Adrenocortical Carcinoma Xenograft in Zebrafish Embryos as a Model To Study the In Vivo Cytotoxicity of Abiraterone Acetate
Alessandra Gianoncelli,1* Michela Guarienti,1* Martina Fragni,1 Michela Bertuzzi,1 Elisa Rossini,1 Andrea Abate,1 Ram Manohar Basnet,1 Daniela Zizioli,2 Federica Bono,1 Massimo Terzolo,3 Maurizio Memo,1 Alfredo Berruti,4 and Sandra Sigala1
1Section of Pharmacology, Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy; 2Section of Biotechnology, Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy; 3Department of Clinical and Biological Sciences, University of Turin, Internal Medicine 1, San Luigi Gonzaga Hospital, 10043 Orbassano, Italy; and 4Oncology Unit, Department of Medical and Surgical Specialties, Radiological Sciences and Public Health, University of Brescia and ASST Spedali Civili di Brescia, 25123 Brescia, Italy
ORCID numbers: 0000-0002-0816-5163 (A. Gianoncelli); 0000-0002-0565-5570 (M. Guarienti); 0000-0001-6641-8985 (M. Bertuzzi); 0000-0002-4171-2851 (M. Terzolo); 0000-0002-7543-0289 (M. Memo); 0000-0002-3956-0043 (A. Berruti); 0000-0003-3294-4121 (S. Sigala).
Abiraterone acetate (AbiAc) inhibits tumor growth when administered to immunodeficient mice engrafted with the in vitro cell model of human adrenocortical carcinoma (ACC). Here, we de- veloped and validated a zebrafish model engrafted with cortisol-secreting ACC cells to study the effects of AbiAc on tumor growth. The experimental conditions for AbiAc absorption in AB zebrafish embryos including embryo number, AbiAc concentration, and absorption time curve by liquid chromatography-tandem mass spectrometry were set up. The AbiAc effect on steroid production in AB zebrafish embryos was measured as well. ACC cells (the NCI-H295R cell line, the primary cell ACC29, and the negative control cell SW13) were treated with drug-induced liver injury fluorescent dye, and ~240 cells per 4 nL was injected in the subperidermal space of the yolk sac of AB zebrafish embryos (n = 80 ± 10). The cell area was measured with Noldus DanioScope™ software. AbiAc absorption in AB zebrafish embryos was stage dependent. Abiraterone (Abi) concentration decreased, whereas its main metabolite, 44A, increased. Accordingly, we demonstrated that zebrafish expressed mRNA encoding the enzyme 30-hydroxysteroid dehydrogenase, which converts Abi in 44A. Furthermore, ABiAc reduced cortisol production and increased progesterone in zebrafish embryos. Three days after cell injection, the cortisol-secreting ACC cell area in solvent-treated embryos was significantly higher than that in 1 p.M AbiAC-treated embryos, whereas no AbiAc effect was observed in SW13 cells, which lack the Abi target enzyme CYP17A1.Zebrafish embryos xenografted with ACC tumor cells could be a useful, fast, and reproducible experimental model to preclinically test the activity of new drugs in human ACC. (Endocrinology 160: 2620-2629, 2019)
A drenocortical carcinoma (ACC) is a rare tumor with an estimated incidence between 0.7 and 2.0 per one million people per year (1). Surgery is the only potentially
curative treatment. Systemic therapies have limited efficacy, and the prognosis of patients with locally ad- vanced or metastatic ACC is often dismal. Mitotane is the
*A.G. and M.G. contributed equally to the work.
Abbreviations: 30-Hsd, 3ß-hydroxysteroid dehydrogenase; Abi, abiraterone; AbiAc, abiraterone acetate; ACC, adrenocortical carcinoma; ESI, electrospray ionization; hpf, hours postfertilization; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MeOH, methanol; MSn, tandem mass spectrometer; PTU, 1-phenyl-2-thiourea; PVDF, polyvinylidine fluoride; T0, 2 hours after treatment; T3, 3 days of treatment; UPLC, ultra- performance liquid chromatography.
only drug approved to treat ACC in both the adjuvant setting and metastatic disease (2, 3); however, the drug’s pharmacokinetics and safety profile limit its efficacy (4). Mitotane can be administered alone or in association with etoposide, doxorubicin, and cisplatin (5, 6). The overall 5-year survival rate of patients with metastatic ACC treated with etoposide, doxorubicin, and cisplatin plus mitotane is ~15%. In this scenario, the introduction of new potentially effective drugs in ACC or the dem- onstration of efficacy of available drugs is of paramount importance. However, evaluation of new targets and drugs using established cell lines is limited by the inexact correlation between responsiveness observed in the cell lines and that elicited in the patient. Tumor cell xeno- grafts in athymic mice, generated from fresh tumor specimens, recapitulate the diversity of malignancies and represent the in vivo model used most often over the last 50 years (7). However, this model is time-consuming and expensive; in addition, there are important limitations in the early phase of drug screening, when a large number of drugs need to be evaluated for their potential antitumor activity. It is therefore mandatory, especially in rare diseases such as ACC, to identify and validate reliable and faster experimental preclinical in vivo models that offer a robust demonstration of the antitumor efficacy of new drugs.
The zebrafish (Danio rerio) model was recently de- veloped and has become a widely used experimental vertebrate model in many fields of scientific research. In particular, zebrafish embryos represent a valuable tool for studying human diseases, including cancer (8, 9) and developing in vivo toxicological and pharmacological screenings (10-16).
Zebrafish xenograft models of different tumor cells have been validated for preclinical anticancer drug screening (17-20). Indeed, very low amounts of cancer cells and drugs are needed to conduct the experiments; in addition, results can be obtained in only a few days in zebrafish embryos/larvae, as opposed to several weeks in mice models. No immunosuppressant treatment is necessary because lymphocytes mature in ~7 days after fertilization (21), and the transparency of zebrafish embryos and larvae allows easy observation of engrafted tumor cells. Taken together, these findings affirm that the zebrafish xenograft models developed thus far are fast, simple, and reproducible (17-20).
The aim of this study was to provide evidence that the zebrafish embryo model is a useful tool for evaluating the in vivo cytotoxicity of drugs with potential efficacy in ACC. To do so, we validated in zebrafish embryos results already obtained in immunodeficient mice xenografted with NCI-H295R cells treated with abiraterone acetate (AbiAc) (22). AbiAc is an irreversible inhibitor of 17a-
hydroxylase/17,20-lyase (CYP17A1), a key enzyme for steroid hormone synthesis (23). Because AbiAc inhibits androgen synthesis throughout the body, it is effective in the management of metastatic prostate cancer (24, 25). In addition to reducing androgen levels, the drug rapidly impairs cortisol synthesis (26) and appears to be po- tentially effective in the management of Cushing syn- drome, which is often associated with ACC. Our group demonstrated in preclinical models that AbiAc not only inhibited cortisol secretion but also exerted cytotoxic activity in the ACC cell line and in ACC primary cell cultures (22) owing to drug-induced increases in pro- gesterone levels (22, 27). When administered daily for 16 days in NCI-H295R cells xenografted in immuno- deficient mice, AbiAc inhibited tumor growth, thus confirming in vitro findings (22).
In the current study, we reproduced in zebrafish embryos the in vivo cytotoxic effect observed with AbiAc in the murine model and demonstrated that zebrafish embryos xenografted with ACC cells are a valuable in vivo preclinical model to screen drugs with potential efficacy in ACC.
Materials and Methods
Chemicals
AbiAc was purchased from Selleckchem (DBA Italia, Seg- rate, MI, Italy) and resuspended in 100% ethanol (stock so- lution: 25 mM). When required, AbiAc was directly added to the fish water at different concentrations (0.5, 1, and 2.5 p.M) according to experimental conditions. Control embryos were treated with solvent alone.
Reagent-grade methanol (MeOH) LC-MS CHROMASOLV® and formic acid (98%) were purchased from Sigma Italia (Milan, Italy). Ultra-pure water was prepared using a Milli-Q Purification System (Millipore Corporation, Billerica, MA). Abi standard and Abi deuterated standard solutions were purchased from Sell- eckchem; cortisol (1 mg/mL in MeOH), progesterone, and 44A standard were purchased from Sigma-Aldrich (Sigma Italia, Milan, Italy). Abi was resuspended in 100% dimethyl formamide; progesterone, Abi deuterated, and A4A were resuspended in 100% dimethyl sulfoxide. Drugs were subsequently diluted in MeOH.
Cell culture and labeling
The human cell lines NCI-H295R and SW13 were obtained from the American Type Culture Collection and cultured as suggested. The NCI-H295R cell line was established from se- creting human ACC and represents the most widely used ex- perimental cell model for studying ACC in vitro (28). The human SW13 cell line was established from small cell carci- noma in the adrenal cortex. These cells do not produce steroids, and their exact histopathologic characteristics are still under investigation (28). The human ACC primary cells, namely ACC29, were derived from a female patient who underwent surgery for ACC (29) and were established as previously de- scribed (22, 27). Cells were characterized as having adrenal
origin, measuring Steroidogenic Factor 1 gene expression (30, 31) by quantitative reverse transcription PCR and measuring cortisol production, as described in Fiorentini et al. (22). The local ethical committee approved the project, and written in- formed consent was obtained from the patient. ACC culture conditions were as indicated for NCI-H295R cells. Conditioned media from ACC29 were obtained as described in Fiorentini et al. (22). The doubling-time was calculated according to American Type Culture Collection indication with the fol- lowing formula: DT = T In2/In(Xe/Xb), where T is the in- cubation time in any units, Xb is the cell number at the beginning of the incubation time, and Xe is the cell number at the end of the incubation time.
Cell viability was evaluated by trypan blue exclusion test. Briefly, cell suspensions containing 0.25% trypan blue were dropped in a hemocytometer chamber, and the viable cells were counted under a phase contrast microscope by two different operators. Cells (3 × 106 cells) were treated overnight with the vital red fluorescent dye CellTracker™M CM-Dil (final con- centration 0.66 ng/ml; Thermo Fisher Scientific, Milan, Italy), then detached with trypsin/EDTA, washed in PBS, resuspended in 50 ML of PBS, and kept at 4℃ until use.
Measurement of cell viability
ACC29 cell viability was measured using the luminescence assay ATPlite™M (Perkin Elmer Italia, Milan, Italy), which measures the ATP production of viable cells. Cells were plated at the density of 5 x 103 cells per well in 96-well plates and treated with increasing concentrations of AbiAc (1 to 200 nM); the viability of untreated and AbiAc-treated cells was measured according to the manufacturer’s instructions. Experiments were conducted at least three times, with each point run in triplicate.
Fish maintenance and egg collection
All zebrafish embryos were handled according to national and international guidelines, following protocols approved by the local committee (OPBA protocol no. 211B5.24) and au- thorized by the Ministry of Health (authorization no. 393/ 2017-PR).
Healthy adult wild-type zebrafish (AB strain) were used for egg production. Fish were maintained under standard labora- tory conditions as described (32), at 28℃ on a constant 14-hour light/10-hour dark cycle. Fish were fed thrice daily with a combination of granular dry food and fresh artemia (Special Diet Services, SDS Diets; LBS Biotech, Horley, UK). Nine-month- old male and female zebrafish were put in the breeding tank overnight in a 1/2 ratio. Immediately after spawning, fertilized eggs were harvested, washed, and placed in 10-cm petri dishes in fish water. The developing embryos were incubated at 28℃ and maintained in 0.003% (w/v) 1-phenyl-2-thiourea (PTU; Sigma Italia) to prevent pigmentation. Preliminary experiments were performed using different numbers of embryos at different stages of development to obtain the best experimental conditions.
AbiAc absorption quantification by liquid chromatography-tandem mass spectrometry
Because of the rapid hydrolysis of AbiAc in abiraterone (Abi) (33), AbiAc absorption from embryos was evaluated by quan- tifying the concentration of Abi and its main metabolite 44A (34) by using liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Ultra-performance liquid chromatography (UPLC) was performed using a Dionex™M UltiMate™M 3000 (Thermo Fisher Scientific) equipped with an LPG-3400SD quaternary analytical pump, a WPS-3000SL analytical autosampler, and a TCC- 3000SD thermostatted column compartment. Chromato- graphic separation was performed using an XSELECT CSH C18 column (150 × 2.1-mm ID; particle size 3.5 um) (Waters, Milan, Italy). Mobile phase (A) was water containing 0.1% formic acid. Mobile phase (B) was methanol containing 0.1% formic acid. An isocratic mobile phase was used with 70% of (B) and a run time of 15 minutes. The UPLC flow rate was 0.3 mL/min. The column temperature was 40°C.
Collision-induced dissociation-tandem mass spectrometer (MSn) experiments were performed on an electrospray ioni- zation mass spectrometer (LCQ Fleet Ion Trap MSn; Thermo Fisher Scientific). The positive electrospray ionization (ESI) conditions were as follows. The source voltage was set at 4.5 kV, and the source current was set at 100 p.A. The capillary voltage was set at 7 kV, and the capillary temperature was 350℃. The spray was stabilized with a nitrogen sheath gas (35 arb), and the auxiliary gas was set at 15 arb. The isolation width of precursor ions was 1 mass unit. Ions were obtained in the range of m/z 300 to 400. For all selected reaction monitoring analyses, the scan time was equal to 100 ms, the collision energy was fixed at 50%, and the isolation width of precursor ions was 2.5 mass units. Data were analyzed with Xcalibur software (Version 4.0; Thermo Fisher Scientific).
The calibration curves for the quantification of both Abi and 44A were obtained as follows. Twenty-five embryos for each batch [up to 120 hours postfertilization (hpf)] were put at 4℃; 100 µL of internal standard (300 nM) was added to each batch with 100 µL of Abi or 44A (depending on the calibration curve) at different dilutions to obtain the final concentrations, 5 to 1000 nM of Abi and 12.5 to 500 nM of A4A, respectively. Samples were broken up with a pestle, homogenized using a pipette, vortexed for 1 minute, centrifuged at 15,000 rpm for 1 minute at 4℃, sonicated for 15 minutes, and centrifuged at 15,000 rpm for 10 minutes at 4℃. The supernatant was transferred into a tube after filtration through a 0.2-um poly- vinylidine fluoride (PVDF) filter, and 5 uL was injected into the LC-MS/MS system. The selected reaction monitoring quantifier transitions were 350-156 for Abi and 348-156 for A4A.
Linearity was determined by least-squares regression (data not shown)
Each batch of 25 embryos (treated as previously indicated) was kept at 4℃; 100 µL of Abi deuterated standard solution (300 nM) was added to each batch with 100 uL of MeOH. Samples were homogenized using a pipette, vortexed for 1 minute, centrifuged at 15,000 rpm for 1 minute at 4℃, sonicated for 15 minutes, and centrifuged at 15,000 rpm for 10 minutes at 4℃. The supernatant was transferred into a tube after filtration through a 0.2-um PVDF filter. Five uL of each sample was analyzed by UPLC interfaced with ESI MSn.
Cortisol and progesterone extraction and quantification
by LC-MS/MS. Cortisol and progesterone extraction was performed as indicated in Fiorentini et al. (22). For ACC29- conditioned media, samples were reconstituted in 20 uL of MeOH, and a volume of 10 uL was directly injected into the LC- MS/MS system. For AB zebrafish embryos, a batch each of 100
solvent-treated or AbiAc-treated embryos was prepared. Briefly, 70 µL of MeOH was added to each batch. Samples were ho- mogenized using a pipette, vortexed for 1 minute, centrifuged at 14,000 rpm for 1 minute at 4℃, sonicated for 15 minutes, and centrifuged at 15,000 rpm for 10 minutes at 4℃. The super- natant was transferred into a tube after filtration through a 0.2-um PVDF filter. Ten microliters of each sample was analyzed.
UPLC was performed using a Dionex™M UltiMate™M 3000 (Thermo Fisher Scientific) equipped with an LPG-3400SD quaternary analytical pump, a WPS-3000SL analytical auto- sampler, and a TCC-3000SD thermostatted column compart- ment. Chromatographic separation was performed with an XSELECT CSH C18 column (150 × 2.1-mm ID; particle size 3.5 um; Waters). Mobile phase (A) was water containing 0.1% formic acid. Mobile phase (B) was MeOH containing 0.1% formic acid. A gradient mobile phase was used. The gradient program was as follows: 58% B for 2 minutes; then from 58% to 100% B in 6 minutes; then 100% B for 2 minutes, from 100% to 58% B in 2 minutes and re-equilibration to 58% B for 8 minutes. All analyses were performed at 30℃.
Collision-induced dissociation-MSn experiments were per- formed on an ESI mass spectrometer (LCQ Fleet Ion Trap MSn; Thermo Fisher Scientific). Positive ESI conditions were as fol- lows: The source voltage was set at 3.8 kV, and the source current was set at 100 p.A. The capillary voltage was set at 11 V, and the capillary temperature was 300℃. The spray was sta- bilized with a nitrogen sheath gas (35 arb), and the auxiliary gas was set at 15 arb. The isolation width of precursor ions was 1 mass unit. Ions were obtained in the range of m/z 250 to 400. The calibration curves for the quantification of cortisol and progesterone were obtained by diluting both reference stan- dards as follows: 500 ng/ml, 250 ng/ml, 100 ng/ml, 50 ng/mL. Ten microliters of each standard dilution was analyzed by LC- MS/MS as previously described. Linearity was determined by least-squares regression (data not shown).
Tumor xenograft
AB zebrafish embryos at 48 hpf were dechorionated, anesthetized with 0.042 mg/mL tricaine (ethyl 3-aminobenzoate methane sulfo- nate salt; Sigma-Aldrich), and microinjected with the labeled tumor cells into the subperidermal space of the yolk sac (8, 18).
Microinjections were performed with the electronic micro- injector FemtoJet coupled with the InjectMan N12 manipulator (Eppendorf Italia, Milan, Italy). Approximately 240 cells in a volume of 4 nL were injected into each embryo, which was then maintained in fish water plus PTU in a 32℃ incubator to allow tumor cell survival and growth. A picture of each injected embryo was acquired under a Leica MZ16F fluorescence ste- reomicroscope 2 hours after treatment (T0). AbiAc or solvent was directly added to the fish water. After 3 days of treatment (T3), pictures were taken as described previously. A schema of the AbiAc protocol is shown in Fig. 1. The tumor areas of AbiAc-treated and untreated groups at TO and T3 were mea- sured with Noldus DanioScope™M software (Noldus Information Technology) and analyzed by GraphPad Prism software ver- sion 6.01.
In silico analysis
Human 3ß-hydroxysteroid dehydrogenase (3ß-Hsd) protein information collected in the UniProt database (35) was used to obtain human 3ß-HSD ensemble gene entry (36). Ensemble full- length protein sequence of the human 3B-Hsd protein was used to search the zebrafish assembly on BLAST. The sequences of the zebrafish and human enzymes were aligned by using Clustal Omega online software (37, 38).
RNA extraction and real-time PCR
Total RNA was extracted from a batch of 30 embryos at five different stages of development (24, 48, 72, 96, and 120 hpf) using the RNAeasy Kit (Qiagen Italia, Milan, Italy). RNA was quantified by mySPEC microvolume spectrophotometer (VWR International, Milan, Italy). One microgram of each sample was transcribed into cDNA using M-MLV reverse transcription (Promega Italia, Milan, Italy). The relative gene expression of 3B-HSD was evaluated by quantitative RT-PCR with the ViiA7 Real-Time PCR System (Applied Biosystems, Milan, Italy) using the iQTMSYBR Green Supermix method (Bio-Rad, Segrate, MI, Italy), according to the manufacturer’s instructions.
The zebrafish full-length 36-HSD transcript was used to design zebrafish-specific primers for PCR by using Pri- mer3web software version 4.1.0 (http://primer3.ut.ee/) (39).
Cells injection
+ solvent
T3
Area measurement
120 hpf embryos
TO 48 hpf embryos
2h
In fish water
+ 1 µM AbiAc
T3
Area measurement
Area measurement
120 hpf embryos
The oligonucleotide sequences for zebrafish-3ß-HSD were as follows: F: 5’-CTTTCAACGCAGCGCTCTAC-3’; R: 5’- TCTTCCAGCAACAGTCGGAC-3’, and the sequence for the zebrafish ß-actin (housekeeping gene) were as follows: F 5’-AATCCCAAAGCCAACAGAGA-3’; R 5’-TCACACCATC- ACCAGAGTCC-3’.
Reactions were conducted under the following conditions: 1 cycle at 95℃ for 10 minutes, 40 cycles at 95℃ for 15 seconds, and 62℃ for 1 minute. Differences in the threshold cycle Ct values between the ß-actin housekeeping gene and zebrafish- 3B-HSD were then calculated as an indicator of the amount of mRNA expressed. Analysis was performed in triplicate for each sample using different groups of embryos.
Statistical analyses
Statistical analyses were performed using GraphPad Prism software version 6.01. One-way ANOVA followed by the Dunnett test was performed to identify statistically significant differences among different groups of data, considering a P value <0.05 as the threshold for a significant difference.
Results
Cytotoxic and antisecretive effect of AbiAc in ACC29 primary cells
ACC29 primary cells were exposed to increasing concentrations of AbiAc (1 to 200 nM) for 4 days and analyzed for cell viability. AbiAc exerted a concentration-dependent reduction in cell viability that reached the plateau of 59.75% + 2.3% at 25-nM concentration and did not increase further. The calcu- lated IC50 was 6 nM (95% CI: 2.9 to 11.3 nM) (40). The conditioned media of ACC29 were then analyzed for cortisol and progesterone production. Results demon- strated that cortisol production of untreated ACC29 cells was reduced from 2.24 ± 0.29 ng/ml/106 cells to 1.55 + 0.29 ng/ml/106 cells in 100 nM AbiAc-treated cells (P < 0.05 vs untreated cells). As expected, progesterone was undetectable in untreated cells and increased to 1.3 ng/ml/106 cells when exposed to AbiAc.
ACC cell viability and doubling time
The viability and doubling time of ACC cells main- tained at 32℃ were investigated to evaluate whether these cells are a suitable model for zebrafish embryo xenograft. Cell viability was evaluated by the trypan blue exclusion test at both 37℃ and 32℃; the results dem- onstrated that at the lower temperature ACC cells were viable (not shown), although with a reduction in cell proliferation rate (Table 1).
Determination of AbiAc treatment concentration
To set up the method, AB strain zebrafish embryos were treated with increasing concentrations of AbiAc to determine the optimal drug concentration for use during in vivo experiments. Embryos maintained in fish water
| Cell Line/Primary Culture | DT at 37ºC | DT at 32ºC |
|---|---|---|
| NCI-H295R | 52 h | 90 h |
| SW13 | 21 h | 26 h |
| ACC29 | 50 h | 74 h |
The doubling time (DT) was calculated according to the American Type Culture Collection indication with the following formula: DT = T In2/ ln(Xe/Xb), where T is the incubation time in any unit, Xb is the cell number at the beginning of the incubation time, and Xe is the cell number at the end of the incubation time.
plus PTU were manually dechorionated at 48 hpf and divided into five groups (n = 90 ± 10 for each). Groups 1, 2, and 3 were treated with 0.5, 1, and 2.5 uM of AbiAc, respectively, added directly to the fish water. In the fourth group, the solvent alone (MeOH) was added to the fish water plus PTU (control group), whereas the last group was left untreated. Embryos were kept at 32℃. After 3 days of treatment, exposure of embryos to 2.5 MM of AbiAc resulted in death or deformity (41). Embryos of the other two treated groups as well as embryos of the solvent-treated and control groups de- veloped normally, and their phenotype was similar to that of untreated embryos (41). On the basis of these findings, the concentration of 1 p.M of AbiAc was chosen for subsequent experiments.
Determination of AbiAc measurement
To determine the optimal embryo number for measure- ment of absorbed drug concentration, 1 µM AbiAc- treated embryos at 24 hpf were divided into groups of 10, 15, 20, 25, and 30 embryos each and analyzed by LC- MS/MS. As indicated in “Materials and Methods,” AbiAc is rapidly hydrolyzed to Abi (30); indeed, AbiAc could not be detected in embryos, whereas Abi was measurable. The LC-MS/MS analysis revealed that in samples obtained from groups of 10 to 20 embryos, Abi was not detectable, whereas samples obtained from a group of 25 embryos gave measurable and reproducible results, as shown in Fig. 2a, where a time curve of the absorbed Abi concentration was performed. AB zebra- fish embryos maintained in fish water plus PTU were divided into five groups (n = 120 ± 10); each group was treated with 1 µM of AbiAc added directly to the fish water, and embryos were collected at different time points, up to 24 hours, for the quantification of Abi. In particular, experiments were conducted in both 24-hpf embryos and 48-hpf embryos.
The LC-MS/MS results reported in Fig. 2 show that Abi absorption increased with the stage of embryo development. Of note, in 48-hpf embryos, the Abi
b)
500
a)
48hpf embryos
400
24hpf embryos
[Abi] nM
300
200
100
0
1.5
3
6
24
1.5
3
6
24
TIME (hrs)
concentration after 24 hours of incubation was ~179 ± 29.5 nM, which is very close to the highest Abi con- centration used in in vitro experiments with NCI-H295R cells, which was 200 nM (22). The time courses at both development stages displayed typical kinetics of the time- dependent concentration curve observed in humans (42), suggesting the capability of embryos to metabolize Abi. This hypothesis was supported by gene expression results demonstrating that zebrafish expressed mRNA encoding the enzyme 3B-HSD; indeed, by quantitative RT- PCR, we demonstrated that the 4Ct was 4.8 + 1.2 in 24-hpf embryos and 5.52 ± 0.75 in 48-hpf embryos. The 3B- HSD gene was transcribed in its protein, as demonstrated by the capability of 48-hpf embryos to metabolize Abi in its main metabolite 44A (Fig. 3a and 3b). Of note, by in silico analysis, we observed a high level of similarity (63%) and identity (46%) with the human counterpart (38). Owing to these results, the cell xenograft was performed in 48-hpf embryos because of the Abi con- centration time-course, to technical limits in injecting 24- hpf embryos (in particular, at this stage of development microinjection is not recommended due to tissue fragility) and according to the protocol of Nicoli et al. (8). Finally, the Abi metabolism time course was conducted at dif- ferent hpfs of embryos, up to 120 (Table 2), and results indicated that the capability of AB zebrafish embryos to metabolize Abi increased with increasing hpf.
AbiAc at a 1-uM concentration also modified the cortisol level of the AB zebrafish embryo. Results are reported in Table 3. After 3 days of exposure, the irre- versible binding of AbiAc to CYP17A1 induced a sig- nificant reduction in cortisol production in AbiAc-treated
Abiraterone
RT: 0.00 - 14.99 SM: 7G
RT: 5.69
100
AA: 505885
90
NL: 2.59E4
80
m/z= 349.40-354.50 F:
ITMS + c ESI Full ms
Relative Abundance
70
[300.00-400.001 MS
60
ICIS Embryos 48 hpf
50
40
30
RT: 0.94
Δ4Α
AA: 89002
20
RT: 4.08
a)
10
AA: 31291
0
0
1
2
3
4
5
6
7
8
Time (min)
9
EMBRYOS 48 HPF
ABI
44A
500
CONCENTRATION (nM)
400
300
I
200
100
0
0
5
10
15
20
25
30
TIME (h)
b)
embryos. Accordingly, as expected, progesterone became measurable in treated embryos, whereas it did not reach detection in solvent-treated embryos.
ACC cell xenograft in AB zebrafish embryos
NCI-H295R cell line. AB zebrafish embryos at 48 hpf were manually dechorionated, anesthetized with tricaine, and microinjected with NCI-H295R-labeled cells into the subepidermal space of the yolk sac (n = 80 ± 10). A picture of each injected embryo was acquired under a fluorescence stereomicroscope at T0, and then embryos were divided into two groups (n = 40 ± 5): one maintained in fish water/PTU plus solvent (solvent- treated), and the other maintained in fish water/PTU plus 1 µM of AbiAc without medium change during 3 days of treatment. Embryos were then incubated at 32℃. At T3, pictures were taken as described, and the injected cell area in solvent-treated and 1 p.M AbiAc- treated embryos was measured and analyzed as de- scribed in “Materials and Methods.” The injected cell area at T3 in solvent-treated embryos was 38.390 ± 1.432 µM2 compared with 24.891 + 1.302 u.M2 in
| Embryos hpf | ABI, nM | ||||
|---|---|---|---|---|---|
| 24 | 48 | 72 | 96 | 120 | |
| T1 = 1.5 h | 242.68 ± 12.12 | 316.99 ± 21.82 | 328.62 ± 12.30 | 341.37 ± 13.10 | 308.65 ± 33.33 |
| T2 = 3 h | 285.74 ± 43.14 | 415.92 ± 35.19 | 394.10 ± 15.53 | 363.49 ± 19.67 | 303.35 ± 14.71 |
| T3 = 6 h | 272.69 ± 17.30 | 334.35 ± 24.67 | 378.25 ± 15.72 | 274.04 ± 17.14 | 363.21 ± 16.07 |
| T4 = 24 h | 111.33 ± 21.44 | 179.48 ± 29.54 | 12.12 ± 2.66 | 4.18 ± 0.60 | 8.19 ± 4.36 |
Time course of Abi quantification was conducted on batches of 25 embryos each by LC-MS/MS, as described in “Materials and Methods,” starting from 24-hpf embryos up to 120-hpf embryos.
AbiAc-treated embryos (P < 0.01) (Fig. 4a). The tumor area in solvent-treated embryos displayed a 1.63 ± 0.07-fold increase at T3 compared with TO, whereas in AbiAc-treated embryos the tumor area was almost unchanged, with a 1.01 ± 0.03-fold increase at T3 compared with T0. A representative image is shown in Fig. 4b. Experiments were repeated, changing zebrafish embryo medium every day after xenograft, thus adding fresh AbiAc every day. Results demonstrated that the injected cell area measured was superimposable on that reported previously (not shown).
ACC29 primary cell culture. The cytotoxic effect of 1 p.M of AbiAc was also evaluated in a primary culture obtained from a patient diagnosed with cortisol-secreting ACC. Results are shown in Fig. 5 and demonstrated that al- though ACC29 cells seemed to be less sensitive to AbiAc both in vitro (see previous text) and in vivo at T3, the injected ACC29 cell area was significantly reduced in 1 µM AbiAc-treated embryos compared with solvent- treated embryos. The area was indeed 56.987 + 2.83 p.M2 in solvent-treated embryos vs 39.776 + 4.516 u.M2 in AbiAc-treated embryos (P < 0.05).
SW13 cell line. The nonsteroidogenic AbiAc-insensitive (22, 28) cell line SW13 was used as the internal negative control. The tumor xenograft area of SW13 cells was not modified by exposure to 1 µM of AbiAc (Fig. 6); indeed, the area of solvent-treated tumor was 52.842 +
| Solvent Treated | AbiAc Treated | |
|---|---|---|
| AB Zebrafish Embryos (n = 100) | AB Zebrafish Embryos (n = 100) | |
| Cortisol | 9.35 ± 0.1 ng | 4.25 ± 0.06 ngª |
| Progesterone | Undetectable | 3.53 ± 0.17 ngª |
Hormone determinations were performed as described in “Materials and Methods” and analyzed by LC-MS/MS as described.
ªP < 0.001 vs solvent-treated AB zebrafish embryos.
5.163 µM2, whereas the area of AbiAc-treated tumor was 49.665 + 3.705 p.M2.
Discussion
Because of its distinctive characteristics, the zebrafish model is being progressively applied in the design of in vivo experimental studies in a number of human diseases (43), including malignancies [reviewed in (44)]. In par- ticular, zebrafish embryos provide a powerful tool to develop functional cancer models for drug discovery and development and drug toxicity. In a rare and severe disease such as ACC, the finding of reproducible and reliable in vivo preclinical models is an open challenge, especially in light of the number of variables that must be taken into account when studying ACC xenografts in the mouse model (45). The timing of drug administration, the drug metabolism, and the solvent in which the drug is dissolved (that per se may affect animal safety and the reproducibility of results) are among the most relevant issues (43). The few available cell lines and their genomic
Tumor Size (um2) &
TO E
40000-
*
30000
T3-AbiAc
20000
10000
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0
TO
T3 -AbiAc
T3 +AbiAc
Tumor Size ( um2)
60000
*
50000
40000
30000
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0
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T3 -AbiAc
T3 +AbiAc
instability, the immune competence of the host, and site of implantation are additional important drawbacks (45).
Here, we validated a NCI-H295R cell xenograft in AB zebrafish embryos as an in vivo model of ACC. We confirmed the in vivo cytotoxicity of AbiAc using an animal model that offers several advantages over other models, such as mice (17-21). It should be underlined that AbiAc was directly added into the fish water and was significantly adsorbed by embryos, reaching concentrations capable of exerting a cytotoxic effect, thus simplifying the treatment procedure. Furthermore, the ACC xenograft in zebrafish was fast, as 3 days of AbiAc treatment was sufficient to demonstrate a substantial difference in the NCI-H295R cell proliferation rate. The AbiAc effect was due to direct binding on its target en- zyme, as the tumor area of xenografts with non- steroidogenic (28), CYP17A1-negative (22) SW13 cells was not modified after 3 days of exposure to 1 µM of AbiAc; this confirmed the insensitivity of these cells to the cytotoxic effect of AbiAc treatment, as observed in in vitro experiments (22).
Tumor Size ( um2)
60000
50000
40000
30000
20000
10000
0
TO
T3 -AbiAc
T3 +AbiAc
Of note, the effect of AbiAc lasted up to 3 days without the addition of fresh drug, probably because of the irreversible binding of Abi to CYP17A1. The in- hibition of NCI-H295R cell area growth in AB zebrafish embryos was about 60% after 3 days, which is even higher than what we observed in immune incompetent mice, where it reached 34% inhibition ~60 days after cell injection and 15 days after the end of 16 days of treatment (22). Furthermore, another finding that supports the use of zebrafish embryos as a useful model for in vivo animal studies on ACC is the expression of enzymes of the steroidogenic pathways in embryos, similar to what was observed in more evolved animal models. Indeed, zebrafish embryos express the 3ß-HSD enzyme that converts Abi to its active metabolite 44A, which inhibits additional enzymes involved in ste- roidogenesis, including CYP17A1, 36-HSD, and the 5a reductase SRD5A (34, 46). The combined effect of Abi and its main metabolite on CYP17A1 induced a de- crease in cortisol level and, as expected, an increase in progesterone level.
In this era of personalized medicine, the small number of cells needed and the shortened duration of experiments make the zebrafish model a potential candidate for preparation of human ACC patient-derived xenografts for real-time selection of the most appropriate cytotoxic drugs for each patient. On these bases, we reproduced results obtained with the NCI-H295R cell line in a patient-derived xenograft, obtained from primary cells from a cortisol-secreting ACC, thus supporting the possible development of an in vivo method to screen available therapeutic options for cancers such as ACC, which have a poor prognosis and few therapeutic options.
We believe that the validation of this animal model offers a useful tool for preclinical first screening of a large number of drugs. This is advantageous in a rare and ag- gressive disease such as ACC, in which treatment strategies are limited. We are aware that this animal model cannot completely replace the others already in use; however, we believe that our findings offer a suitable and expeditious model for the initial selection of potentially effective drugs, identification of dose toxicity, and determination of the most promising compounds for more advanced preclinical phases.
Acknowledgments
We thank the FIRM Foundation (Cremona, Italy) for its sup- port and generosity
Financial Support: This work was supported by Asso- ciazione Italiana per la Ricerca sul Cancro Project IG17678 (to M.T.) and by local grants from the University of Brescia.
Additional Information
Correspondence: Sandra Sigala, MD, PHD, Section of Pharmacology, Department of Molecular and Translational Sciences, Viale Europa 11, 25123 Brescia, Italy. E-mail: sandra.sigala@unibs.it.
Disclosure Summary: The authors have nothing to disclose.
Data Availability: All data generated or analyzed during this study are included in this published article or in the data repositories listed in References.
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