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Molecular and Cellular Endocrinology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m c e
Review
Xenograft models for adrenocortical carcinoma Constanze Hantel *, Felix Beuschlein Endocrine Research Unit, Medizinische Klinik und Poliklinik IV, Ludwig-Maximilians-Universität, Munich, Germany
A R T I C L E
I N F O
Article history: Received 12 March 2015 Received in revised form 26 May 2015 Accepted 26 May 2015 Available online Keywords: Adrenocortical carcinoma Xenograft Preclinical animal model
A B S T R A C T
Adrenocortical carcinomas (ACCs) are rare, heterogeneous and very malignant endocrine tumors with a poor prognosis. An important prerequisite to optimize existing therapeutic regimens and to develop novel therapeutic strategies are preclinical disease models. In recent years molecular and genetic profiling of surgical tumor specimen led to the identification of novel interesting markers. However, precise involvement of these markers in tumorigenesis and their functional relevance in therapeutic outcome is still under investigation. Xenograft models are important tools for such functional studies as they bear the potential to mimic the complexity of solid tumors including tumor cells, stroma and blood vessels. Thus, for the successful and safe development of novel therapeutic strategies xenograft models remain to be indispensable experimental tools. Here we provide an overview on currently existing xenograft models for ACC, their tissue origins, establishment, implications as well as limitations. © 2015 Elsevier Ireland Ltd. All rights reserved.
Contents 1. 2. 3.
4. 5. 6.
Introduction ............................................................................................................................................................................................................................................................. Xenograft models of murine origin ................................................................................................................................................................................................................. Xenograft models of human origin ................................................................................................................................................................................................................. 3.1. NCI-H295 ..................................................................................................................................................................................................................................................... 3.2. SW-13 ........................................................................................................................................................................................................................................................... 3.3. Xenograft models based on primary cultures ................................................................................................................................................................................ 3.4. SJ-ACC3 ........................................................................................................................................................................................................................................................ 3.5. Patient-derived tumor xenografts (PDTXs) from adult ACCs .................................................................................................................................................... Cell-culture derived vs tissue-based xenograft models for ACC ............................................................................................................................................................ Other variables to be considered in preclinical ACC models .................................................................................................................................................................. Conclusions .............................................................................................................................................................................................................................................................. References ................................................................................................................................................................................................................................................................
1. Introduction Adrenocortical carcinoma (ACC) is a rare tumor affecting both adults and children with an annual incidence of 0.7–2.0 cases per million population. The disease is highly malignant with only 16–38% of patients surviving more than 5 years after diagnosis (Abiven et al., 2006; Allolio and Fassnacht, 2006; Fassnacht et al., 2013; Libe et al., 2007). The only drug approved for adjuvant ACC treatment as well as for advanced tumor stages is mitotane. Furthermore, a recent prospective randomized interventional trial for late stage ACC patients
* Corresponding author. Endocrine Research Unit, Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Ziemssenstr. 1, D-80336 Munich, Germany. Tel.: +49 89 4400 52943; fax: +49 89 4400 54467. E-mail address: [email protected] (C. Hantel).
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has demonstrated that combination of the cytostatic drugs etoposide, doxorubicin and cisplatin together with mitotane (EDP-M) resulted in a higher response rate and longer progression free survival in comparison to streptozotocin and mitotane (Fassnacht et al., 2012). However, even in the setting of a clinical study systemic treatments achieved only partial responses and even after radical resection as many as 85% of patients relapse (Allolio and Fassnacht, 2006). Thus, novel treatment options for patients with ACCs are urgently needed, but clinical translation of novel therapeutic strategies often fails indicating that the currently utilized models only poorly predict clinical success (Johnson et al., 2001). One reason for this shortcoming is that only a few cell lines are available thereby lacking patient heterogeneity and specific therapeutic responses of individual tumors (Leibovitz et al., 1973; Logie et al., 2000; Pinto et al., 2013). Secondly, even though these tumor models can be utilized
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as xenografts in vivo and are, thus, indispensable tools, they originate from cell suspensions. There is good evidence that selection during cell culture passages change biologic properties compared to the original patient tumor (Siolas and Hannon, 2013). In fact, genomic instability of cell-lines has been observed during cell propagation so that resulting xenografts can carry alterations in chromosomal arrangements, karyotype and subsequent alterations in gene expression, the presence of differentiation markers and growth rates (Lum et al., 2012). This review aims to provide an overview on commonly used xenograft models for ACC as well as on alternatives and recently achieved progress. 2. Xenograft models of murine origin The first transplantable model for ACC arose in 1951 on a group of inbred mice of the LAF1 strain. Upon exposure to the irradiation of an atomic test bomb explosion 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 developed spontaneously, necropsy of the mouse revealed numerous small lung metastases as proof of the malignant phenotype of this adrenocortical tumor (Fig. 1) (Cohen et al., 1957). The primary tumor was excised, minced and subsequently inoculated intramuscularly in the thigh of male and female LAF1 mice where it displayed a 100% take on-rate in male and 92% in female hosts, respectively (Cohen et al., 1957). The average tumor latency of the original passages ranged between 106 (males) and 133 (females) days, which decreased in further passages down to 16–23 days. Histological examination revealed well vascularized tumors spotted with necrotic areas. In tumor bearing mice profound polyuria and polydipsia were observed beginning with a tumor size of 2–3 cm in diameter. The fact that these symptoms were also detectable in adrenalectomized and gonadectomized animals indicated that these were signs of functional activity of the xenografts. Consequently, a clone of steroid producing tumor cells was established as a continuous cell line named Y1 which retained the ability of tumor formation in the syngeneic LAF1 mouse strain in vivo (Cohen and Furth 1959; Humphreys et al., 1965; Yasumura et al., 1966). The fact of availability of a syngeneic host represents an important advantage of this tumor model as the adrenal xenografts can be maintained in immune competent animals. In the past, this murine xenograft model aided for example in the understanding of the role of ACTH-receptor (Melanocortin 2 receptor (MC2-R)) on adrenocortical growth and tumorigenesis (Zwermann et al., 2005). For this purpose, the human ACTH receptor was introduced in an ACTH unresponsive sub-clone of Y1 cells (Y6 cells (Frigeri et al., 2002)). 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 (Zwermann et al., 2005). 3. Xenograft models of human origin 3.1. NCI-H295 The most commonly used in vitro and in vivo adrenocortical model is that of the human ACC cell line NCI-H295 (Fig. 1). Gazdar et al. reported in 1990 on the establishment and characterization of a continuous human adrenocortical cell line originated from an invasive primary adrenocortical carcinoma (Gazdar et al., 1990). The original tumor was derived from a 48-year-old woman who showed symptoms of weight loss, acne, facial hirsutism, edema, diarrhea and secondary amenorrhea. Diagnostic work-up revealed elevated
levels of serum cortisol as well as urinary cortisol, aldosterone and 17-ketosteroids and a right-sided adrenal mass (Gazdar et al., 1990). After tumor removal a surgical specimen was minced, seeded in micro-well plates and further passaged. The resulting cell line has been demonstrated to retain its ability to produce all of the major adrenal steroids (Gazdar et al., 1990). Injections of 5 × 106 NCIH295 cells revealed the general tumorigenicity of these tumor cells in athymic nude mice. A more detailed characterization of the NCI-H295 xenograft model was followed by Logie et al. (2000). Upon subcutaneous injection of 6 × 106 cells into the flanks of female nude mice tumor take-on rate has been reported in the range of 90% with a medium doubling time of 12 days. Plasma steroid levels have been shown to be elevated in the in vivo model and histological examination demonstrated that morphological features of the xenografts were furthermore comparable to that of the original patient tumor. Moreover, the tumors were characterized by dysregulation of the insulin like growth factor system including an overexpression of IGF2 and IGF-binding protein-2 similar to that demonstrated in primary human tumor specimen. In accordance, a significant inhibition of tumor growth and increase in survival time of NCI-H295 tumor bearing mice could be observed after treatment with IGF1R antagonistic compounds indicating the functional relevance of targeting this dysregulated pathway (Barlaskar et al., 2009; Hantel et al., 2012). Subcutaneous NCI-H295 xenografts have also been utilized upon genetic modification to study the role of beta catenin pathway in the context of adrenocortical tumorigenesis (Gaujoux et al., 2013), for preclinical evaluation of existing clinical treatment regimens (Doghman and Lalli 2013; Hantel et al., 2014) as well as for the development of novel therapeutic strategies (Gaujoux et al., 2013; Hantel et al., 2012; Szabó et al., 2014). 3.2. SW-13 Another example for a preclinical ACC model bases on the subcutaneous injection of SW13 cells which had been established in the 1970s from a non-secreting small-cell carcinoma of the adrenal cortex (Fig. 1) (Leibovitz et al., 1973). 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. Injection of 10 × 106 cells gave rise to the development of SW-13-xenografts in the flank of female nude mice. Upon the established gene therapeutic approach oncolytic effects through replicating adenoviral vectors could be demonstrated that was followed by tumor reduction and significant increase in animal survival (Wolkersdorfer et al., 2002). Utilizing subcutaneous SW-13 xenografts the importance of angiogenic pathways in ACCs could be demonstrated by investigating the anti-tumoral efficacy of the VEGF receptor tyrosine kinase inhibitors sorafenic and everolismus (Mariniello et al., 2012). Another recent example for therapeutic intervention is the identification of nanoparticle albumin-bound paclitaxel (nab-paclitaxel) as a potential treatment for ACC. This compound showed greater antitumoral effects than the adrenolytic substance mitotane in SW-13 tumor bearing mice (Demeure et al., 2012). Following a different approach Zeng et al. reported very recently the enrichment of cancer stem cells from this tumor model and their characterization in three generations of SW-13 xenografts (Zeng et al., 2014). 3.3. Xenograft models based on primary cultures Hornsby and colleagues reported on a technique in which a suspension of bovine or human adrenocortical cells is introduced under the kidney capsule of SCID mice. Upon xenograft establishment these cells can replace the essential steroidogenic functions of the hosts’
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Fig. 1. Schematic illustration of the different existing xenograft models for ACC from cell-line based murine (Y1/Y6) and human (NCI-H295 and SW-13) to the novel tissue based models SJ-ACC3 and MUC-1.
adrenal glands (Thomas et al., 2003). In fact, as structure and function of the transplant are dependent on circulating pituitary hormones from the host transplantation experiments are usually performed in adrenalectomized animals (Thomas et al., 2003). Utilizing this transplantation model Thomas and colleagues could in a series of experiments demonstrate that introduction of telomerase reverse transcriptase (hTERT) into bovine adrenocortical cells resulted in
a tissue phenotype similar to that of un-transfected cells and animals that received these transplants survived despite adrenalectomy. Moreover, the developing xenografts showed no signs of enhanced proliferation rates or of malignant transformation (Thomas et al., 2000). In contrast, forced expression of SV40 large T antigen (SV40 TAg) and oncogenic RasG12V together with hTERT lead to the induction of a malignant phenotype of the transgenic tissue that
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gave rise to a tumor resembling many features of ACC (Thomas et al., 2002). 3.4. SJ-ACC3 Recently, Pinto et al. reported the establishment of the first pediatric tumor model for ACC (Fig. 1) (Pinto et al., 2013). In this case, the original patient tumor derived from an 11-year-old boy with an incidentally found right adrenal mass resected and subsequently confirmed as an ACC. Laboratory tests revealed endocrine functionality even though the patient showed no remarkable signs of virilization or hypercortisolism. Immunohistochemical and Western blot analyses of the patient tumor revealed positivity for inhibin A, keratin 8, synaptophysin, a strong nuclear p53 staining and a Ki-67 index of approximately 60%, while no specific staining was detectable for chromogranin, HMB-45 and S-100. For inoculation of tumor xenografts surgical specimen were implanted as tumor pieces without mincing in CB17 scid−/− mice. Histological examination, DNA fingerprinting and gene expression profiling of developed xenografts, referred as SJ-ACC3, demonstrated pathomorphological similarity with the original patient tumor (Pinto et al., 2013). Even though tissue and not cell-line based, SJACC3 represents a quite standardizable tumor model. To this end, successful re-implantation was feasible in our laboratory following a simple de-freezing protocol (Fig. 1).
well as applicability of stably expressing GFP or luciferase transfectants allowing in vivo imaging approaches (Mariniello et al., 2012). 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. The intra-lienal injection of stably luciferase expressing NCI-H295 cells results for example in the spread of NCI-H295 liver metastases in athymic nude mice. Therefore, a median laparotomy was followed by intrasplenic injection of 5 × 106 stably luciferase transfected tumor cells. A subsequent incubation time of 10 minutes enabled the cells to distribute via the portal vein. Afterwards, mice were splenectomized to avoid the growth of a large primary tumor in the spleen. In vivo bioluminescence imaging has been carried out after intraperitoneal injection of 3 mg luciferin. (Fig. 2A–E; Hantel, unpublished data). However, the development and routinely utilization of such cell-line based orthotopic and metastasis models for ACC is as limited by the complexity of the required surgery (Luconi and Mannelli, 2012). While tissue-based transplantation lacks the possibility of tumor inoculation by an exact cell number, it provides the important advantage of reflecting tumor heterogeneity and no further processing such as mincing and sub-culturing as described for the other available ACC tumor models. Even though cell suspensions as Y1, NCIH295 and SW-13 form xenografts in vivo, recent data suggest that selection pressure in cell cultures changes biologic properties compared to the original patient tumors (Siolas and Hannon, 2013).
3.5. Patient-derived tumor xenografts (PDTXs) from adult ACCs 5. Other variables to be considered in preclinical ACC models One of the most frequently cited reasons for the high failure rate of new therapeutic regimens in oncology is the lack of preclinical models to reflect patient heterogeneity (Johnson et al., 2001; Tentler et al., 2012). As outlined earlier also for ACC only a few cell lines are available (Gazdar et al., 1990; Leibovitz et al., 1973; Yasumura et al., 1966) which furthermore do not reflect heterogeneous functional properties and specific therapeutic response rates of individual tumors. In an attempt to amend the poor availability of preclinical in vivo models and to furthermore facilitate personalized treatment approaches, our workgroup aimed in recent years at the establishment of patient-individual tumor models for adult ACC. Similar approaches have been described for other tumor entities (Malaney et al., 2014; Morton and Houghton, 2007; Ruggeri et al., 2014). For this purpose, pieces of surgical tumor specimen were implanted subcutaneously in athymic NMRI nude mice (Fig. 2F–I). To investigate whether morphological and functional characteristics between tumor samples after mouse engraftment in comparison to the original tumor would be comparable, we started examination of implanted material and original patient tumor by histology and immunohistochemistry (Hantel et al., manuscript in preparation). Moreover, we utilized patient-tumor bearing mice to investigate putative applicability in therapeutic short-term settings (Hantel et al., 2014). In contrast to the majority of the adrenal PDTXs, one of the implanted tumors referred as MUC-1 showed a large increase in tumor size, increasing take-on rates and a decrease in effective take-on times indicating that these xenografts could be utilized as standardizable tumor model for multiple preclinical settings. As the currently existing standard models NCI-H295R and SW-13 of human origin are both cell-line based (Gazdar et al., 1990; Leibovitz et al., 1973), MUC-1 xenografts may be established as the first tissue based human xenograft model for adult ACC in the future. 4. Cell-culture derived vs tissue-based xenograft models for ACC An important advantage of cell-culture derived xenograft models is the putative establishment of orthotopic or metastasis models as
In addition to the origin of tumor xenograft, immune-competence of the host and site of implantation many other variables have to be taken into account for in vivo experiments. These include among others the chosen endpoints, type and timing of drug administration as well as drug metabolism (Luconi and Mannelli, 2012). One example for the large impact of variables in xenograft-studies is the differently reported therapeutic outcome of mitotane treatments in the same xenograft model (NCI-H295R (Barlaskar et al., 2009; Doghman and Lalli, 2013; Lindhe and Skogseid, 2010)). The choice of solvent might represent also a critical issue in xenograft studies. In a very recent experiment, 8 week old male H295R xenografted SCID-mice received intraperitoneal treatments of either mitotane (solved in 10% Tween) or the diluent alone. Six to eight days after continuous administration, all mice of both control and treatment groups (n = 8 each) died unexpectedly. The repetition of this experiment utilizing the same xenograft model but per oral administrations and corn-oil as solvent avoided the previously observed lethality, suggesting that Tween could have been responsible for the sudden death of mice (personal communication with Péter Igaz, Semmelweis University, Budapest, Hungary). Altogether, this observation underlines the importance of standardization not only of the utilized xenograft models, but also for treatment protocols utilized for in vivo experiments. As outlined earlier, also the origin of a specific xenograft model (cell-line based vs. tissue-based) might be of high impact (Lum et al., 2012; Siolas and Hannon, 2013; Tentler et al., 2012). Moreover, while tremendous technological advances led to important insights into the molecular and genetic determinants that drive human cancers in general and ACC in particular, successful translation into effective therapeutic strategies is hampered by the difficulty in predicting therapeutic outcome in the context of different mutational backgrounds (Assie et al., 2014; Nardella et al., 2011). RNA-knockdown approaches (Gaujoux et al., 2013; Guillaud-Bataille et al., 2014) are first steps taken these mechanisms into account, but tumor models reflecting different genetic backgrounds within ACC are urgently needed to improve the clinical prediction of xenograft models for ACC.
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Fig. 2. Exemplary pictures of the surgery (A), intra-lienal injection and splenectomy necessary for the induction of NCI-H295 liver metastasis and follow up utilizing in vivo bioluminescence imaging (D and E). Patient tumor (F) and prepared tissue specimen for subsequent subcutaneous implantation in the neck of athymic nude mice (H and I).
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6. Conclusions Preclinical xenograft models of human cancers are important tools for the investigation of tumorigenesis and the development of novel anti-cancer agents. Even though indispensable for preclinical drug development also these tumor models have specific limitations and variables such as tumor origin, sub-culturing, genetic background, immune-competence of the murine host, hormonal activity or site of implantation. These have to be individually taken into account for the design of preclinical studies on ACC. References Abiven, G., Coste, J., Groussin, L., Anract, P., Tissier, F., Legmann, P., et al., 2006. Clinical and biological features in the prognosis of adrenocortical cancer: poor outcome of cortisol-secreting tumors in a series of 202 consecutive patients. J. Clin. Endocrinol. Metab. 91, 2650–2655. Allolio, B., Fassnacht, M., 2006. Clinical review: adrenocortical carcinoma: clinical update. J. Clin. Endocrinol. Metab. 91, 2027–2037. Assie, G., Letouze, E., Fassnacht, M., Jouinot, A., Luscap, W., Barreau, O., et al., 2014. Integrated genomic characterization of adrenocortical carcinoma. Nat. Genet. 46, 607–612. Barlaskar, F.M., Spalding, A.C., Heaton, J.H., Kuick, R., Kim, A.C., Thomas, D.G., et al., 2009. Preclinical targeting of the type I insulin-like growth factor receptor in adrenocortical carcinoma. J. Clin. Endocrinol. Metab. 94 (1), 204–212. doi:10.1210/jc.2008-1456; [Epub 2008 Oct 14]. Cohen, A.I., Furth, J., 1959. Corticotropin assay with transplantable adrenocortical tumor slices: application to the assay of adrenotropic pituitary tumors. Cancer Res. 19, 72–78. Cohen, A.I., Furth, J., Buffett, R.F., 1957. Histologic and physiologic characteristics of hormone-secreting transplantable adrenal tumors in mice and rats. Am. J. Pathol. 33, 631–651. Demeure, M.J., Stephan, E., Sinari, S., Mount, D., Gately, S., Gonzales, P., et al., 2012. Preclinical investigation of nanoparticle albumin-bound paclitaxel as a potential treatment for adrenocortical cancer. Ann. Surg. 255, 140–146. Doghman, M., Lalli, E., 2013. Lack of long-lasting effects of mitotane adjuvant therapy in a mouse xenograft model of adrenocortical carcinoma. Mol. Cell. Endocrinol. 381 (1–2), 66–69. doi:10.1016/j.mce.2013.07.023; [Epub 2013 Jul 30]. Fassnacht, M., Terzolo, M., Allolio, B., Baudin, E., Haak, H., Berruti, A., et al., 2012. Combination chemotherapy in advanced adrenocortical carcinoma. N. Engl. J. Med. 366, 2189–2197. Frigeri, C., Tsao, J., Cordova, M., et al., 2002. A polymorphic form of steroidogenic factor-1 is associated with adrenocorticotropin resistance in y1 mouse adrenocortical tumor cell mutants. Endocrinol. 143, 4031–4037. Fassnacht, M., Kroiss, M., Allolio, B., 2013. Update in adrenocortical carcinoma. J. Clin. Endocrinol. Metab. 98, 4551–4564. Gaujoux, S., Hantel, C., Launay, P., Bonnet, S., Perlemoine, K., Lefèvre, L., et al., 2013. Silencing mutated beta-catenin inhibits cell proliferation and stimulates apoptosis in the adrenocortical cancer cell line H295R. PLoS ONE 8 (2), e55743. doi:10.1371/ journal.pone.0055743; [Epub 2013 Feb 7]. Gazdar, A.F., Oie, H.K., Shackleton, C.H., Chen, T.R., Triche, T.J., Myers, C.E., et al., 1990. Establishment and characterization of a human adrenocortical carcinoma cell line that expresses multiple pathways of steroid biosynthesis. Cancer Res. 50 (17), 5488–5496. Guillaud-Bataille, M., Ragazzon, B., de Reynies, A., Chevalier, C., Francillard, I., Barreau, O., et al., 2014. IGF2 promotes growth of adrenocortical carcinoma cells, but its overexpression does not modify phenotypic and molecular features of adrenocortical carcinoma. PLoS ONE 9, e103744. Hantel, C., Jung, S., Mussack, T., Reincke, M., Beuschlein, F., 2014. Liposomal polychemotherapy improves adrenocortical carcinoma treatment in a preclinical rodent model. Endocr. Relat. Cancer 21 (3), 383–394. doi:10.1530/ERC-13-0439; Print 2014 Jun. Hantel, C., Lewrick, F., Reincke, M., Süss, R., Beuschlein, F., 2012. Liposomal doxorubicin-based treatment in a preclinical model of adrenocortical carcinoma.
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Molecular and Cellular Endocrinology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / m c e
Review
Xenograft models for adrenocortical carcinoma Constanze Hantel *, Felix Beuschlein Endocrine Research Unit, Medizinische Klinik und Poliklinik IV, Ludwig-Maximilians-Universität, Munich, Germany
A R T I C L E
I N F O
Article history: Received 12 March 2015 Received in revised form 26 May 2015 Accepted 26 May 2015 Available online Keywords: Adrenocortical carcinoma Xenograft Preclinical animal model
A B S T R A C T
Adrenocortical carcinomas (ACCs) are rare, heterogeneous and very malignant endocrine tumors with a poor prognosis. An important prerequisite to optimize existing therapeutic regimens and to develop novel therapeutic strategies are preclinical disease models. In recent years molecular and genetic profiling of surgical tumor specimen led to the identification of novel interesting markers. However, precise involvement of these markers in tumorigenesis and their functional relevance in therapeutic outcome is still under investigation. Xenograft models are important tools for such functional studies as they bear the potential to mimic the complexity of solid tumors including tumor cells, stroma and blood vessels. Thus, for the successful and safe development of novel therapeutic strategies xenograft models remain to be indispensable experimental tools. Here we provide an overview on currently existing xenograft models for ACC, their tissue origins, establishment, implications as well as limitations. © 2015 Elsevier Ireland Ltd. All rights reserved.
Contents 1. 2. 3.
4. 5. 6.
Introduction ............................................................................................................................................................................................................................................................. Xenograft models of murine origin ................................................................................................................................................................................................................. Xenograft models of human origin ................................................................................................................................................................................................................. 3.1. NCI-H295 ..................................................................................................................................................................................................................................................... 3.2. SW-13 ........................................................................................................................................................................................................................................................... 3.3. Xenograft models based on primary cultures ................................................................................................................................................................................ 3.4. SJ-ACC3 ........................................................................................................................................................................................................................................................ 3.5. Patient-derived tumor xenografts (PDTXs) from adult ACCs .................................................................................................................................................... Cell-culture derived vs tissue-based xenograft models for ACC ............................................................................................................................................................ Other variables to be considered in preclinical ACC models .................................................................................................................................................................. Conclusions .............................................................................................................................................................................................................................................................. References ................................................................................................................................................................................................................................................................
1. Introduction Adrenocortical carcinoma (ACC) is a rare tumor affecting both adults and children with an annual incidence of 0.7–2.0 cases per million population. The disease is highly malignant with only 16–38% of patients surviving more than 5 years after diagnosis (Abiven et al., 2006; Allolio and Fassnacht, 2006; Fassnacht et al., 2013; Libe et al., 2007). The only drug approved for adjuvant ACC treatment as well as for advanced tumor stages is mitotane. Furthermore, a recent prospective randomized interventional trial for late stage ACC patients
* Corresponding author. Endocrine Research Unit, Medizinische Klinik und Poliklinik IV, Klinikum der Universität München, Ziemssenstr. 1, D-80336 Munich, Germany. Tel.: +49 89 4400 52943; fax: +49 89 4400 54467. E-mail address: [email protected] (C. Hantel).
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has demonstrated that combination of the cytostatic drugs etoposide, doxorubicin and cisplatin together with mitotane (EDP-M) resulted in a higher response rate and longer progression free survival in comparison to streptozotocin and mitotane (Fassnacht et al., 2012). However, even in the setting of a clinical study systemic treatments achieved only partial responses and even after radical resection as many as 85% of patients relapse (Allolio and Fassnacht, 2006). Thus, novel treatment options for patients with ACCs are urgently needed, but clinical translation of novel therapeutic strategies often fails indicating that the currently utilized models only poorly predict clinical success (Johnson et al., 2001). One reason for this shortcoming is that only a few cell lines are available thereby lacking patient heterogeneity and specific therapeutic responses of individual tumors (Leibovitz et al., 1973; Logie et al., 2000; Pinto et al., 2013). Secondly, even though these tumor models can be utilized
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as xenografts in vivo and are, thus, indispensable tools, they originate from cell suspensions. There is good evidence that selection during cell culture passages change biologic properties compared to the original patient tumor (Siolas and Hannon, 2013). In fact, genomic instability of cell-lines has been observed during cell propagation so that resulting xenografts can carry alterations in chromosomal arrangements, karyotype and subsequent alterations in gene expression, the presence of differentiation markers and growth rates (Lum et al., 2012). This review aims to provide an overview on commonly used xenograft models for ACC as well as on alternatives and recently achieved progress. 2. Xenograft models of murine origin The first transplantable model for ACC arose in 1951 on a group of inbred mice of the LAF1 strain. Upon exposure to the irradiation of an atomic test bomb explosion 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 developed spontaneously, necropsy of the mouse revealed numerous small lung metastases as proof of the malignant phenotype of this adrenocortical tumor (Fig. 1) (Cohen et al., 1957). The primary tumor was excised, minced and subsequently inoculated intramuscularly in the thigh of male and female LAF1 mice where it displayed a 100% take on-rate in male and 92% in female hosts, respectively (Cohen et al., 1957). The average tumor latency of the original passages ranged between 106 (males) and 133 (females) days, which decreased in further passages down to 16–23 days. Histological examination revealed well vascularized tumors spotted with necrotic areas. In tumor bearing mice profound polyuria and polydipsia were observed beginning with a tumor size of 2–3 cm in diameter. The fact that these symptoms were also detectable in adrenalectomized and gonadectomized animals indicated that these were signs of functional activity of the xenografts. Consequently, a clone of steroid producing tumor cells was established as a continuous cell line named Y1 which retained the ability of tumor formation in the syngeneic LAF1 mouse strain in vivo (Cohen and Furth 1959; Humphreys et al., 1965; Yasumura et al., 1966). The fact of availability of a syngeneic host represents an important advantage of this tumor model as the adrenal xenografts can be maintained in immune competent animals. In the past, this murine xenograft model aided for example in the understanding of the role of ACTH-receptor (Melanocortin 2 receptor (MC2-R)) on adrenocortical growth and tumorigenesis (Zwermann et al., 2005). For this purpose, the human ACTH receptor was introduced in an ACTH unresponsive sub-clone of Y1 cells (Y6 cells (Frigeri et al., 2002)). 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 (Zwermann et al., 2005). 3. Xenograft models of human origin 3.1. NCI-H295 The most commonly used in vitro and in vivo adrenocortical model is that of the human ACC cell line NCI-H295 (Fig. 1). Gazdar et al. reported in 1990 on the establishment and characterization of a continuous human adrenocortical cell line originated from an invasive primary adrenocortical carcinoma (Gazdar et al., 1990). The original tumor was derived from a 48-year-old woman who showed symptoms of weight loss, acne, facial hirsutism, edema, diarrhea and secondary amenorrhea. Diagnostic work-up revealed elevated
levels of serum cortisol as well as urinary cortisol, aldosterone and 17-ketosteroids and a right-sided adrenal mass (Gazdar et al., 1990). After tumor removal a surgical specimen was minced, seeded in micro-well plates and further passaged. The resulting cell line has been demonstrated to retain its ability to produce all of the major adrenal steroids (Gazdar et al., 1990). Injections of 5 × 106 NCIH295 cells revealed the general tumorigenicity of these tumor cells in athymic nude mice. A more detailed characterization of the NCI-H295 xenograft model was followed by Logie et al. (2000). Upon subcutaneous injection of 6 × 106 cells into the flanks of female nude mice tumor take-on rate has been reported in the range of 90% with a medium doubling time of 12 days. Plasma steroid levels have been shown to be elevated in the in vivo model and histological examination demonstrated that morphological features of the xenografts were furthermore comparable to that of the original patient tumor. Moreover, the tumors were characterized by dysregulation of the insulin like growth factor system including an overexpression of IGF2 and IGF-binding protein-2 similar to that demonstrated in primary human tumor specimen. In accordance, a significant inhibition of tumor growth and increase in survival time of NCI-H295 tumor bearing mice could be observed after treatment with IGF1R antagonistic compounds indicating the functional relevance of targeting this dysregulated pathway (Barlaskar et al., 2009; Hantel et al., 2012). Subcutaneous NCI-H295 xenografts have also been utilized upon genetic modification to study the role of beta catenin pathway in the context of adrenocortical tumorigenesis (Gaujoux et al., 2013), for preclinical evaluation of existing clinical treatment regimens (Doghman and Lalli 2013; Hantel et al., 2014) as well as for the development of novel therapeutic strategies (Gaujoux et al., 2013; Hantel et al., 2012; Szabó et al., 2014). 3.2. SW-13 Another example for a preclinical ACC model bases on the subcutaneous injection of SW13 cells which had been established in the 1970s from a non-secreting small-cell carcinoma of the adrenal cortex (Fig. 1) (Leibovitz et al., 1973). 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. Injection of 10 × 106 cells gave rise to the development of SW-13-xenografts in the flank of female nude mice. Upon the established gene therapeutic approach oncolytic effects through replicating adenoviral vectors could be demonstrated that was followed by tumor reduction and significant increase in animal survival (Wolkersdorfer et al., 2002). Utilizing subcutaneous SW-13 xenografts the importance of angiogenic pathways in ACCs could be demonstrated by investigating the anti-tumoral efficacy of the VEGF receptor tyrosine kinase inhibitors sorafenic and everolismus (Mariniello et al., 2012). Another recent example for therapeutic intervention is the identification of nanoparticle albumin-bound paclitaxel (nab-paclitaxel) as a potential treatment for ACC. This compound showed greater antitumoral effects than the adrenolytic substance mitotane in SW-13 tumor bearing mice (Demeure et al., 2012). Following a different approach Zeng et al. reported very recently the enrichment of cancer stem cells from this tumor model and their characterization in three generations of SW-13 xenografts (Zeng et al., 2014). 3.3. Xenograft models based on primary cultures Hornsby and colleagues reported on a technique in which a suspension of bovine or human adrenocortical cells is introduced under the kidney capsule of SCID mice. Upon xenograft establishment these cells can replace the essential steroidogenic functions of the hosts’
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Fig. 1. Schematic illustration of the different existing xenograft models for ACC from cell-line based murine (Y1/Y6) and human (NCI-H295 and SW-13) to the novel tissue based models SJ-ACC3 and MUC-1.
adrenal glands (Thomas et al., 2003). In fact, as structure and function of the transplant are dependent on circulating pituitary hormones from the host transplantation experiments are usually performed in adrenalectomized animals (Thomas et al., 2003). Utilizing this transplantation model Thomas and colleagues could in a series of experiments demonstrate that introduction of telomerase reverse transcriptase (hTERT) into bovine adrenocortical cells resulted in
a tissue phenotype similar to that of un-transfected cells and animals that received these transplants survived despite adrenalectomy. Moreover, the developing xenografts showed no signs of enhanced proliferation rates or of malignant transformation (Thomas et al., 2000). In contrast, forced expression of SV40 large T antigen (SV40 TAg) and oncogenic RasG12V together with hTERT lead to the induction of a malignant phenotype of the transgenic tissue that
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gave rise to a tumor resembling many features of ACC (Thomas et al., 2002). 3.4. SJ-ACC3 Recently, Pinto et al. reported the establishment of the first pediatric tumor model for ACC (Fig. 1) (Pinto et al., 2013). In this case, the original patient tumor derived from an 11-year-old boy with an incidentally found right adrenal mass resected and subsequently confirmed as an ACC. Laboratory tests revealed endocrine functionality even though the patient showed no remarkable signs of virilization or hypercortisolism. Immunohistochemical and Western blot analyses of the patient tumor revealed positivity for inhibin A, keratin 8, synaptophysin, a strong nuclear p53 staining and a Ki-67 index of approximately 60%, while no specific staining was detectable for chromogranin, HMB-45 and S-100. For inoculation of tumor xenografts surgical specimen were implanted as tumor pieces without mincing in CB17 scid−/− mice. Histological examination, DNA fingerprinting and gene expression profiling of developed xenografts, referred as SJ-ACC3, demonstrated pathomorphological similarity with the original patient tumor (Pinto et al., 2013). Even though tissue and not cell-line based, SJACC3 represents a quite standardizable tumor model. To this end, successful re-implantation was feasible in our laboratory following a simple de-freezing protocol (Fig. 1).
well as applicability of stably expressing GFP or luciferase transfectants allowing in vivo imaging approaches (Mariniello et al., 2012). 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. The intra-lienal injection of stably luciferase expressing NCI-H295 cells results for example in the spread of NCI-H295 liver metastases in athymic nude mice. Therefore, a median laparotomy was followed by intrasplenic injection of 5 × 106 stably luciferase transfected tumor cells. A subsequent incubation time of 10 minutes enabled the cells to distribute via the portal vein. Afterwards, mice were splenectomized to avoid the growth of a large primary tumor in the spleen. In vivo bioluminescence imaging has been carried out after intraperitoneal injection of 3 mg luciferin. (Fig. 2A–E; Hantel, unpublished data). However, the development and routinely utilization of such cell-line based orthotopic and metastasis models for ACC is as limited by the complexity of the required surgery (Luconi and Mannelli, 2012). While tissue-based transplantation lacks the possibility of tumor inoculation by an exact cell number, it provides the important advantage of reflecting tumor heterogeneity and no further processing such as mincing and sub-culturing as described for the other available ACC tumor models. Even though cell suspensions as Y1, NCIH295 and SW-13 form xenografts in vivo, recent data suggest that selection pressure in cell cultures changes biologic properties compared to the original patient tumors (Siolas and Hannon, 2013).
3.5. Patient-derived tumor xenografts (PDTXs) from adult ACCs 5. Other variables to be considered in preclinical ACC models One of the most frequently cited reasons for the high failure rate of new therapeutic regimens in oncology is the lack of preclinical models to reflect patient heterogeneity (Johnson et al., 2001; Tentler et al., 2012). As outlined earlier also for ACC only a few cell lines are available (Gazdar et al., 1990; Leibovitz et al., 1973; Yasumura et al., 1966) which furthermore do not reflect heterogeneous functional properties and specific therapeutic response rates of individual tumors. In an attempt to amend the poor availability of preclinical in vivo models and to furthermore facilitate personalized treatment approaches, our workgroup aimed in recent years at the establishment of patient-individual tumor models for adult ACC. Similar approaches have been described for other tumor entities (Malaney et al., 2014; Morton and Houghton, 2007; Ruggeri et al., 2014). For this purpose, pieces of surgical tumor specimen were implanted subcutaneously in athymic NMRI nude mice (Fig. 2F–I). To investigate whether morphological and functional characteristics between tumor samples after mouse engraftment in comparison to the original tumor would be comparable, we started examination of implanted material and original patient tumor by histology and immunohistochemistry (Hantel et al., manuscript in preparation). Moreover, we utilized patient-tumor bearing mice to investigate putative applicability in therapeutic short-term settings (Hantel et al., 2014). In contrast to the majority of the adrenal PDTXs, one of the implanted tumors referred as MUC-1 showed a large increase in tumor size, increasing take-on rates and a decrease in effective take-on times indicating that these xenografts could be utilized as standardizable tumor model for multiple preclinical settings. As the currently existing standard models NCI-H295R and SW-13 of human origin are both cell-line based (Gazdar et al., 1990; Leibovitz et al., 1973), MUC-1 xenografts may be established as the first tissue based human xenograft model for adult ACC in the future. 4. Cell-culture derived vs tissue-based xenograft models for ACC An important advantage of cell-culture derived xenograft models is the putative establishment of orthotopic or metastasis models as
In addition to the origin of tumor xenograft, immune-competence of the host and site of implantation many other variables have to be taken into account for in vivo experiments. These include among others the chosen endpoints, type and timing of drug administration as well as drug metabolism (Luconi and Mannelli, 2012). One example for the large impact of variables in xenograft-studies is the differently reported therapeutic outcome of mitotane treatments in the same xenograft model (NCI-H295R (Barlaskar et al., 2009; Doghman and Lalli, 2013; Lindhe and Skogseid, 2010)). The choice of solvent might represent also a critical issue in xenograft studies. In a very recent experiment, 8 week old male H295R xenografted SCID-mice received intraperitoneal treatments of either mitotane (solved in 10% Tween) or the diluent alone. Six to eight days after continuous administration, all mice of both control and treatment groups (n = 8 each) died unexpectedly. The repetition of this experiment utilizing the same xenograft model but per oral administrations and corn-oil as solvent avoided the previously observed lethality, suggesting that Tween could have been responsible for the sudden death of mice (personal communication with Péter Igaz, Semmelweis University, Budapest, Hungary). Altogether, this observation underlines the importance of standardization not only of the utilized xenograft models, but also for treatment protocols utilized for in vivo experiments. As outlined earlier, also the origin of a specific xenograft model (cell-line based vs. tissue-based) might be of high impact (Lum et al., 2012; Siolas and Hannon, 2013; Tentler et al., 2012). Moreover, while tremendous technological advances led to important insights into the molecular and genetic determinants that drive human cancers in general and ACC in particular, successful translation into effective therapeutic strategies is hampered by the difficulty in predicting therapeutic outcome in the context of different mutational backgrounds (Assie et al., 2014; Nardella et al., 2011). RNA-knockdown approaches (Gaujoux et al., 2013; Guillaud-Bataille et al., 2014) are first steps taken these mechanisms into account, but tumor models reflecting different genetic backgrounds within ACC are urgently needed to improve the clinical prediction of xenograft models for ACC.
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Fig. 2. Exemplary pictures of the surgery (A), intra-lienal injection and splenectomy necessary for the induction of NCI-H295 liver metastasis and follow up utilizing in vivo bioluminescence imaging (D and E). Patient tumor (F) and prepared tissue specimen for subsequent subcutaneous implantation in the neck of athymic nude mice (H and I).
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6. Conclusions Preclinical xenograft models of human cancers are important tools for the investigation of tumorigenesis and the development of novel anti-cancer agents. Even though indispensable for preclinical drug development also these tumor models have specific limitations and variables such as tumor origin, sub-culturing, genetic background, immune-competence of the murine host, hormonal activity or site of implantation. These have to be individually taken into account for the design of preclinical studies on ACC. References Abiven, G., Coste, J., Groussin, L., Anract, P., Tissier, F., Legmann, P., et al., 2006. Clinical and biological features in the prognosis of adrenocortical cancer: poor outcome of cortisol-secreting tumors in a series of 202 consecutive patients. J. Clin. Endocrinol. Metab. 91, 2650–2655. Allolio, B., Fassnacht, M., 2006. Clinical review: adrenocortical carcinoma: clinical update. J. Clin. Endocrinol. Metab. 91, 2027–2037. Assie, G., Letouze, E., Fassnacht, M., Jouinot, A., Luscap, W., Barreau, O., et al., 2014. Integrated genomic characterization of adrenocortical carcinoma. Nat. Genet. 46, 607–612. Barlaskar, F.M., Spalding, A.C., Heaton, J.H., Kuick, R., Kim, A.C., Thomas, D.G., et al., 2009. Preclinical targeting of the type I insulin-like growth factor receptor in adrenocortical carcinoma. J. Clin. Endocrinol. Metab. 94 (1), 204–212. doi:10.1210/jc.2008-1456; [Epub 2008 Oct 14]. Cohen, A.I., Furth, J., 1959. Corticotropin assay with transplantable adrenocortical tumor slices: application to the assay of adrenotropic pituitary tumors. Cancer Res. 19, 72–78. Cohen, A.I., Furth, J., Buffett, R.F., 1957. Histologic and physiologic characteristics of hormone-secreting transplantable adrenal tumors in mice and rats. Am. J. Pathol. 33, 631–651. Demeure, M.J., Stephan, E., Sinari, S., Mount, D., Gately, S., Gonzales, P., et al., 2012. Preclinical investigation of nanoparticle albumin-bound paclitaxel as a potential treatment for adrenocortical cancer. Ann. Surg. 255, 140–146. Doghman, M., Lalli, E., 2013. Lack of long-lasting effects of mitotane adjuvant therapy in a mouse xenograft model of adrenocortical carcinoma. Mol. Cell. Endocrinol. 381 (1–2), 66–69. doi:10.1016/j.mce.2013.07.023; [Epub 2013 Jul 30]. Fassnacht, M., Terzolo, M., Allolio, B., Baudin, E., Haak, H., Berruti, A., et al., 2012. Combination chemotherapy in advanced adrenocortical carcinoma. N. Engl. J. Med. 366, 2189–2197. Frigeri, C., Tsao, J., Cordova, M., et al., 2002. A polymorphic form of steroidogenic factor-1 is associated with adrenocorticotropin resistance in y1 mouse adrenocortical tumor cell mutants. Endocrinol. 143, 4031–4037. Fassnacht, M., Kroiss, M., Allolio, B., 2013. Update in adrenocortical carcinoma. J. Clin. Endocrinol. Metab. 98, 4551–4564. Gaujoux, S., Hantel, C., Launay, P., Bonnet, S., Perlemoine, K., Lefèvre, L., et al., 2013. Silencing mutated beta-catenin inhibits cell proliferation and stimulates apoptosis in the adrenocortical cancer cell line H295R. PLoS ONE 8 (2), e55743. doi:10.1371/ journal.pone.0055743; [Epub 2013 Feb 7]. Gazdar, A.F., Oie, H.K., Shackleton, C.H., Chen, T.R., Triche, T.J., Myers, C.E., et al., 1990. Establishment and characterization of a human adrenocortical carcinoma cell line that expresses multiple pathways of steroid biosynthesis. Cancer Res. 50 (17), 5488–5496. Guillaud-Bataille, M., Ragazzon, B., de Reynies, A., Chevalier, C., Francillard, I., Barreau, O., et al., 2014. IGF2 promotes growth of adrenocortical carcinoma cells, but its overexpression does not modify phenotypic and molecular features of adrenocortical carcinoma. PLoS ONE 9, e103744. Hantel, C., Jung, S., Mussack, T., Reincke, M., Beuschlein, F., 2014. Liposomal polychemotherapy improves adrenocortical carcinoma treatment in a preclinical rodent model. Endocr. Relat. Cancer 21 (3), 383–394. doi:10.1530/ERC-13-0439; Print 2014 Jun. Hantel, C., Lewrick, F., Reincke, M., Süss, R., Beuschlein, F., 2012. Liposomal doxorubicin-based treatment in a preclinical model of adrenocortical carcinoma.
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