REVIEW
Cancer cell xenografts in zebrafish embryos as an experimental tool in drug screening for adrenocortical carcinoma
Mariangela TAMBURELLO *, Andrea ABATE, Sandra SIGALA
Section of Pharmacology, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy
*Corresponding author: Mariangela Tamburello, Section of Pharmacology, Department of Molecular and Translational Medicine, University of Brescia, 25123 Brescia, Italy. E-mail: mariangela.tamburello@unibs.it
ABSTRACT
Despite the widespread use of murine models in in-vivo experiments, the zebrafish (Danio rerio) offers unique advan- tages that make it a versatile and faster preclinical model for drug screening, particularly for adrenocortical carcinoma (ACC), a rare malignancy with limited preclinical models that reflect patient heterogeneities. Over the past decade, significant progress has been made with models like cell lines, organoids, and murine models, which are crucial for advancing disease understanding and treatment development. However, recent reviews have overlooked zebrafish model for ACC. This mini review aims to fill this gap by detailing the advancements of the zebrafish model in ACC research. Re- cent studies have utilized zebrafish embryos xenografted with ACC cells as a novel approach to studying drug effects on tumor growth and metastasis, consistent with studies regarding other tumors. Specifically, it was demonstrated the ability of abiraterone acetate, trabectedin and progesterone to significantly reduce the tumor area at non-toxic-concentrations. Interestingly, this model allowed to confirm in vivo that metastasis-derived cells were able to metastasize and that trabect- edin and progesterone reduced the rate of embryos with metastasis. One more study showed that metastasis formation was significantly reduced in H295R/TR-SF-1-xenografted embryos after fascin1 knock-out or inhibition with G2-044. Even with some limitations, the zebrafish xenografts offer a suitable and expeditious animal model for the screening of potentially effective drugs, identification of dose toxicity, and determination of the most promising compounds for more advanced preclinical phases, especially in rare diseases with limited therapeutic options such as ACC.
(Cite this article as: Tamburello M, Abate A, Sigala S. Cancer cell xenografts in zebrafish embryos as an experimental tool in drug screening for adrenocortical carcinoma. Minerva Endocrinol 2025;50:182-93. DOI: 10.23736/S2724-6507.24.04270-2) KEY WORDS: Adrenocortical carcinoma; Heterografts; Neoplasm metastasis.
A drenocortical carcinoma (ACC) is an ex- tremely rare and aggressive malignancy. Surgery is the mainstay of therapy in case of local disease, however, most radically operated ACC patients are destined to undergo disease recur- rence. For patients with locally advanced/meta- static ACC, not amenable to surgery, mitotane in association with chemotherapy (etoposide, doxorubicin, and cisplatin) (EDP-M) is the elec- tive first-line treatment.1 However, this scheme’s
overall efficacy is limited and no standard sec- ond-line therapies are available. There is an ur- gent need to improve the efficacy and tolerability of standard therapy and find new treatment strat- egies. Management of ACC is dependent on dis- ease stage with complete surgical resection as the only potentially curative option.2 The scarcity of preclinical models that reflect patient heteroge- neities - considering various genotypes, secre- tion profiles, and drug-resistant phenotypes - has
hampered the progress and development of new therapies. Fortunately, the last decade has seen tremendous advancements in this field with the development of several preclinical models, such as cell lines, organoids, and murine models. They are essential for improving our basic knowledge of disease and developing treatments. While these models are well described in the more re- cent reviews,3-6 they have overlooked a newly developed preclinical model for ACC. Indeed, recent studies from our group have reported re- sults obtained from zebrafish embryos and larvae xenografted with ACC cells, which are used to investigate the effects of different drugs on tu- mor growth and metastasis formation. This mini review aims to address this gap by describing the knowledge and advancements of the zebrafish model in the ACC field. Additionally, the use of zebrafish model in ACC and its methods will be discussed and compared with existing technolo- gies used for other cancers to highlight both its benefits and limitations (Supplementary Digital Material 1: Supplementary Figure 1).
The zebrafish xenograft models in cancer therapeutics investigations
Cancer drug discovery involves in-vitro screen- ing with human cell models and in-vivo testing with animal models. Genetically engineered mouse models and patient-derived xenografts (PDX) in immunocompromised mice are the gold standards for evaluating drug safety and efficacy, as they maintain tumor biology and predict drug efficacy through direct comparisons with patient responses.7-9 PDX is especially valuable for pre- serving the original tumor characteristics. Recent advancements in humanized PDX models incor- porating human immune systems have enhanced the ability to mimic tumor-immune system in- teractions, making them ideal for studying im- munotherapy. These models retain the tumor’s genetic landscape and clonal heterogeneity, en- abling accurate evaluation of therapeutic effects and drug resistance, closely reflecting the clini- cal behavior of cancer.10
However, they are limited in studying early tumor dissemination and microenvironment changes at the cellular level and are unsuitable
for large-scale molecule screening.11 In recent years, the zebrafish (Danio rerio) tumor cell xe- notransplantation model has gained increasing attention as a complementary system to mouse xenografts and popular cost-effective alternative with high-throughput capabilities.12-14 One im- portant advantage of zebrafish is that the adults are small and prefer to be housed in large groups, or “shoals.” As a result, they require much less space and are cheaper to maintain than mice. An- other advantage of adult zebrafish is their ability to breed readily, producing approximately 50 to 300 eggs every 10 days. This contrasts with mice, which typically produce litters of one to ten pups and can only bear about three litters in their life- time. Scientific experiments often require mul- tiple repetitions to ensure accuracy, making an animal model that can produce a large number of offspring repeatedly highly beneficial. Zebraf- ish also meet the criteria for high-throughput and low-cost research. Their embryos can be kept in petri dishes or individually in 96-well plates, al- lowing for easy handling and maintenance dur- ing various experimental phases, thereby accel- erating the preclinical development process.15 An intriguing aspect is that the zebrafish genome project revealed a high degree of interspecies conservation, with zebrafish orthologs identi- fied for 70% of total genes and 82% of genes associated with human disease. Crucially for oncology research, the major players in human cancer-related pathways have homologs in the zebrafish genome.16 Furthermore, although ze- brafish are not mammals, within approximately two days post-fertilization, they develop a com- plement of orthotopic organs and tissues with functions similar to those of mammals. These include the musculoskeletal and cardiovascular systems, eyes, brain, liver, heart, gastrointesti- nal tract, pancreas, thyroid, interrenal, and chro- maffin cells (corresponding to mammal adrenal gland).17-19 In cancer research, a key advantage of zebrafish is the absence of a fully developed adaptive immune system in early life, which only matures 30 days post-fertilization. This reduces the rejection of patient-derived cancer cells and eliminates the need to establish immunodefi- cient animals typically used in murine cancer models.20 Additionally, zebrafish transplantation
requires only hundreds of cells or fewer, while mouse xenografts require several times as many. This is particularly important when cancer cell numbers are finite, such as those derived from primary patient tissue samples. Moreover, the optical clarity of embryo-larva zebrafish allows for precise and non-invasive observation of tu- mor shape and size, drug effect evaluation, and detailed imaging of single-cell movement and interactions. In contrast, working with mice is much more complicated. Mouse embryos are not clear and develop inside the mother, so the ob- servation of live embryo development like that in zebrafish is not possible, and accessing or ma- nipulating them requires sacrificing the mother.21 Not least, zebrafish can survive at temperatures from 32 ℃ to 36 ℃, closer to human cell culture conditions, despite they prefer an environmental temperature of 28 ℃.22, 23
Another advantage is that are available well- established zebrafish transgenic lines with fluo- rescently labeled tissues can add new insights into cancer cell growth, dissemination, and tu- mor microenvironment in real time.13, 24 Thus, zebrafish have joined the mouse as a new model for xenograft assays.14
However, there are also limitations and chal- lenges of using zebrafish as a model in cancer research, including high mortality after injection, disparity in protocols among laboratories and different body temperatures between fish and
humans. Vantages and disadvantages are sum- marized in Table I. Despite these shortcomings, zebrafish are still being applied as a promising model in tumor research. 12
Focus on ACC study involving xenografted zebrafish embryos
The first evidence that the zebrafish embryo model is a useful tool for evaluating the in-vivo cytotoxicity of drugs with potential efficacy in ACC was published by our group.25 We vali- dated the results previously obtained in immu- nodeficient mice xenografted with cortisol-se- creting NCI-H295R cells (a widely recognized experimental cell model of ACC) and treated with abiraterone acetate in zebrafish embryos. Abiraterone acetate is an irreversible inhibitor of 17a-hydroxylase/17,20-lyase (CYP17A1), a crucial enzyme for steroid hormone synthesis that impairs the production of androgens and cortisol. This drug appears to be potentially ef- fective in managing Cushing syndrome, which is often associated with ACC. In a prior study, we demonstrated that abiraterone acetate not only inhibited cortisol secretion but also exerted cytotoxic activity in the ACC cell line and ACC primary cell cultures,26 due to the drug-induced increase in progesterone levels.26, 27 When ad- ministered daily for 16 days in immunodeficient mice xenografted with NCI-H295R cells xeno-
| TABLE I .- Vantages and disadvantages of zebrafish embryos as a research animal model in cancer. | |
|---|---|
| Vantages | Disadvantages |
| Small in size and easy to house in greater numbers with low cost. | The rapid and continuous development of early embryonic stages restricts cancer studies. |
| High fecundity. One female can produce around 200 to 300 eggs per week. | Anatomical differences from mammals, such as the absence of mammary and prostate glands, joints, limbs, and lungs, make certain tumor models impossible to develop. |
| Extra-utero and rapid development facilitating manipulation at different stages and yielding results in just a few days. | Immune suppression required to grow xenografts in adult stage |
| Optical transparency of zebrafish embryos and larvae allowing for non-invasive observation of engrafted tumor cells. | The degree of interaction between zebrafish and human cells is not well established. |
| Use of transgenic lines facilitates studies on interaction with human cells and specific host factors | Transplanted cells are exposed to a different host niche and different environmental factors than human |
| No immune rejection since they develop a functional adaptive immune system only after 4-6 weeks post-fertilization. | Zebrafish often have multiple copies of genes (paralogs) - meaning some genes may not be functional |
| Zebrafish are permeable and easily absorb drugs, which can be directly dissolved in their water. | Zebrafish have a preferred temperature of around 28℃, which can limit their use in studies where mammalian temperatures are important. |
| Fewer legal restriction on research. | Reproducibility issues may arise due to disparities in protocols among laboratories. |
grafted in immunodeficient mice, abiraterone ac- etate inhibited tumor growth and interfered with steroid production, thus confirming the in-vitro findings.26 The ACC xenograft in zebrafish vali- dated the results obtained in mice, as just 3 days of abiraterone acetate treatment was sufficient to demonstrate a substantial reduction in the NCI- H295R cell proliferation rate. The inhibition of NCI-H295R cell area growth in AB zebrafish embryos was about 60% after 3 days, which is even higher than the 34% inhibition observed in immunodeficient mice approximately 60 days after cell injection and 15 days after the end of the 16-day treatment. The effect of abiraterone acetate was due to direct binding to its target en- zyme, as the tumor area of xenografts with non steroidogenic, CYP17A1-negative SW13 cells was not affected after 3 days of abiraterone ace- tate exposure. This confirmed the insensitivity of these cells to the cytotoxic effect of abiraterone acetate treatment, as observed in in-vitro experi- ments.26 Furthermore, another finding that sup- ports the use of zebrafish embryos as a useful model for in-vivo animal studies on ACC is the expression of enzymes of the steroidogenic path- ways in embryos, similar to what was observed in more evolved animal models. Indeed, zebraf- ish embryos express the 3ß-HSD enzyme that converts abiraterone acetate to its active metabo- lite 44A, which inhibits additional enzymes in- volved in steroidogenesis, including CYP17A1, 3B-HSD, and the 5a reductase SRD5A.28, 29 As previously mentioned, zebrafish embryos have the adrenal/interrenal gland starting at 24 hpf.30 The combined effect of abiraterone acetate and its main metabolite on CYP17A1 induced a significant reduction in cortisol production in treated embryos. Consistent with in-vitro results, progesterone became measurable in treated em- bryos, whereas it did not reach detection limit in solvent-treated embryos.25
The zebrafish model was subsequently used routinely to confirm the drug effects observed in vitro. Specifically, the cytotoxic and antip- roliferative effect of trabectedin31 and proges- terone32 were observed in vivo in zebrafish em- bryos xenografted with NCI-H295R, TVBF-7, and MUC-1 ACC cells. Besides being a useful tool for initial in-vivo drug screening, this animal
model demonstrated that trabectedin and proges- terone inhibit metastasis formation in zebrafish embryos xenografted with metastatic cell lines such as MUC-1 and TVBF-7.31, 32
Indeed, according to their metastatic origin, MUC-1 and TVBF-7 cells were capable of me- tastasis, with differences in rate and localization. Both trabectedin and progesterone reduced the rate of embryos with MUC-1 cells migrating to the caudal region. TVBF-7 cells formed metas- tases at a lower rate compared to MUC-1 cells, with metastases mostly localized in the pericar- dial zone. No embryos with metastases were found in the treated groups.31, 32
Further investigations using in-vitro ap- proaches, such as the transwell assay and wound healing assay, validated the in-vivo results. MUC-1 cells displayed high invasive capability, which was significantly reduced by trabectedin and progesterone. A similar antimetastatic effect of progesterone was observed in TVBF-7 cells, confirming their low invasion ability both in vi- tro and in vivo.31, 32
Finally, the zebrafish model was used to in- vestigate the role of the actin-bundling protein fascin (FSCN1) that has been shown to enhance the invasion properties of ACC cancer cells.33, 34 Based on those results, it was investigated the effects of FSCN1 inactivation by CRISPR/Cas9 or pharmacological blockade by G2-044 on the invasive properties of H295R/TR SF-1 GFP luc (a H295R subclone with Dox-inducible over- expression of the transcription factor SF-135). Indeed, FSCN1 is a transcriptional target for ß-catenin in H295R ACC cells and its inactiva- tion resulted in defects in cell attachment and proliferation. FSCN1 knock-out modulated the expression of genes involved in cytoskeleton dynamics and cell adhesion. When SF-1 dosage was upregulated in H295R cells, activating their invasive capacities, FSCN1 knock-out reduced the number of filopodia, lamellipodia/ruffles and focal adhesions, while decreasing cell invasion in Matrigel. Similar effects were produced by the FSCN1 inhibitor G2-044, which also diminished the cell invasiveness. In the zebrafish model, me- tastases formation was significantly reduced in FSCN1 knock-out cells and G2-044 significantly reduced the number of metastases formed by
ACC cells contributing to indicate that FSCN1 is a new druggable target for ACC.35
Method: strengths and limitations
Zebrafish maintenance
Zebrafish are maintained and used according to EU Directive 2010/63/EU for animal use follow- ing protocols approved by the local committee (OPBA). Healthy adult zebrafish are used for egg production. Fish are maintained under standard laboratory conditions at 28 ℃ on a constant 14- hour light/10-hour dark cycle. Fish are fed thrice daily with a combination of granular dry food and fresh artemia. Breeding of adult male and female zebrafish is carried out through natural crosses, and embryos were collected and raised in fish water with incubation at 28.5 ℃ until the experiments. Embryos at 24 hpf are treated with 0.003% 1-phenyl-2-thiourea (PTU) to prevent pigmentation. After the conclusion of the experi- ments, that is 5 days post fertilization, the zebraf- ish larvae are euthanized with 400 mg/L tricaine as they start to feed and are no longer are not considered embryos or larval form.32
Cell labeling
Fluorescent proteins are a valuable tool for long- term in-vivo cell-tracking experiments, allow- ing researchers to monitor cell behavior under physiological conditions. Stable expression of fluorescent proteins can be achieved through ma- nipulation, such as transfection or transduction, commonly used with established cell lines.36 However, this approach is often impractical for primary cells like hematopoietic stem cells or human cancer cells, as it requires labor-intensive in-vitro engineering. To address this, gene-trans- fer-free cell-labeling methods have been devel- oped for rapid, efficient, and uniform ex-vivo labeling of cells, particularly for those that are difficult to culture. These methods also support high-throughput screening of transplanted cells for in-vivo imaging.37 An alternative approach is labeling cancer cells with lipophilic dyes, which offer ease of use and a wide range of colors, but come with challenges compared to endogenous- ly expressed fluorescent proteins.38 The chemi-
cal compounds used can be subdivided based on the cell compartment they label: cytoplasm, nucleus and cell membrane, each class with its own specific advantages and disadvantages. Cytoplasmic and nuclear dyes offer potential for in-vivo single-cell tracking due to their abil- ity to provide uniform staining. However, their high cytotoxicity limits their use in-vivo micros- copy (IVM). Dyes like CFSE and CFDA-SE are widely used in flow cytometry to track cell divi- sion but are less suitable for IVM due to pho- tobleaching and reduced fluorescence in deeper tissues.39 Nuclear dyes, although ideal for track- ing densely clustered cells, often interfere with key cellular processes such as DNA replication and transcription. For example, Hoechst 33342, while effective for tracking lymphocyte migra- tion and apoptosis, can inhibit cell proliferation at high concentrations.40
Lipophilic carbocyanine dyes, like PKH and Di dyes, are preferred for cell tracking due to their lower cytotoxicity. PKH dyes, such as PKH26, are commonly used to label plasma membranes for lymphocyte and hematopoietic stem and pro- genitor cell (HSPC) tracking. However, issues with uneven labeling and dye transfer between daughter cells can complicate tracking. 37, 41 DiO, Dil, DiD, and DiR dyes, which cover a broad spectral range, have gained popularity for track- ing multiple cell populations in vivo, offering strong, photostable staining with minimal cyto- toxicity. CM-Dil, a Dil variant resistant to fixa- tion, has proven useful for labeling tumor cells in zebrafish xenograft models.42
In the published papers, ACC cells (3×106 cells) are treated overnight with red fluorescent dye CellTrackerTM CM-Dil (final concentration 0.66 ng/ml; Thermo Fisher Scientific, Milan, Italy), then detached with trypsin/EDTA, washed in PBS, resuspended in 50 µL of PBS, and kept at 4 ℃ until use.25, 31, 32, 35
Dil is a commonly used lipophilic dye in can- cer research and animal xenograft models. 13, 43, 44 It integrates into cellular membranes due to its similarity to membrane lipids45 and it is non- toxic, it does not alter cell proliferation, and it retains fluorescence for extended periods.46-48 However, Dil has limitations, including trans- fer to unlabeled cells and retention in the mem-
branes of dead cells, which can lead to overes- timating the number of viable labeled cells,49 leading different authors to affirm that the tumor growth assessment based solely on Dil labeling could be inaccurate and needs to be corroborated with other methods.38, 49
However, these critical challenges can be ad- dressed. Before drawing definitive conclusions about the suitability of lipophilic dyes as cell tracers, it is crucial to optimize the labeling pro- tocol to minimize cell death. This involves care- fully balancing staining duration, washing steps, and timing of injection. Additionally, cross-val- idation experiments should be conducted to as- sess the impact of dye transfer, such as through double labeling of the injected cells. Finally, the viability of the labeled cells must be function- ally confirmed by testing long-term engraftment, performing live/dead cell assays, or ideally, har- vesting and functionally testing the labeled cells after imaging.37
Injection
To evaluate the effect of the drugs on tumor growth and metastasis formation, zebrafish em- bryos at 48 hpf were dechorionated, anesthetized with 0.042 mg/mL tricaine, and microinjected with the labeled ACC tumor into the subperider- mal space of the yolk sac. Approximately 250 cells/4 nL were injected into each embryo using an electronic microinjector.25, 31, 32, 35
While yolk sac is a popular injection site for xenotransplantation of tumor cells into zebraf- ish due to its accessibility, some studies have observed better tumor formation and growth at the perivitelline site or when injected orthotopi- cally into the brain. This suggests that certain zebrafish-derived growth-promoting factors may be specific to particular microenvironments.49
The yolk sac provides a nutrient-rich environ- ment, abundant in cholesterol, lipids, and phos- phatidylcholine, that supports the proliferation and migration of human cancer cells.50 Notably, it appears to perfectly mimic the adrenal cortex environment, making it the most suitable injec- tion site for ACC cells. In general, many differ- ent human cancer cell lines, including breast,51 neuroblastoma,52 melanoma,42, 53 leukemia,54-56 prostate,57 testis58, 59 and ovarian cancers, 60
among numerous others60 can survive and prolif- erate in the zebrafish yolk.
The optimal timepoint for yolk sac injections is 48 hpf, a method first established by Haldi et al.42 At 48 hpf, the yolk sac’s large size fa- cilitates the transplantation process, providing an easy-to-identify and accessible injection site for microinjectors and cancer cells are less likely to diffuse passively throughout the developing or- ganism since the gastrulation is completed.
With transplantation into the yolk, many dif- ferent cancer cell characteristics can be assessed with the most predominant endpoints being growth, survival, invasion, and metastasis. Can- cer cell proliferation can be prolific as malignant cells can quadruple in three days while inside the zebrafish yolk.42 Aggressive cells can migrate from the yolk sac into the bloodstream, mimick- ing metastasis.61 Zebrafish xenograft experiments have successfully replicated results from mouse models, with cancer cell lines that metastasize in mice showing similar behavior in zebrafish, while non-metastasizing lines did not.62 Microin- jection is typically performed in research labs us- ing manual systems involving a stereo dissecting microscope, an air-pressure-based microinjector, and a micromanipulator. However, this method presents significant challenges due to the small and delicate nature of zebrafish larvae, the need for tiny needle tips to inject cancer cells, and the difficulty of precisely targeting specific sites without causing damage. As a result, the process requires extensive training, it is time-intensive, and it has low throughput. Even after training, variability in results persists, due to differences in skill levels, protocol interpretation, instrument handling, and environmental factors.63 Efforts have been made to improve larvae preparation64 and develop robotic microinjection systems.65-67 However, these systems have not been validated for xenograft assays or widely tested by research- ers. While aqueous liquids are relatively easy to inject due to their low viscosity and uniformity, injecting living cells presents significant chal- lenges. The larger size of cells requires needles with larger diameters, which can cause more damage and reduce the survival rate of larvae. Additionally, cells tend to sediment, clump to- gether, and are more sensitive to temperature and
time, leading to increased variability in viscos- ity, fluidity, and injection volume under constant pressure.63
Incubation at 32 ℃
Zebrafish is a poikilothermic fish with a preferred temperature around 28 ℃. This might be adverse in studies where the mammalian homeostatic temperature would be important. However, in short time periods, zebrafish can tolerate temper- atures ranging from 6 ℃ to 38 ℃.68 The viabil- ity and doubling time of ACC cells maintained at 32°C were investigated to evaluate whether these cells are a suitable model for zebrafish embryo xenograft. Cell viability was evaluated by the try- pan blue exclusion test at both 37 ℃ and 32 ℃; the results demonstrated that at the lower tem- perature ACC cells were viable, although with an increase in their doubling time.25 Other authors showed that to support human cell engraftment, embryos can be raised at 34 ℃,43 which, while below the normal physiological temperature for human cells, is the upper limit tolerated by ze- brafish embryos and larvae.69 Only adult fish can tolerate living at 37 ℃ for months if the tempera- ture is slowly raised from 28.5 to 37 ℃ by about 0.5-1 ℃ per day.70
Drug toxicity evaluation
Systemic toxicity in zebrafish can be quickly as- sessed by adding treatments directly to the fish media, with observable effects including reduced survival and phenotypic abnormalities, and ad- ditional developmental and behavioral endpoints can be monitored non-invasively.71
Thus, preliminary experiments evaluated drug toxicity on wild-type zebrafish embryos to deter- mine safe doses for treating xenografted embry- os. Below are two examples (namely abiraterone acetate and progesterone) of the procedure ap- plied: embryos were manually dechorionated at 48 hpf, treated with various concentrations of abiraterone acetate (0.5-2.5 µM)25 and progester- one (10-100 µM),32 and kept at 32 ℃.
After 3 days of treatment, abiraterone acetate at 2.5 µM caused death or deformity, while 1 µM was chosen as a safe dose for subsequent experi- ments.25 For progesterone, doses above 25 uM
caused 100% mortality, 25 µM caused pericar- dial edema and yolk sac edema, and 10 µM was safe. Doses of 6.25 µM and 12.5 uM were initial- ly chosen, but 12.5 uM caused high mortality in embryos xenografted with the MUC-1 cell line.32
Evaluation of drug administration, intake and metabolism
Major advantages of the larval zebrafish xeno- graft model are the possibility to administer the compounds directly into the surrounding water and that their small size allows them to fit into 96-well plates to treat quickly and easily with multiple concentrations of different small mole- cules.49 Furthermore, each embryo-larva requires only microliter volumes of media, allowing treatments to be conducted with minimal quan- tities of test chemicals. Additionally, images of the entire fish can be captured using a wide-field objective, enabling the rapid acquisition of phe- notypic effects. With high-content microscopes, this process allows for the collection of data on a large number of xenografts efficiently.72
In ACC studies, the tested drugs were directly added to the fish water, achieving cytotoxic con- centrations and simplifying the treatment pro- cess. The concentration of progesterone needed to reduce tumor mass area in ACC cell lines was significantly lower than in-vitro levels32 likely due to the lipophilic nature of the yolk sac, which may contributes to the retention of the drug. Con- versely, the dose of trabectedin in fish water was 100 times higher than in vitro, but LC-MS/MS results showed embryo uptake levels comparable to the in-vitro concentrations.31
Abiraterone acetate, similarly added to fish water, reached cytotoxic concentrations within 24 hours and maintained its effect for up to three days, due to irreversible binding to CYP17A1.26
LC-MS/MS results showed that abiraterone acetate absorption increased with embryo devel- opment, displaying typical time-dependent con- centration kinetics similar to those in humans. The ability of zebrafish embryos to metabolize abiraterone acetate increased with developmental stages up to 120 hpf. This was supported by gene expression results showing zebrafish embryos expressed mRNA for the enzyme 3ß-HSD, which has high similarity (63%) and identity (46%) to
the human counterpart.25 Indeed, several studies indicate that zebrafish larvae metabolize drugs similarly to humans, supported by proteomics and transcriptomics analyses showing high con- servation of metabolic enzymes, including cyto- chrome P450 (CYP) genes.73, 74 Zebrafish larvae possess liver, kidney, and blood-brain barriers. 15 Studies on testosterone metabolism suggest adult zebrafish have more metabolic enzymes, with the main testosterone metabolite being identical to humans.75 Paracetamol clearance rates in zebraf- ish larvae are comparable to higher vertebrates and young humans.76 While further research is needed, pharmacological effects between ze- brafish and mammals are well-conserved, with 22 out of 23 known cardiotoxic drugs showing similar toxicity in zebrafish embryos and 8 out of 10 compounds identified in zebrafish producing expected effects in rodents. 14, 15
Evaluation of drugs effect
The effects of the drug on cancer growth were usually scored by taking pictures using a Zeiss Axiozoom V13 fluorescence microscope, 2 h af- ter cell injection (TO) and after 3 days of treat- ment (T3).25, 31, 32 Fluorescent cell area is used to measure cancer growth allowing for direct com- parisons within and between animals over time, enhancing statistical power and increasing sam- ple size.13, 22 Changes in area and intensity are relied upon for growth information and because increases in both can be correlated to cancer growth. However, cells cannot be counted indi- vidually as they are typically indistinguishable in a large mass. High-content imaging microscopes can automate this process13 but so far these have not been broadly applied likely due to remaining technical challenges.49
Alternative methods involve euthanizing ze- brafish before and after treatment, enzymatically dissolving them to produce unicellular suspen- sions, and counting fluorescent cells using a he- mocytometer or flow cytometry. This approach provides accurate proliferation rates and allows for live/dead cell and protein expression analy- ses using fluorescent antibodies. However, it precludes time-course comparisons and is la- bor-intensive, limiting high-throughput screen- ing.42, 54 Furthermore, euthanizing zebrafish for
cell counting precludes time-course comparisons for each animal, leading to less statistically pow- erful unpaired group comparisons.77 Variations in transplanted cell numbers due to microinjec- tion can cause significant differences between xenografts.
Consequently, imaging fluorescent cell areas and intensities in live zebrafish is the preferred method, enabling continuous, within-subject comparisons without euthanasia and facilitating more robust statistical analyses.13
Evaluation of metastasis formation
One of the most interesting aspects of using trans- plantations into zebrafish embryos and larvae is the ability to directly observe tumor cells in the transparent host. This allows for monitoring not only their proliferation but also their migratory behavior via live microscopy. Additionally, the interaction of tumor cells with the host environ- ment, including biological processes such as neovascularization, can be investigated.78-80 Ze- brafish blood vessels provide the essential struc- ture to evaluate metastatic behaviors, such as in- travasation and extravasation, of tumor cells. In metastatic cancers, tumor cells must intravasate into the bloodstream and then extravasate out to spread to other organs and tissues. Tumor cells transplanted into the zebrafish bloodstream can extravasate, as evidenced by their attachment to the endothelium and exit from the capillaries.81 Intravasion can also be seen, as tumor cells trans- planted into the yolk can invade into the blood stream, travel to the tailfin and form microme- tastases.62 Indeed, in the ACC studies, it was demonstrated that progesterone,32 trabectedin31 and NP-G2-044 as well as the FSCN1 knock- out35 were able to reduce the number of embryos with metastases, meaning the presence of at least one fluorescence dot outside the site of injec- tion. To distinguish authentic metastases from cells that may have been inadvertently injected into the vessels, the embryos are observed under Zeiss Axiozoom V13 fluorescence microscope two hours after injection. Embryos exhibiting fluorescent spots outside the injection site are discarded, as are those with tumor masses that are either too large or too small. In these studies the genetically-modified zebrafish line kdrl:GFP
was used. In this line, the blood vessels can be tracked using confocal microscopy due to the ex- pression of green fluorescence protein (GFP) un- der the promoter of vascular endothelial growth factor receptor (VEGFR2), also known as Flk-1 (fetal liver kinase 1) or KDR (kinase insert do- main receptor) gene and which is expressed in endothelial cells.79 This model allows as well im- portant insights into the mechanisms of sprouting angiogenesis,80, 82 vessel guidance, and vascular endothelial growth factor signaling,78, 83 among other angiogenic processes.13
Concluding remarks
In conclusion, as technology and methodology advance, zebrafish will become a vital part of cancer drug discovery, providing a bridge for translating studies from in vitro to in vivo. The validation of this animal model offers a useful tool for the preclinical first screening of a large number of drugs, particularly advantageous in rare and aggressive diseases such as ACC, where treatment strategies are limited. Zebrafish offer vertebrate anatomy with a hospitable in-vivo en- vironment containing relevant structures, such as the extracellular matrix and flowing blood ves- sels, necessary for human tumor development. They are relatively inexpensive in-vivo models for early drug testing, allowing the assessment of drug effectiveness and toxicity. Moreover, ze- brafish enable high-throughput testing, reducing the time- and money-consuming aspects.
While this model cannot completely replace others already in use, our review suggest that ze- brafish xenografts provide a suitable and expedi- tious model for the initial selection of potentially effective drugs, identification of dose toxicity, and determination of the most promising com- pounds for more advanced preclinical phases. Despite some limitations, and various technical improvements needed to optimize the results, this model is particularly valuable for screening drugs potentially effective in ACC. Additionally, we hope that future research in the ACC field can fully take advantage from zebrafish as a tool to study various aspects of the disease and drug re- sponses.
Future prospective
There are several key areas to address for ad- vancing zebrafish models in the context of ACC and other malignancies.
Optimization of zebrafish models for ACC:
· tumor microenvironment recapitulation: fu- ture studies could focus on enhancing the fidel- ity of the zebrafish model to better replicate the human tumor microenvironment. Approaches could involve incorporating stromal, immune, and vascular components to study the interac- tions between the tumor and its microenviron- ment, as well as mechanisms underlying immune evasion and metastasis in ACC.
High-throughput drug discovery and valida- tion:
· identification of therapeutic targets and combinatorial approaches: zebrafish models of- fer unique opportunities for large-scale in-vivo drug screening. Future research could leverage this capability to identify novel therapeutic tar- gets and optimize drug combinations specifically for ACC treatment. Screening libraries of small molecules, including repurposed drugs, could lead to the discovery of novel anticancer agents or synergistic drug combinations that enhance treatment efficacy;
· personalized medicine approaches: devel- oping zebrafish xenograft models using patient- derived tumor cells could support personalized therapeutic strategies. Zebrafish models could be used to rapidly assess patient-specific drug responses, providing a preclinical platform for precision medicine in ACC and other cancers.
Cross-species validation and translational rel- evance:
· comparative oncology approaches: a critical next step would involve cross-species validation of zebrafish findings in established murine mod- els and human cell lines. Comparative studies that align zebrafish-based data with murine and human systems will be essential for establish- ing the robustness and translational applicability of the zebrafish model in cancer research. Such efforts could solidify the zebrafish as a comple- mentary preclinical platform, particularly for cancers where traditional models are insufficient or slow to develop.
These research directions would broaden the utility of zebrafish models, advancing our under- standing of ACC and potentially other malignan- cies, while accelerating drug discovery and pre- cision oncology efforts.
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Conflicts of interest
The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manu- script.
Funding
This research was funded by the AIRC IG27233 project (PI S.S.) and by the University of Brescia local grants.
Authors’ contributions
Conceptualization, writing-original draft preparation: Mariangela Tamburello. Writing-review and editing: Mariangela Tambu- rello, Andrea Abate, and Sandra Sigala. Supervision: Sandra Sigala. Funding acquisition: Sandra Sigala. All authors have read and agreed to the published version of the manuscript.
Congresses
This paper was presented as poster at the WCP2023 - World Congress of Basic and Clinical Pharmacology was held in Glasgow, Scotland on 2-7 July, 2023; and as an oral communication at the 22nd ENS@T and 2nd COST Harmonis@tion meeting that take place in Dubrovnik, Croatia, 11-13 October 2023.
History
Article first published online: February 25, 2025. - Manuscript accepted: December 3, 2024. - Manuscript revised: November 11, 2024. - Manuscript received: July 16, 2024.
Supplementary data
For supplementary materials, please see the HTML version of this article at www.minervamedica.it