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Molecular and Cellular Endocrinology
journal homepage: www.elsevier.com/locate/mce
Metodutse and Celular Endocrinology
Review
Xenograft models for preclinical drug testing: Implications for adrenocortical cancer
Michaela Luconi *, Massimo Mannelli
Endocrinology Unit, Dept. of Clinical Physiopathology, DENOTHE Center of Excellence for Research, Transfer and High Education, University of Florence, Florence 50139, Italy
ARTICLE INFO
Article history:
Available online 26 October 2011
Keywords:
In vivo animal model
Adrenocortical carcinoma
IGF system
Xenograft
Orthotopic
ABSTRACT
Adrenocortical carcinoma (ACC) is a very rare but aggressive tumor, whose biological and cellular fea- tures and processes underlying the development, progression and metastatic evolution are still obscure. Despite many attempts to use general cytostatic and cytotoxic drugs, the only available drug therapy for advanced ACC is still represented by mitotane (MTT). However, the mechanism of action of this adreno- lytic derivative of the pesticide DDT has still been poorly characterized. In this context, the development of more specific drugs for ACC treatment is based on the knowledge of the molecular pathways involved in the tumor growth. Xenograft models for the screening of such drugs at preclinical levels is mandatory. In the first part of this review, we will summarize the “pro” and “con” of the different xenograft models available for anticancer drug testing in different types of tumors in general and in the last part, we will focus on the preclinical evidence obtained so far with the use of such models applied to drug screening for anticancer effects in ACC.
@ 2011 Elsevier Ireland Ltd. All rights reserved.
Contents
1. Introduction
71
2. Xenograft models
72 73
3. Orthotopic xenograft
4. Endpoints
5. Follow-up of xenograft tumors
73 73 74 74
6. Pharmacology and pharmacokinetics in xenograft models
7. ACC xenograft models for screening anticancer drug activity
8. Conclusion 76
Acknowledgments 76
References
76
1. Introduction
Adrenocortical carcinoma (ACC) is a rare and aggressive endo- crine tumor with an annual estimated incidence of 1 out of 4 × 106. Its prognosis is poor because of a limited response to radio/chemotherapy (Allolio and Fassnacht, 2006). At present, the only valuable option for ACC cure is an early diagnosis followed by total surgical resection of the tumor. Prognosis also depends on the tumor stage at surgery: in fact, overall mean survival rate at 5 years is 16-38% (Allolio and Fassnacht, 2006), but in case of
metastatic disease (stage IV), survival rate at 5 years drops to less than 10% (Fassnacht et al., 2009). Mitotane is the only specific phar- macological treatment indicated for metastatic ACC (Luton et al., 1990; Terzolo et al., 2007) either alone or in combination with other chemotherapic drugs, but its use has led to variable and often dis- appointing results (Khan et al., 2000; Berruti et al., 2005). Therefore, the development of new drugs specific for ACC to be eventually combined with mitotane (Barlaskar et al., 2009) is mandatory.
The development of novel specific drugs relies on several con- secutive steps such as molecular biology studies investigating on the mechanisms underlying malignant tumor transformation and progression, in vitro studies on cell lines and in vivo experiments on animal models to test the efficacy of potentially effective new drugs. ACC xenograft models represents an obligatory step in this sequential drug validation.
* Corresponding author. Address: Dept. Clinical Physiopathology, University of Florence, Viale Pieraccini 6, 50139 Florence, Italy. Tel .: +39 055 4271369; fax: +39 055 4271371.
A. SUBCUTANEOUS ACC XENOGRAFT MODEL
1. GFP-transfected ACC cells
TUMOR EXPANSION THROUGH MULTIPLE MOUSE PASSAGES
2. ACC cells
3. ACC
B. ORTHOTOPIC ACC XENOGRAFT MODEL
1. GFP-transfected ACC cells
TUMOR EXPANSION THROUGH MULTIPLE MOUSE PASSAGES
2. ACC cells
3. ACC
In general, the cost of the initial phases of screening of drugs, the majority of which doomed to be further abandoned, is one of the main contribute to the total budget required for drug develop- ment, estimated to be around 400 million USD/molecule (DiMasi et al., 2003). Therefore, the sooner the drug is found to be ineffective or toxic, the less is the overall costs of new agent development.
2. Xenograft models
Since 1969 when the first evidence of human tumor growth in immunodepressed mice has been published (Rygaard and Povlsen, 1969), human tumor xenografts in athymic or in severe combined immunodeficient (SCID) mice have been developed for the main tumor types and represents the major model for preclinical drug development and screening.
Xenografts can be established either by direct implant of patient biopsy or by inoculation of human tumor cell lines (Fig. 1). More- over, from the initial model of subcutaneous implant (Fig. 1A), also the orthotopic model, in which the tumor/tumor cells are im- planted in the organ the tumor derives from (Fig. 1B), has been achieved. One of the main contributes of testing anticancer drug efficacy in xenografts is to compare the drug activity in the patient with the effects in vivo in the xenograft obtained with the patient tumor as well as in vitro in the established parallel cell lines. A large panel of xenografts obtained from several types of tumor has been used to compare drug response between the xenograft
and the individual patient (Fiebig and Burger, 2002), resulting in a xenograft potency greater than 90% in predicting correctly the clinical response. The model potency to predict clinical activity has been validated so far for cytotoxicity, as demonstrated by the extensive retrospective analysis from the NCI, where both Phase II and xenograft data were available for 39 screened compounds (Johnson et al., 2001). In this study, preclinical activity in at least 33% of the xenografts tested predicted for clinical activity (re- sponse in at least two different tumor types in Phase II trials). Several compounds tested with a positive response in such study are currently used standard chemotherapics (e.g., paclitaxel, doxorubicin).
A more preclinical screening approach for anticancer molecules is to compare the response obtained in in vitro cell lines and in the corresponding xenografts. However, it is not only very difficult to obtain cell lines from xenografts, but for some tumors including ACC, it is even harder to obtain primary cultures or cell lines, resulting in the use of the very few available lines (H295, H295R, SW13) to compare in vivo and in vitro effects.
In the past, several anticancer compounds have been developed, based on their general cytotoxic or cytostatic activity on different tumors (e.g. alkylating agents) obtained in animal models. Nowa- days, the majority of new drugs are directed to interfere with spe- cific molecular pathways that impact tumor biology. Thus, when using a xenograft model, the molecular characterization of the im- planted cells or tumor is mandatory to confirm the maintenance of the drug targets in the engrafted tumor. Gene expression profiling
and proteomic/metabolomic analyses of the graft tumor compared to the original biopsy along with the follow up of the tumor re- sponse to the drug is now feasible for helping molecularly targeted therapy (Table 1).
3. Orthotopic xenograft
The orthotopic xenograft model has been set up to overcome some of the main limits of the subcutaneous xenograft model. In orthotopic xenografts, the human tumor cell suspension or the his- tologically intact tumor fragment are microsurgically inserted in their natural location in the organ of origin (Fig. 1B). Since the first report of human metastatic colon cancer model (Fu et al., 1991), orthotopic xenografts have been developed for nearly all human solid tumors (Hoffman, 1999). Indeed, a different preclinical drug activity has been reported whether screened on subcutaneous or orthotopic xenografts (Bibby, 2004; Fidler and Ellis, 1994). One of the main reasons for such discrepancy may relay on the contribute played by the non physiological subcutaneous microenvironment (stroma, vascularization, macrophages, etc.) to tumor development and progression as well as to the drug response. This point is par- ticularly evident for the metastatic potential, which is poorly ex- pressed in the subcutaneous environment, thus potentially affecting tumor response to drug. Interestingly, tumor metastatic potential is deeply influenced by the architecture of the tumor mass, as suggested by the scarce metastatic rate observed when using orthotopic transplantation of tumor cell suspension com- pared to implantation of the original tumor (Hoffman, 1999). Large cohorts of xenograft mice for drug screening could be easily con- structed from a single patient tumor specimen by an amplification procedure (Fig. 1B). This method consists of a primary orthotopical implantation of small tumor pieces obtained from the biopsy in at least 20 isogenic immunodepressed recipient mice. The tumors are thus orthotopically expanded in this first passage, then collected, fragmented and re-implanted orthotopically in a larger cohort of isogenic mice for drug reliable screening. This serially “sub implan- tation “procedure is not only very efficient (nearly 100% implanta- tion rate) but it also results in an extreme stability of the original tumor phenotype (Hoffman, 1999).
One of the main challenging and limiting aspects of producing orthothopic xenografts is the complexity of the surgery. In fact, variability in the taking rate of the tumors mainly depends on the organ to be implanted. This is also the case of surgery in mouse adrenal gland in which the small size of the organ makes the sur- gery and the implant challenging. Another critical point when using tumor cells for adrenal orthotopic xenografts is to retain the implanted cells inside the organ. Hornsby’s group recently developed an optimized protocol for orthotopic adrenal transplan- tation of ACC cells in immunodepressed mice (Cardoso et al., 2010).
| Variable | Type |
|---|---|
| Origin of tumor graft | Cell lines or patient tumor |
| Site of implantation | Subcutaneous or orthotopic |
| Type of endpoints | Tumor growth, molecular pathways, metastases |
| Drug administration | Gavage, bolus, intraperitoneal, intravenous, agent formulation |
| Drug dosage | Toxic effects, non therapeutic range |
| Timing of drug administration | Regression or prevention studies |
| Type of drug metabolism | Liver, kidney |
| Bioavailability to the tumor | Drug metabolism, graft take rate |
| Xenograft engraftment | Mouse metabolism, mouse environment of the human tumor |
| Involvement of | Athymic or SCID mice |
| immunocompetent system |
In order to minimize cell leakage from the injection site, the authors injected the cells in a fibrinogen/thrombin suspension, which immediately clotted after injection, trapping cells inside the adrenal. Evidently, a number of technical challenges have to be met before orthotopic ACC models could be utilized routinely in a preclinical setting.
4. Endpoints
When choosing a xenograft model to test anticancer drug activ- ity two main kinds of endpoints can be selected: modulation of tumor growth or impact on molecular targets. The effect on tumor growth can be expressed as the percentage of treated versus control tumor weight or dimension calculated on each day of fol- low-up (%T/C, where the optimal %T/C is the lowest calculated ratio observed over time corresponding to the maximal effect of the drug), tumor growth delay, net logarithmic cell kill, median days to reach an assessed tumor mass or a specified tumor doubling or tumor regression. Moreover, for tumor models which enable metastatic spread, also the number of metastatic lesions is a com- mon endpoint for testing drug anticancer activity. Drug-related deaths and body weight loss are used as toxicological parameters.
Two different endpoints of cell growth could be considered in xenograft studies depending whether the drug treatment is initi- ated before tumor development or after the tumor nodule ap- peared. The former model enables the study of drug ability to prevent tumor formation (prevention study), whereas the latter model to regulate tumor growth once the tumor has developed (regression study).
5. Follow-up of xenograft tumors
Subcutaneous tumor growth can be followed in general by mea- suring tumor diameter by a caliper during the treatment period. Conversely, orthothopic and metastatic/disseminated malignant tumor can be monitored in vivo only by non invasive imaging tech- niques. In particular, recently developed new imaging techniques applied to small animals, allows not only to follow tumor growth, but even to measure a variety of tumor-related variables, which are crucial to better define the anti-tumor effect of the tested drug.
Essentially, two types of marker techniques associated with noninvasive microimaging revelation systems are used:
(1) Injection of tumor cell lines preventively labeled by engi- neered expression of reporter proteins have been obtained using green fluorescent protein (Hoffman, 2002), beta-galac- tosidase (lacZ) gene (Lin et al., 1990) or firefly luciferase (El Hilali et al., 2002). This technique allows to follow cell dis- semination and tumor response to treatment with several types of fluorescence imaging instruments, and is essential to distinguish tumor cells from the host stromal, vascular and infiltrating compartments.
(2) In vivo tumor cell labeling by using radioactive or fluorescent probes which are selectively captured or selectively interact with specific structures of the tumor. Magnetic resonance imaging and positron emission tomography/SCAN can also be used to visualize tumor and metastasis progression, although with different sensitivity and resolution (for rev see Lyons, 2005; Henriquez et al., 2007).
(3) Moreover, being an endocrine tumor, ACC burden and the response to the treatment can be followed up by monitoring the blood levels of hormones specific of human adrenal activ- ity (cortisol) as well as the variation in the levels of adreno- cortical hormones (adrenal androgens) in the implanted mice versus the sham-operated controls.
6. Pharmacology and pharmacokinetics in xenograft models
Despite the enormous efforts put in preclinical studies for anti- cancer drug screening on the basis of xenograft models, very few efficacious agents are clinically relevant at well-tolerated doses (Suggitt and Bibby, 2005). Moreover, many compounds with prom- ising activity in xenografts when progressed to the clinic revealed disappointing results, fueling a controversial debate on the general value of xenograft screening.
Major limitations in the predictability of tumor treatment re- sponse can be intrinsic to xenograft model itself, because of species differences in pharmacokinetics of the drug, leading to inappropri- ate drug dosing or non physiological xeno interaction between hu- man and mouse cells (Table 1). The xenograft seems in some cases to no longer retain the original molecular and histological charac- teristics of the patient tumor, when using cell lines cultured in vitro for long time. In contrast, when using fresh tumor explants, there is a better correlation between drug activity in the xenograft and clinical response (Fiebig et al., 2004). This is true, in particular, when the xenografts have been molecularly characterized, con- firming the expression of specific molecular markers by the patient tumor (Fichtner et al., 2004).
In general, the main problem in translating data obtained from xenograft mice to patients, is represented by the fact that the “liv- ing machinery” which sustains tumor growth and metabolizes and delivers the drug to the graft is a mouse and even an immunode- pressed one. Thus, this non physiological situation may affect the results at two levels: (1) drug bioavailability, depending on its administration, absorption, metabolism and delivery; (2) interac- tion between human cancer cells in the graft and the mouse stro- ma, vasculature and infiltrating cells (Table.1).
In order to predict pharmacology and pharmacokinetics of an agent in humans it is necessary to take into account the putative differences between men and mice and the conventional approach for that is to evaluate the absorption, distribution, metabolism and excretion properties of the compound (ADME). Conventional allo- metric scales (CAS) are available for predicting interspecies vari- ables in several parameters (Mahmood, 1999). Animal data are scaled-up to predict pharmacokinetics and pharmacodynamics in men using this empirical allometric method. However, this ap- proach can be generally used for compounds metabolized and ex- creted by the kidney (Ritschel et al., 1992). For compounds metabolized in the liver and excreted in the bile other models may allow a better prediction, taking into account physiological properties of the species and biochemical characteristics of the drugs (Poulin and Theil, 2002). This point is particularly important for drugs such as mitotane, which is an extremely potent activator of hepatic mitochondrial enzymes. Mitotane has been demon- strated to produce a strong and long-lasting inducing effect on the mitochondrial enzyme CYP3A4, which metabolizes and acti- vates many anticancer drugs. This effect may affect the bioavail- ability of these compounds, resulting in a reduced exposure of the tumor, as recently shown in two ACC patients who had been simultaneously treated with the tyrosine kinase inhibitor sunitinib and mitotane (van Erp et al., 2011). In this context, it is mandatory to evaluate the pharmacokinetic of different drugs to be combined, in order to avoid possible negative interference and to potentiate possible positive cooperation. Interestingly, some of these enzymes are not only involved in activation of mitotane but even represent a target for mitotane action in the adrenal (Lindhe et al., 2002; Vey- tsman et al., 2009). On the other hand, in a large prospective study, Berruti and colleagues (Berruti et al., 2005) demonstrated that combination of mitotane with other chemotherapics (etoposide, doxorubicin and cisplatin = EDP) in advanced adrenocortical carcinoma, resulted in a significant increase in response rate and
survival compared with either mitotane alone or other chemother- apy schemes, (Van Slooten and van Oosterom, 1983; Decker and Kuehner, 1991; Schlumberger et al., 1991; Bukowski et al., 1993; Bonacci et al., 1998; Khan et al., 2000; Williamson et al., 2000; Abraham et al., 2002). This enhancing effect could partially be ex- plained by the ability of MTT to hamper tumor resistance mecha- nisms such as chemotherapic extrusion from the cell.
Therapeutic plasma levels of mitotane between 14 and 20 mg/L have now been considered protective from ACC recurrence without being toxic, as they significantly lengthen the time to recurrence (Hermsen et al., 2011). However, since MTT and its metabolites can accumulate in several tissues, and in particular in the fat (Hermansson et al., 2008; De Francia et al., 2006), it could be worth reconsidering the possibility that MTT plasma levels do not linearly correlate with MTT accumulation at the adrenal level where it can directly affect primary tumor, or in other organs where it may af- fect the metastatic process.
Another very critical point in MTT bioavailability in the xeno- graft is the way of administration. Indeed, a different bioavailabil- ity is associated to oral or intraperitoneal administration.
7. ACC xenograft models for screening anticancer drug activity
The first ACC xenograft model to be molecularly characterized for therapeutic screening was obtained by subcutaneous injection of H295 cell line in female nude (nu/nu) mice (Logié et al., 2000). The tumor implant rate was nearly 100%, the tumor was detectable (4-5 mm3) after about 6 weeks and tumorigenicity was main- tained approximately for 2 years after five passages. As expected, the model was not able to produce metastases. It was deeply char- acterized and shown to maintain the main histological and bio- chemical features of the original adrenocortical carcinoma. In particular, the xenograft reproduced the dysregulation in the IGF system found in malignant human tumors and in the H295R cell line. Interestingly, the xenograft was demonstrated to produce and secrete steroids and human IGFBP-2. IGFII/IGF-IR system upregulation has been demonstrated to be one of the main features of adrenocortical cancers (Fig. 2, Gicquel et al., 1994; Logie et al., 2000). Consequently, the most important signaling pathways downstream of the activated receptor have been investigated first in ACC cell models (Cantini et al., 2008; Barlaskar et al., 2009) and represent an interesting and promising target for anti-tumor drugs.
A previous paper reported the effect on ACC xenograft tumor growth of gossypol, a natural product of cottonseed oil which af- fects membrane-associated enzyme activity. In SCID mice, this compound was able to slow down the appearance of tumor in pre- vention experiments as well as to significantly inhibit the tumor growth when administered after the tumor appearance (Wu et al., 1989). However, the xenograft was obtained by subcutane- ous injection of SW13 cells and neither the histology nor the molecular properties of the developed tumor had been character- ized, making it difficult to define the specific mechanism of action of this compound. Indeed, currently used ACC xenografts are ob- tained by H295R injection, as this is so far the only well established and characterized reproducible model.
Since then, the ACC xenograft model obtained by subcutaneous injection of H295 cell line has been extensively used in studies evaluating new and established anticancer drugs. Taking into ac- count that IGFII/IGF-IR signaling is so far the most clearly dysregu- lated pathway in ACC, inhibitors of this axis have been tested first in in vitro experiments and then in in vivo xenograft models. Bar- laskar and colleagues (Barlaskar et al., 2009), reported that two dif- ferent inhibitors of the IGF-IR (IMC-A12 antibody and NVP- AEW541 inhibitor) significantly affected H295R cell proliferation
IGF-II
IGF-
IGF-IIR
IGF-IR
PDK1
PI3K
IRS-I
P
P
SOS
SHC
P
Ras
NO SIGNALING
AKT
P
PIP 3 PIP 2
P
P
BAD/Bcl2
SGK1
mTORC2
mTORC1
Raf
P
mTOR
mTOR
APOROSIS
RAPTOR
MEK
RICTOR
P
P
S6
S6K
CELL SURVIVAL
PROTEIN
ERK 1/2
SYNTHESIS
CELL PROLIFERATION
Fig.2
in vitro and that the monoclonal antibody inhibited tumor growth in the xenograft by interfering with Akt phosphorylation down- stream of the IGF-IR and reducing VEGF and tumor vascularization. Moreover, when the antibody was combined with intraperitone- ally administered MTT, the inhibition of tumor growth was enhanced, causing an additive effect higher than the small one pro- duced by MTT alone. Similarly, the combined therapy was much more effective than MTT alone in reducing VEGF expression and microvessel density in the tumor. Curiously, in the xenograft, tu- mor growth inhibition was only tested with the antibody alone or combined with MTT, while the in vitro effects were obtained by using NVP-AEW541. Based on these results, two ongoing clini- cal trials on advanced adrenocortical cancer, using IGF-IR inhibi- tors (IMC-A12 versus MTT and OSI-906 versus placebo) are actively recruiting ACC patients.
Other compounds acting as more downstream inhibitors in the signaling cascade of the IGF-IR, have been tested in ACC xenograft models for their anticancer activity. Among them, the mammalian target of rapamycin (mTOR) signaling is strictly interconnected with the IGF pathway. In fact, the mTOR and IGF-1/AKT pathways are two evolutionarily conserved pathways that play critical roles in the reg- ulation of cell proliferation, survival and energy metabolism (Fig. 2, Feng and Levine, 2010). Because of the effects of mTOR signaling on tumor cell growth and proliferation, mTOR inhibitors derived from the macrolide rapamycin are now used in the chemotherapy of dif- ferent cancers (Guertin and Sabatini, 2007). Everolimus, a mTOR inhibitor widely used in the clinic as immunosuppressive agent, can dose-dependently inhibit in vitro cell growth of different ACC cancer cell lines and of ACC primary cultures (Doghman et al., 2010). When ACC xenograft mice were orally treated with everoli- mus at 10 mg/Kg/day, as soon as the tumor was detectable, there was a significant reduction of cell tumor mass compared to the
placebo-treatment, starting from the very first days (Doghman et al., 2010). This effect was strictly correlated with the ability of ever- olimus to potently reduce phospho-RPS6 expression and blood ves- sel bed extension in the tumor (Doghman et al., 2010). These data strongly suggest that angiogenesis is one of the main processes in- volved in the IGF system support to tumor growth and that therefore should be adequately targeted by different therapies.
To achieve more potent anticancer effects acting on the proan- giogenic properties of the tumor, a combined therapy using everol- imus and the tyrosine kinase inhibitory sorafenib was tested in both SW13 and H295 xenograft models. These xenografts have been obtained by subcutaneous injection of luciferase-transduced H295R and SW13, to allow tumor growth analysis by non invasive bioluminescence imaging (Mariniello et al., 2011). The combined action of the two molecules have been first evaluated in vitro on SW13 and H295 cell growth. Interestingly, sorafenib has already been demonstrated to inhibit growth of different tumors, by di- rectly inhibiting Raf-MEK-ERK signaling and multiple receptor tyrosine kinase activity, including VEGFR-2, (Wilhelm et al., 2006, 2004). In xenograft mice, the two drugs in monotherapy only slightly affected tumor growth and animal survival, with very little effect in H295R xenograft model for sorafenib treatment alone. Conversely, combination of the two drugs significantly affected tu- mor growth and increased animal survival in both models, sug- gesting a synergistic activity (Mariniello et al., 2011).
The synergistic effects of the combination therapy might de- pend not only on a complementary direct action of the two drugs, but also on the effect that one drug might possibly exert on the bio- availability of the other.
The PPARgamma ligands rosiglitazone (RGZ) and pioglitazone, used as insulin-sensitizer in type 2 diabetes therapy, have been demonstrated to induce apoptosis and differentiation as well as
inhibition of proliferation and invasiveness in human ACC cell lines (Ferruzzi et al., 2005; Betz et al., 2005; Cantini et al., 2008).
Oral administration of RGZ 5 mg/day to xenografts obtained by subcutaneous injection of H295R, significantly reduced tumor growth compared to placebo treated controls, with a maximal reduc- tion already achieved at 17 day treatment (1 - T/C = 75.4%, 43.7- 93.8% CI) (Luconi et al., 2010). Notably, RGZ inhibitory effects were also histologically validated in tumor extracts, showing inhibition of cell mitosis, reduction in ki67 staining and induction of apoptosis. In addition, at the molecular level, the tumors of RGZ-treated mice showed a significantly higher reduction in the expression of angio- genic (human VEGF and murine CD31), proliferative (BMI-1) and anti-apoptotic (Blc-2) markers than the tumors of the control group. The RGZ dose used was significantly lower than the one currently administered in xenograft models (Heaney et al., 2002; Cellai et al., 2010), resulting in less toxic effects. Interestingly, RGZ effects are somehow interfering with IGF-IR downstream signaling, since both ERK and PI3K/Akt transduction cascade was significantly affected by RGZ in SW13 and H295R cultures, with no additional effects com- pared with NVP-AEW541 inhibitor (Cantini et al., 2008, Fig. 2).
All these findings indicate that all the drugs screened so far in ACC xenograft models interfere with the main tumor activating pathway, represented by the IGFII/IGF-IR signaling. A higher inhibi- tion might be hypothetically achieved by the association of the above proposed compounds with different drugs affecting parallel signaling pathways not involving the IGF-IR axis.
Finally it is worth taking into account the limits when interpret- ing the results, that the immunodeficiency of mice used as xeno- graft model might somehow modify the antitumoral and antiangiogenic response to these drugs in these animals.
8. Conclusion
In conclusion, both the subcutaneous and the orthotopic xeno- graft model can still be regarded as a valuable and predictive tool in the era of target-driven anticancer drug discovery if used appro- priately and taking into account the limits of this model. In partic- ular, in order to improve the affordability of the system, the tumor should be always characterized in the xenograft to ensure that the molecular drug targets are maintained also in the xenograft tumor growth. Moreover, results obtained on tumor growth in the xeno- graft model must be always integrated and evaluated in the light of pharmacokinetic and pharmacodynamic investigation of the drug in that system (Suggitt and Bibby, 2005; Kelland et al., 1992; Kel- land, 2004; Sausville and Feigal, 1999; Kerbel, 2003; Peterson and Houghton, 2004). These preclinical in vivo models represent an even more valuable tool for rare and aggressive tumors such as ACC, not only for the screening of potential drugs, but also for investigating the molecular mechanisms underlying tumor growth and progression, whose knowledge will enable us to design more targeted and efficacious anticancer molecules.
Acknowledgments
We thank Dr. Giulia Cantini (University of Florence) for graph- ical help.
The research leading to these results has received funding from the Seventh Framework Programme (FP7/2007-2013) under Grant Agree- ment No. 259735 (ENS@T-CANCER), from FIRB No. 2010RBAP1153LS and from Malattie Rare Regione Toscana 2009-10FF11.
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