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Best Practice & Research Clinical Endocrinology & Metabolism
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Clinical Endocrinology & Metabolism
2
Mouse models of adrenal tumorigenesis
Constanze Hantel, MSc, Felix Beuschlein, MD*
Department of Medicine, Endocrine Research, University Hospital Innenstadt, Ludwig Maximilians University, Ziemssenstr. 1, D-80336 Munich, Germany
Keywords: adrenocortical carcinoma adrenal tumor model xenotransplantation model inhibin Gata4 Sf1 ACTH receptor
Adrenocortical carcinomas (ACCs) are heterogeneous tumors with a poor prognosis. The rarity of this disorder causes a lack of treatment experience and material availability which is necessary to optimize existing treatments and to develop novel therapeutic strategies. Although surgery is still the treatment of choice, adjuvant therapies are urgently needed as the rate of recurrence for these tumors is high. In recent years molecular characterization of surgical tumor specimen has aided in the understanding of disease mechanisms and definition of therapeutic targets also in adrenocortical carcinoma. However, most of the functional properties of potential target molecules are still unpredictable from pure expression and sequence analysis. For functional studies of gene products, mouse models remain to be intensively utilized as an experimental system due to the similarity to humans with respect to genome organization, development and physiology. Here we give an overview on rodent models that have been described to either have adrenocortical tumors as part of their phenotype or have been utilized for therapeutic screens as adreno- cortical tumor models.
@ 2010 Elsevier Ltd. All rights reserved.
Introduction
Mouse models exhibit a wide range of possibilities for the investigation of adrenocortical tumori- genesis. Incidental discovery of adrenal tumors in genetically modified animals can provide clues on pathways involved in adrenal tumorigenesis that would not have been predicted on the basis of structural analysis or in vitro exploration. Mouse models can also be used to verify functional signifi- cance of a given gene for adrenal growth and steroidogenesis in vivo through targeted genetic
* Corresponding author. Tel .: +49 (0)89 5160 2110 (2116); fax: +49 (0)89 5160 4467. E-mail address: felix.beuschlein@med.uni-muenchen.de (F. Beuschlein).
modification. Furthermore, high incidence of adrenal tumors in inbred mouse strains can serve as the starting point for genetic approaches to identify the underlying genetic cause. Similarly, chemically or radiation based mutagenesis could be utilized to create informative mouse models, known as forward (phenotype-driven) genetics. Finally, well-defined tumor models have been successfully used for preclinical intervention trials to screen for novel therapeutic approaches. In this overview we will provide an overview on adrenocortical tumor models relevant for either mechanistic studies or preclinical screening approaches.
Mouse models with spontaneous or induced adrenal tumor growth
Cohen and co-worker in 1951 reported the time course of a group of inbred mice of the LAF1 strain upon exposition to the irradiation of an atomic test bomb explosion (Operation Greenhouse). One male animal developed a tumor in the right adrenal cortex while the left adrenal gland was atrophic. Although it remained uncertain whether the incidence of the adrenal tumor was in fact induced by the irradiation or arose spontaneously, necropsy of the mouse revealed numerous small metastases of the lung as proof of the malignant phenotype of this adrenocortical tumor.1 The tumor was excised and implanted intramuscularly in the thigh of male and female LAF1 from which it was maintained as a transplantable tumor. While metastatic spread was initially noted on a regular basis in host animals upon later passages the tumors lost their metastatic properties.2 Histological and laboratory evidence of steroid secretion by the tumor was evident including thymic involution and adrenal atrophy, increase in serum sodium and decrease of serum potassium as well as eosinopenia and lymphopenia.1 Tumor-bearing animals were even utilized to assay for ACTH bioactivity from transplanted cortico- troph tumors.3 Consequently, a clone of steroid producing tumor cells was established by Yasamura and colleagues as a continuous cell line named Y1.4
Another early documented case of adrenal tumorigenesis in mice has been reported for the inbred mouse strain CE after surgical gonadectomy.5 This observation could be reproduced not only in the CE/J strain6 but also in other inbred mouse strains including DBA/2J,7 C3H, BALB/c8 and NU/J animals,9 while other strains such as C57BL/6J10 and FVB/N7 were found to be resistant to tumor formation. As both surgical gonadectomy and xenografting of hCG producing tumors9 were able to induce adrenocortical tumor growth in the described mouse strains it was suggested that chronic elevation of gonadotropins represents a major determinant of adrenocortical tumorigenesis. Thus, gonadectomy-induced adrenal tumorigenesis in susceptible mouse strains was characterized in more detail: The investigated tumors were of a benign or semi-malignant phenotype as metastasis was usually not present. The occurrence of these tumors, also when transplanted into littermates,11 was accompanied by morphological changes in hormone responsive organs such as the uterus or the mammary glands indicative of sex steroid production.12 Parts of the adrenal tumors were described to be reminiscent of seminiferous tubules of the testis or follicular structures similar to that found in the cortex of the ovary.5 In accordance with early morphological findings, later functional and molecular studies in fact revealed the expression of markers which are otherwise restricted to the gonad including receptors for LH and Mullerian inhibiting substance (MIS) as well as steroidogenic enzymes such as P450cyp17 and P450cyp19.6, 7, 9 Accordingly, an adaption to the gonad’s ability to secret sex steroids was detected.6 Interestingly, this functional change was also accompanied by a switch in the expression of the transcription factor Gata6 to that of Gata4.6, 7, 9 Gata4 has been implicated in the regulation of tissue-specific gene expression and cellular proliferation in the gonad.13, 14 Moreover, over expression of Gata4 is sufficient to induce expression of gonadal markers in adrenocortical tumor cells in vitro.15 Thus, these findings provide indirect evidence that induction of Gata4 expression is linked to the phenotypic shift observed in gonadectomy-induced adrenal tumors (Fig. 1A).
To identify genetic markers that are associated with gonadectomy-induced adrenal tumorigenesis Bernichtein and colleagues performed a genome-wide association study in non-susceptible C57BL/6J and susceptible DBA/2J animals.16 Linkage analysis identified a major locus on chromosome 8 con- taining 31 candidate genes. Among these genes Sfrp1, a dominant negative regulator of the Wnt sig- nalling pathway which is down-regulated in a number of tumor entities through epigenetic modifications was highlighted as a promising candidate.16 However, the exact contribution of the genes identified on the basis of genetic association studies remains to be determined. In addition, it has been
SF-1S172 ? Sfrp1 ?
A
sex steroids
gonadectomy
LH 1 - GATA-4 1 -+ LH-R 1-+
adrenal tumor
susceptible strain [no gonadal tumor]
adrenal growth dysregulation
SV40TAg
B
sex steroids
gonadectomy
LH 1 - GATA-4 1 -+ LH-R +-+
adrenal tumor
Inha-Tag [gonadal tumor]
adrenal growth dysregulation
dis-inhibition through inhibin deficiency
C
sex steroids +
gonadectomy
LH 1 -+ GATA-4 1 -+ LH-R +-+
activin
adrenal tumor
Inha-/- [gonadal tumor]
adrenal growth dysregulation
recognized that mouse strains with high tumor susceptibility have in common a polymorphism in Steroidogenic Factor -1 (Sf1), a transcription factor necessary for proper development and function of the adrenal cortex.17 This polymorphism which results in a substitution from alanine to serine at residue 172 of the protein appears to influence steroidogenic capacity of adrenocortical cells.18 Since Sf1 dosage has been described as a relevant trigger of adrenocortical tumor growth in childhood ACC19 as well as in a transgenic mouse model20 it is possible that the polymorphism could be associated with higher baseline Sf1 expression levels, thus, predisposing the development of adrenocortical tumor growth.
Mouse models utilizing transplanted adrenal tumor cells
Tumor transplantation models represent well-established tools that can be used to answer specific questions of tumor pathogenesis or can be applied for preclinical screening of anticancer treatments. As only very few instances of successful engraftment of primary adrenocortical tumor material have been reported in the literature21 the number of available adrenal tumor models is limited by the number of well characterized ACC cell lines (Table 1).
One example is the subcutaneous transplantation of adrenocortical SW13 cells which had been established in the 1970s from a non-secreting ‘small cell’ ACC22 and RL251 cells from a chemokine- secreting adrenal tumor23, 24 in immuno-compromised animals.25 Utilizing SW13 cells grown as subcutaneous tumors in immunodeficient mice Wolkersdörfer and colleagues could provide first evidence for the usefulness of local gene transfer therapy of a HSV thymidine kinase expressing adenoviral shuttle followed by ganciclovir treatment. Upon this therapeutic approach oncolytic effects through replicating adenoviral vectors could be demonstrated that was followed by tumor reduction and significant increase in animal survival.25
A more commonly used adrenocortical model is that of human ACC cell line NCI-H295. Upon subcutaneous injection of 6 x 106 cells in athymic nude mice tumor take rate has been reported in the range of 90% with a medium doubling time of 12 days.26 This cell line which had been established from a patient with hormonally active ACC has been demonstrated to retain its ability to produce all of the major adrenal steroids.27 Thereby, adrenal androgens among others have been shown to be elevated in
| Origin | Cell line | Host | Purpose of Model | Reference |
|---|---|---|---|---|
| Non-secreting 'small cell' human ACC | SW13 | Immuno-compromised mouse | - Intervention trials | 22, 25 |
| Chemokine-secreting human ACC | RL251 | Immuno-compromised mouse | - Studies of pathophysiology - Intervention trials | 23, 24 |
| Hormonally active human ACC | NCI-H295 | Immuno-compromised mouse | - Intervention trials | 24, 26, 27 |
| Murine cell lines derived from transplantable adrenocortical tumors | Y1 and Y6 cells | Syngenic mouse model (LAF1 strain) | - Studies of pathophysiology - Intervention trials | 4, 30 |
| Transgenic expression of oncogenes | Bovine and human adrenocortical cells | Immuno-compromised mouse | - Studies of pathophysiology | 36, 38, 69 |
nude mice bearing subcutaneous NCI-H295 tumors.26 Moreover, the tumors were characterized by dysregulation of the insulin-like growth factor system such as over expression of IGF2 and IGF-binding protein-2 similar to that observed in primary human tumor specimen. As insulin-like growth factors have been defined as major contributors of adrenocortical tumor growth28, 29 targeting of type I insulin-like growth factor receptor (IGF1R) dependent pathways represents a promising approach to modulate the proliferative phenotype of ACC cells. In accordance with this notion Barlaskar and colleagues could demonstrate that treatment of animals bearing NCI-H295 derived subcutaneous tumors with IGF1R antagonistic compounds resulted in a significant amelioration of tumor growth and increase in survival time.24
Adrenal tumor cells can also be maintained in immune competent mice as long as the strain is chosen from which the particular cell line had initially been derived. Accordingly, Y1 cells which had been established from LAF1 mice as described above can be re-transplanted in this strain of animals. A possibility to use this model to investigate tumor relevant regulatory pathways is to genetically alter these cells and assay for tumor growth behavior in vivo. Following this approach the human ACTH receptor was introduced in an ACTH unresponsive sub-clone of Y1 cells (Y6 cells; 18). These cells upon transplantation into LAF1 animals could be demonstrated to have a lower proliferative potential in comparison to wild-type Y6 cells under baseline conditions and after ACTH stimulation (Fig. 2).30 Thus, these studies supported clinical evidence that ACTH receptor expression in ACC is associated with a less aggressive adrenal tumor phenotype31 and demonstrated that ACTH dependent anti-proliferative effects can be amplified through stimulation with physiological doses of ACTH.
Genetic modification have also been applied on primary cultures of normal human and bovine adrenocortical cells to recapitulate molecular alterations found in the course of tumorigenesis in vivo and to define those to be required and sufficient to induce malignant tumor growth in transplanted animals. Following this approach Hornsby and colleagues have developed a model in which a suspen- sion of adrenocortical cells is introduced under the kidney capsule.32 Once implanted at this location cells rapidly aggregate followed by invasion by host endothelial cells, formation of a vascular system, and the subsequent survival, growth and function of the transplant tissue.32 As structure and function of the transplant are dependent on circulating pituitary hormones from the host transplantation exper- iments are usually performed in adrenalectomized animals.33 Utilizing this transplantation model, in a number of experiments Thomas and colleagues could demonstrate that introduction of telomerase reverse transcriptase (hTERT) into bovine adrenocortical cells resulted in a tissue phenotype similar to that of untransfected cells.34 In contrast, forced expression of SV40 large Tantigen (SV40 TAg), oncogenic RasG12V together with hTERT were sufficient to induce a malignant phenotype of the transgenic tissue that gave rise to a tumor resembling many features of ACC.35 Consequently, even in the absence of hTERT, SV40 TAg and RasG12V alone was demonstrated to be sufficient to induce a malignant phenotype in bovine and human adrenocortical cells.36 However, these tumors displayed a reduction in growth rate after several consecutive rounds of transplantation which was accompanied with the progressive entry of cells into crisis. These changes could be reversed after transduction with hTERT, thus restoring tumorigenicity of the cells.36
radiation
LAF1 (donor)
Y1 cells (MC2-R+)
Y6 cells (MC2-R-)
pcDNA +
1
stable transfection
+ pcDNA-MC2-R
Y6 pcDNA (MC2-R-)
Y6 MC2-R (MC2-R+)
baseline conditions or ACTH treatment
LAF1 (host)
LAF1 (host)
A similar experimental design was chosen to investigate the role of illicit receptor expression which has been identified as the cause of ACTH independent hypercortisolism in bilateral adrenal macro- nodular hyperplasia as well as adrenocortical tumors.37 Bovine adrenal cells upon transduction with the receptor for the gastric inhibitory polypeptide (GIPR)38 or the luteinizing hormone receptor (LHR)39 in both instances developed into hyperproliferative adenomatous, but not overtly malignant tissue when transplanted under the kidney capsule and resulted in a mild ACTH independent hyper- cortisolism of the host animal. Thus, while it remained uncertain whether the ectopic expression of G protein coupled receptors in human patients resembles the cause or consequence of hyperplastic or adenomatous adrenocortical growth the mouse model experiments inform that expression of the GIPR or the LHR each is sufficient to induce a phenotype similar to those defining the clinical entity.
The standardization of the described tumor models will play an increasing role in the future to establish the ground for early clinical trials in patients with ACC. However, further improvement of the described tumor models is being sought (Fig. 3): From a clinical perspective the subcutaneous tumor niche represents an ‘unphysiological’ manifestation of ACC. More relevant metastatic sites would include local lymph nodes, liver, lung and bones. Although these manifestations could be mimicked in the mouse model through local or intravenous administration of tumor cells these models would have the disadvantage of hindered follow-up examination. To further improve suitability of such a model, ACC cells stably expressing GFP or luciferase could allow in vivo imaging approaches in the future. Another aspect to improve adrenocortical tumor models that should be targeted by future research is
Cell line for ACC
Patient with ACC
Development of stably luciferase expressing cells
Surgery
Tissue preparation
Injection in syngenic or immunocomromised mice
3.5
3.0
Tissue implantation or injection of primary cultures
2.5
2.0
00
1.5
1.0
0.5
0
In vivo bioluminescence
Development of metastasis models
Patient individual tumor xenografts
that of therapeutic testing in the context of personalized medicine. For example tumor material from patients that could be maintained in xenograft models would provide the opportunity to specifically test for the suitability of a specific compound for an individual patient.
Genetically modified mouse models diplaying an adrenal tumor phenotype
Mice that have been designed to harbor specific genetic modifications through transgenic tech- niques or knock-out approaches have been instructive for the identification of molecular mechanisms involved in adrenocortical tumorigenesis. These models can be utilized to provide information of the functional significance of a specific gene or downstream pathway that might have been identified by in
vitro experiments, through expression studies from surgical tumor samples or on the basis of clinical information from patients with rare genetic syndromes. Furthermore, careful phenotypic character- ization of available mouse models in which an adrenal phenotype had been discovered can serve as a starting point for further functional analysis.
Mouse models with transgenic expression of an oncogene inducing adrenal tumors
The Simian Virus 40 large T antigen (SV40) represents a commonly used oncogene which can be expressed in the adrenal cortex upon transgenic introduction under the control of a tissue-specific promotor sequence. Promotors that have been used to target the adrenal cortex include 5’-flanking sequences from the human CYP11A gene,40 the aldose reductase-like (akr1b7) gene41 as well as the inhibin alpha promotor.42 While the former two models have been mainly used to generate cell lines for further in vitro analyses,43 the alpha inhibin promotor driven transgenic animal (inha/Tag) has been phenotypically characterized in great detail:
While originally developed to study their gonadal tumor phenotype44 it became soon clear that surgical gonadectomy not only rescued the animals from gonadal tumor associated death but also introduced another phenotypic facet with the development of adrenocortical tumors.42 Inhibin, a member of the TGFB family of growth factors, which is composed of a unique alpha subunit and beta subunit shared with activin is mainly expressed in the gonad and the adrenal cortex.45 As such, distribution of tumor manifestation in inha/Tag resembles the expression pattern of the endogenous inhibin a subunit. However, further molecular studies revealed an adrenal tumor phenotype very similar to that of inbred mouse strains susceptible for gonadectomy-induced adrenal tumorigenesis. Thus, the inha/Tag model has provided valuable insights in more general mechanisms of adrenal tumorigenesis that also occur without introduction of a viral oncogene. By induction of a hypogonadic state through pharmacological treatment with a GnRH antagonist or long-term testosterone treatment or by cross-breeding the mice into the hypogonadotropic hpg background development of both gonadal and adrenal tumors could be prevented.46, 47 Moreover, proliferation rate of an adrenal tumor cell line derived from a gonadectomized inha/Tag animal could be increased by in vitro treatment with the LH receptor ligand hCG.48 Taken together, these findings indicated that pituitary gonadotropins play an important role for the induction of the adrenal tumor phenotype in this mouse model. Interestingly, chronic elevation of LH levels results in upregulation of adrenal Lhr expression required to induce further LH dependent functional changes. As an important contributor for this direct interrelation of LH and its receptor, Gata4 has been identified as upregulation of Gata4 and LH coincides and co-localizes in adrenal tumors.49 In addition, in vitro49 and in vivo studies50 suggested an LH dependent increase in adrenal Gata4 expression levels. Furthermore, a Gata-responsive element in the Lhr promotor was identified and Gata4 dependent transcriptional activation of the Lhr gene could be demonstrated in in vitro promotor experiments.49 Overall, these findings describe a feed-forward loop of chronic elevated LH levels that induce Gata4 levels which in turn increases expression of the adrenal Lhr which provides an important molecular switch in the initiation of adrenal tumorigenesis in this model (Fig. 1B).51
Upon this detailed molecular characterization gonadectomized inha/Tag animals were further utilized as a model of Lhr targeted therapy. Using a lytic peptide (hecate) which was conjugated to a fragment of the hCG beta chain (hecate-hCG beta) treatment of animals with established adrenal tumors induced a significant reduction in adrenal tumor size in male animals. As hecate-CGbeta conjugate treatment seemed not to affect normal adrenocortical function this finding argued for a cell specific effect, which only targeted Lhr expressing tumor cells.52 Although preliminary in its nature, these efforts provide the rationale to further explore treatment modalities directly or indirectly tar- geting Lhr expressing adrenal tumors.
Another example for a transgenic mouse model characterized by adrenal tumor growth has recently been reported in an animal harboring multiple copies of a yeast artificial chromosome including the Sf1 genetic locus.20 The gain in Sf1 copy number which resulted in an increase in adrenal Sf1 protein levels was associated with the development of macronodular adrenocortical disease that further progressed into adrenal tumors in an age dependent manner. Interestingly, tumors of Sf1 transgenic animals displayed a similar expression pattern in comparison to those of inbred susceptible mouse strains including upregulation of Gata4 and Mis while adrenocortical markers including P450scc were
absent.20 Further gene expression profiling revealed that genes involved in cell adhesion and the immune response and transcription factor signal transducer and activator of transcription-3 (Stat3) were differentially expressed in Sf1 transgenic mouse adrenals in comparison to wild-type littermates. Together with clinical findings from childhood ACC in which the Sf1 is amplified and overexpressed this model presents further evidence that gene dosage of Sf1 can contribute to the phenotype of adreno- cortical tumors. As specific Sf1 inverse agonists have been developed and proofed to be effective in in vitro adrenocortical systems33 the transgenic in vivo model might aid in further preclinical testing of these compounds.
Mouse models with targeted deletions inducing adrenal tumors
In addition to susceptible inbred mouse strains (Fig. 1A) and the inha/Tag transgenic model (Fig. 1B) also animals with a targeted deletion of the inhibin alpha subunit (Inha-/-) are characterized by the phenotype of gonadectomy-induced adrenal tumorigenesis (Fig. 1C). This animal model which was originally introduced by Matzuk and colleagues in 199254 has since been subjected to an in depth phenotypic and molecular characterization. As introduction of a transgenic background with chronically elevation of pituitary LH secretion enhances adrenal tumor growth in gonadectomized Inha-/- animals LH does act as an adrenal growth factor also in the context of inhibin deficiency. Very similar to what had been described in inha/Tag animals adrenal tumors in Inha-/- mice are characterized by a switch from Gata6 to Gata415 and a further change in expression pattern towards a gonadal phenotype.55
One aspect that is not shared by other gonadectomy dependent tumor models is the secretion of high levels of activin both by gonadal and adrenal tumors in Inha-/- animals which are associated with the development of cancer cachexia-like syndrome.56 Interestingly, activin secreted by gonadal tumors has direct functional and morphological consequences on the adrenal cortex which might contribute to the inhibition of adrenal tumor growth before gonadectomy is performed.55 However, genetic removal of Smad3 which is required for activin signalling from Inha-/- mice could be demonstrated to attenuate adrenal tumor progression by uncoupling extracellular mitogenic signals from the cell cycle machinery including cyclin D2.57 These findings are endorsed by studies on gonadectomized Inha-/- animals which had been cross-bred to generate double knockouts for p27 or cyclin D2, respectively. Interest- ingly, while loss of p27 had little effect on adrenal cortical tumor progression in the absence of inhibin the loss of cyclin D2 prolonged the lifespan of double knockouts animals.58
An example in which the occurrence of ACC is indicative of the presence of a genetic syndrome is that of childhood adrenal cancer in the context of specific TP53 mutations.59 p53 acts as a cell cycle check point to regulate DNA repair or induce growth arrest or apoptosis in response to DNA damage and its loss of function has been demonstrated to affect a large array of tumor entities.60 However, only very recently its role as a tumor suppressor gene in ACC has been highlighted in a mouse model of telomere dysfunction in which animals with p53 haploinsufficiency developed ACC in 5% of cases.61 While these tumors exhibited locally invasive growth and a malignant histology, no metastatic spread has been reported.
In patients with Multiple Endocrine Neoplasia Typ 1 (MEN1) in addition to parathyroid adenomas, pancreatic islets tumors and pituitary adenomas development of adrenocortical tumors has been described in up to 40% of patients.62 Accordingly, animals with targeted deletion of the menin gene resembled the clinical features of MEN1 including that of development of adrenocortical nodular disease which progressed into adrenal tumors.63-65 As part of an aging experiment adrenocortical lesions described as microadenomas or tumors developed in 6% of heterozygous animals within the first year of life, and in up to 30% in a cohort of roughly 2 year old animals.65 In addition to these small lesions, adrenal tumors with a more aggressive growth behavior have been reported with an incidence after 18 months of up to 46% of heterozygous animals.64 Notably, other MEN1 defining tumors including pancreatic islet cell tumors and pituitary adenomas developed at an earlier time point and with higher penetrance.63, 65 As homozygous menin knock-out animals die in utero, only heterozygous mice were phenotypically characterized. However, in accordance with a two hit model of a tumor suppressor gene the remaining wild-type menin allele could be demonstrated to be lost in somatic tumor cells. 63-65 This is in striking contrast to human adrenal lesions as part of MEN1, where LOH does not seem to be a dominant tumorigenic mechanism.
Mouse models with defects in adrenal growth and development
In addition to the summarized mouse models defined by an adrenal tumor phenotype mouse models displaying a growth defect of the adrenal cortex either during embryogenesis or in the adult animal are worthwhile to be noticed also from the perspective of adrenal tumor research. This concept is highlighted by Sf1 which had originally been identified as a transcription factor that when absent results in adrenal agenesis. As described above, however, Sf1 when overexpressed or overtly active can be associated with an adrenal growth defect contributing to adrenal tumor growth. Other examples include animal models exploring transcription factors such as Pbx166 or pathways of B-catenin67 or hedgehog68 signalling. Along this line, it is likely that in the future other mouse models with adrenal growth defects will provide novel insights in the pathogenesis of unrestricted growth as observed in ACC.
Research agenda
· Development of inducible adrenocortical tumor models with appropriate time course for screening of effective therapeutic strategies.
· Development of further mouse models that represent a more pathophysiological relevant tumor niche or metastatic site
· Optimization of in vivo detection methods and quantification of tumor load in adrenocortical tumor mouse models.
· Improvement of personalized tumor models utilizing xenotransplantation approaches.
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