the involvement of IGF-II in cell proliferation (8). This cell line is, therefore, a useful in vitro model for adrenal carcinoma. However, cell lines alone are insufficient for therapeutic evaluation, and the main limitation to the development of new treatment strat-
p < 0.005
p < 0.05
17OH-PREG (nmol/L)
50
12
40
DHEA (nmol/L)
10
30
8
20
6
4
10
2
0
control
xenografted
0
control
xenografted
NS
p < 0.005
17OH-P (nmol/L)
14
20
12
10
A (nmol/L)
15
8
6
10
4
2
5
0
control
xenografted
0
control
xenografted
p < 0.005
p< 0.01
75
8
11-DOF (nmol/l)
60
T (nmol/L)
6
45
30
4
15
2
0
control
xenografted
0
control
xenografted
NS
p < 0.01
300
150
B (nmol/L)
250
F (nmol/l)
200
100
150
100
50
50
0
control
xenografted
0
control
xenografted
egies for ACC has been the lack of an appropriate animal model.
In this study we used the H295R cell line, which has abnormalities of the IGF system similar to those of human ACC and characteristics typical of tumorigenic cells (immor- tality, loss of contact inhibition, growth factor-independent proliferation, and anchorage-independent growth) (6-8) to establish and characterize an in vivo murine xenograft model.
We successfully transplanted H295R cells into nude mice with a take rate of almost 100%. The latency period for tumor growth was 6 weeks at the first passage and remained con- stant for sequential passages. Tumorigenicity has to date been maintained for approximately 24 months (five passag- es). The xenografted tumors reproduced the features of hu- man ACC and exhibited histological features similar to those
xenografts
plac
3
5
21
295 ACC
6 kb-
IGF- II
4.8 kb -
2.2 kb -
28S
xenografted mouse plasma
control mouse plasma
xenograft
human plasma
fetal calf serum
A
kDa
42
39
34
WLB
32
B
kDa
34
anti-IGFBP-2 WIB
32
25
of the original tumor (6). No metastases were observed in xenografted mice.
NCI H295R xenografts appear to be a useful ACC model for two major reasons: xenografts reproduce the dysregula- tion of the IGF system found in malignant human tumors and in the H295R cell line. Indeed, most malignant adrenocortical tumors strongly overexpress the IGF-II gene, and efficient translation results in the production of large amounts of IGF-II protein, mostly in precursor forms (5). We showed that IGF-II is involved in adrenocortical tumor cell proliferation
xenograft
3
5
21
H295R
H20
ladder
D cDNA D cDNA D cDNA D cDNA
type 1 IGF receptor
447 bp
type 2 IGF receptor
160 bp
by demonstrating that cell proliferation is reduced by block- ing antibodies directed against the IGF-II peptide or the type 1 IGF receptor (8). IGF-II effects are restricted to tumors via an auto/paracrine pathway, and systemic plasma levels of IGF-II from patients with ACC are in the normal range (22). In this study plasma levels of IGF-II from xenograft-bearing mice are also in the same range as those in control mice.
The high level of IGF-II production by malignant tumors should also be seen in the light of chemoresistance. A recent study suggested that the antiapoptotic function of IGF-II may result in a decrease in sensitivity to various chemotherapeu- tic agents (23). It also demonstrated that sensitivity to che- motherapeutic agents is highly enhanced if IGF-II is blocked. Various antimitotic agents (mostly cisplatin based) have been tested, but partial and transient tumor responses have been observed in only a small percentage of patients (24-29). Clearly, trials with other drug combinations are required, but the rarity of adrenal carcinoma considerably limits the eval- uation of chemotherapy. Thus, H295R xenografts provide a much-needed model for testing adjuvant therapies, partic- ularly therapies directed against growth factors.
Another key advantage of this xenograft model is the production by xenografts and secretion into mouse plasma of IGFBP-2 and steroids that could be used as tumor markers. The xenografts produced human IGFBP-2. IGFBPs locally modulate the actions of IGF (30). We previously showed the specific overexpression of IGFBP-2 in human ACC (5) and the exclusive expression of IGFBP-2 in the H295R cell line (8). Several studies have suggested that IGFBP-2 expression may be associated with malignancy. Both stimulatory and inhib- itory effects of IGFBP-2 on IGF activity have been reported. Among stimulatory effects, IGFBP-2 proteolysis, by decreas- ing IGF-II affinity, may increase IGF-II bioavailability and enhance its proliferative and/or its antiapoptotic effects. Al- though IGFBP-2 proteolysis did not occur in H295R cells (8), it exists in vivo, both in human ACC (5) and in xenografts, suggesting that IGFBP-2 proteolysis requires other cell com- ponents. A recent report (31) suggests that IGFBP-2 enhances cell proliferation in adrenal tumors by an IGF-independent mechanism. Human IGFBP-2 was secreted into mouse plasma. We have previously shown that IGFBP-2 is secreted in human ACC and that IGFBP-2 levels are correlated with
tumor spread (Boulle, N., E. Baudin, C. Gicquel, A. Logié, J. Bertherat, A. Penformis, X. Bertagna, J. Luton, M. Schlum- berger, and Y. Le Bouc, manuscript submitted).
In conclusion, this mouse model, involving the xenograft- ing of H295R cells, should make it possible to study the involvement of the IGF system in chemoresistance and to evaluate new therapeutic agents. Angiostatic therapy seems to be of potential interest, as recent data have suggested that the angiogenic switch is activated during the early prema- lignant stages of adrenocortical tumorigenesis (32).
Acknowledgments
We thank Dr. S. Babajko for assisting us with the animal work.
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