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. Author manuscript; available in PMC: 2014 Apr 8.
Published in final edited form as: Int J Androl. 2011 Jun 9;34(4 0 2):e278–e287. doi: 10.1111/j.1365-2605.2011.01160.x

Spontaneous metastasis in mouse models of testicular germ-cell tumours

J L Zechel *, G T MacLennan †, J D Heaney *, J H Nadeau *
PMCID: PMC3979466  NIHMSID: NIHMS344453  PMID: 21651572

Summary

Testicular germ-cell tumours (TGCTs) are the most common cancer in young men; the incidence is increasing worldwide and they have an unusually high rate of metastasis. Despite significant work on TGCTs and their metastases in humans, absence of a mouse model of spontaneous metastasis has greatly limited our understanding of the mechanisms by which metastatic potential is acquired and on their modes of dissemination. We report a new model of spontaneous TGCT metastasis in the 129 family of mice and provide evidence that these are true metastases derived directly from primary testicular cancers rather than independently from ectopic stem cells. These putative metastases (pMETs) occur at similar frequencies among TGCT-affected males in six genetically distinct TGCT-susceptible strains and were largely found in anatomical sites that are consistent with patterns of TGCT metastasis in humans. Various lines of evidence support their pluripotency and germ-cell origin, including presence of multiple endodermal, mesodermal and ectodermal derivatives as well as cells showing OCT4 and SSEA-1 pluripotency markers. In addition, pMETs were never found in males that did not have a TGCT, suggesting that metastases are derived from primary tumours. Finally, pMETS and primary TGCTs shared several DNA copy number variants suggesting a common cellular and developmental origin. Together, these results provide the first evidence for spontaneous TGCT metastasis in mice and show that these metastases originate from primary TGCTs rather than independently from ectopic stem cells.

Keywords: metastasis, mouse model, testicular cancer

Introduction

Testicular germ-cell tumours (TGCTs) are the most common cancer affecting young men between the ages of 15 and 40 years. Although representing only 1.0–1.5% of all cancers (Akbarian et al., 1995), incidence has increased steadily in the last 30 years (Bosl & Motzer, 1997) and currently TGCTs account for nearly 60% of all cancers in men aged 20–40 years (Di et al., 2005). Although the cure rate is high, presence of metastases reduces survival rates. Interestingly, TGCTs have a disproportionately high number of metastases compared with other tumours, averaging 5.8 metastases per primary tumour (Anant & Davidson, 2001; De Giorgi et al., 2008). Approximately 30% of TGCT patients have metastases at the time of diagnosis (Powles et al., 2005), and 15–20% of patients have subclinical metastases in stage 1 seminoma (Benne et al., 1986), which is the most common presentation (Rodriguez et al., 1992).

Metastasis is an important factor in TGCTs, directly affecting treatment modality, tumour surveillance, and survival. Treatment involves surgery and chemotherapy with BEP (bleomycin, etoposide and cisplatin), which together carries a mortality risk of 2.3–4.5% (Fossa et al., 1998; Williams et al., 1987). Resistance to cisplatin occurs in 20% of patients with metastatic disease, further complicating treatment and worsening prognosis (Piulats et al., 2009). After treatment, patients presenting with metastatic disease have a higher incidence of relapse (Holzik et al., 2008) and lower survival rate (Anant & Davidson, 2001).

Testicular cancer metastases target various tissues, including lymph nodes, lung, liver and spleen (De Giorgi et al., 2008). Differentiation of TGCT metastases leads to morphological diversity, which makes it difficult to distinguish between metastases of primary germ-cell tumours and unrelated secondary cancers (Ulbright, 1999). The OCT4 pluripotency marker is present in 100% of tested primary and metastatic seminomas and embryo-nal carcinomas (Cheng, 2004; Jones et al., 2004; Looijenga et al., 2003) and has become an important diagnostic marker for both primary germ-cell tumours and their metastases (Cheng et al., 2007). OCT4 is a transcription factor associated with the maintenance of pluripotency and is expressed in embryonic cells, germ cells and embryonal carcinoma (EC) cells (Okamoto et al., 1990; Rosner et al., 1990). OCT4 expression is dramatically reduced as both primary tumours and their metastases differentiate (Niwa et al., 2000) and may be absent in cells that possess an EC phenotype (Mueller et al., 2010).

Most mouse models of spontaneous TGCTs are limited to a single genetic background, the 129 family of inbred strains (Stevens & Hummel, 1957), with 5–10% of males affected with a TGCT by 3–4 weeks of age (Maris et al., 2005). Several mutations modify this frequency (Di et al., 2005; Heaney & Nadeau, 2008; Di Cristofano et al., 1998; Kimura et al., 2003). For example, the Ter mutation in the Dead-end1 gene increases the incidence of TGCTs to over 94% in Ter/Ter homozygotes (Clark et al., 2004; Matin et al., 1999). In addition, the 129.MOLF-Chr19 chromosome substitution strain, which has chromosome 19 from the MOLF/Ei inbred strain substituted onto the 129/Sv background, has 82–86% of its males affected with a TGCT (Matin et al., 1999).

Developmental features of TGCT stem cells have led to alternative hypotheses about whether extragonadal germ-cell tumours are true metastases of TGCTs in the testis, or whether they originate independently from ectopic stem cells (Fabre et al., 2004; Fine et al., 1962; Hailemariam et al., 1997; Johnson et al., 1973). Primordial germ cells (PGCs), which are the stem cell of many TGCTs, arise early in development, migrate from the base of the allantois to the urogenital ridge, where fetal gonads subsequently develop (Molyneaux et al., 2001; Upadhyay & Zamboni, 1982). In the 129 inbred strains, PGCs in the fetal gonad transform to EC cells and during the next several weeks develop into TGCTs (Stevens, 1962a, 1967). In some cases, PGCs migrate to ectopic locations instead of the urogenital ridge (Anderson et al., 2000; MacLean et al., 2007; Runyan et al., 2006; Upadhyay & Zamboni, 1982; Zamboni & Upadhyay, 1983) and these cells have been proposed to be the origin for extragonadal germ-cell tumours (Dixon & Moore, 1953; Fine et al., 1962; Hail-emariam et al., 1997). Absence of metastasis in these mouse models has until now precluded tests of these alternative hypotheses about the origins of metastasis and extragonadal germ-cell tumours.

During routine necropsies, we discovered putative metastases (pMETs) in several TGCT susceptible mouse strains. In the literature, only a single instance of a spontaneous mouse TGCT pMET has been described, from a large survey of over 7700 129-TerSv males affected with a primary TGCTs (Stevens, 1973); investigation into that pMET was limited to histological analysis. We therefore undertook a systematic study to estimate the rate and anatomical distribution of pMETs in these strains, to test whether they have the same germ-cell origin as TGCTs (Stevens, 1962b, 1967) and if they were true metastases from a primary TGCT in the testis, or alternatively from germ cells that migrated to ectopic locations where they then transformed to extragonadal germ-cell tumours.

Materials and methods

Mice

129S1/SvImJ (JR002448, previously known as 129/SvImJ) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). 129T1/Sv (known previously as 129T1/Sv-p+ Tyrc-ch Dnd1Ter/J) and 129-Chr 19MOLF CSS (CSS-M19, described previously, Matin et al., 1999) were obtained from our research colony. Mice were maintained in the Case Western Reserve University Animal Resource Center on a 12 : 12-h light : dark cycle and fed Lab Diet 5010 (LabDiet, Richmond, IN, USA). All protocols were approved by the Institutional Animal Care and Use Committee.

Genotyping

DNA for PCR genotyping was extracted from tail tissue. The nucleotide substitution that led to the Ter mutation created a Dde1 site that we used for genotyping (Matsumoto et al., 2006).

Tumour surveys

Male mice between the ages of 3 and 15 months were surveyed for TGCTs and pMETs. pMETs were identified macroscopically and confirmed histologically. Tumour incidence was calculated as the percentage of males with either a unilateral or bilateral TGCT; metastasis incidence was calculated both as the percentage of TGCT-affected males with at least one metastasis, and as the percentage of all males with at least one pMET (not shown).

Tissue processing

Testes and macroscopic pMETs were fixed with 10% formalin for at least 48 h. Tissues were then paraffin embedded and sectioned (5 μm) at the Case Comprehensive Cancer Center's Tissue Procurement and Histology Core facility (TPHC). Haematoxylin and eosin staining was performed in the TPHC facility.

Immunohistochemistry

Paraffin sections (5 μm) were rehydrated and blocked with dilution buffer (1X PBS, 3% BSA, 0.1% NaN3, 0.3% Triton X-100), unconjugated goat anti-mouse IgM (Jackson Immunolabs, West Grove, PA, USA) diluted 1 : 1000 and 5% normal goat serum for 1 h at room temperature, then rinsed in PBS. Sections were incubated with rabbit anti-OCT4 (ab19857; Abcam, Cambridge, MA, USA) antibody diluted 1 : 250 and mouse IgM anti-SSEA1 (ab16285; Abcam) diluted 1 : 50 in dilution buffer with 5% normal goat serum overnight at 4 °C. Sections were washed in PBS, and incubated with DyLight 488 anti-mouse IgM and DyLight 594 anti-rabbit IgG (115-486-020 and 711-516-152, respectively, Jackson Immunoresearch) both 1 : 500 in dilution buffer with 5% normal goat serum for 2 h at room temperature. Nuclei were counterstained with DAPI and sections were visualized using a Leica TCS SP2 AOBS filter-free UV/spectral confocal microscope (Leica Microsystems Inc., Bannockburn, IL, USA).

Array-based CGH

DNA was isolated from normal spleen, primary TGCTs and pMETs using Qiagen DNeasy Blood and Tissue kits (Valencia, CA, USA). In cases where primary TGCTs and pMETs were found in conjunction with normal tissue, tumours were visually dissected to minimize the contribution of normal cells to the tumour sample. Samples were sent to Empire Genomics (Buffalo, NY, USA) where hybridizations were performed with the Agilent 244k mouse aCGH array. CGH Microarray images were processed using Feature Extraction Software (v10.5.1.1) (Agilent Technologies, Palo Alto, CA, USA). DNA gains and losses were identified by importing the Feature Extraction-generated CGH.txt files into DNA Analytics (v4.0.76) (Agilent Technologies), and applying the ADM-1 Aberration Algorithm tool with parameters set at: Threshold range 11.0–23.9, Centralization ON, Bin Size 10 and Centralization Threshold 6.0. Regions of significant gain or loss were annotated to the corresponding chromosomes, cytobands and genes using genome build NCBI36/mm8.

Real-time PCR

Putative metastases were selected for validation with real-time PCR based on the number of DNA copy number changes identified within the pMET and the corresponding primary TGCT. Regions of DNA copy number change were used for real-time PCR validation if three or more aCGH probes were targeted to the area and if there was at least one gene in the segment. Genes used for DNA copy number validation were chosen based on their central location within the altered genomic region. Primer sequences are given in supplemental Table S1. Copy number changes were quantified with the Chromo4 real-time PCR system (MJ Research, Watertown, MA, USA) and the PerfeCTa SYBR Green Supermix kit (VWR Scientific, Radnor, PA, USA) using manufacturer-suggested protocols. Copy numbers were normalized to the POMC gene (Hill-Baskin et al., 2009); normal spleen DNA was used to generate standard curves for each primer set. Cut-off values of 1.5 and below were used to define a copy number value of 1, and values of 2.5 and above were used to define copy number values of 3 and above. As described in previous studies, changes in genomic DNA were considered significant when the copy number (n) was below 1.5 or above 2.5 (Braude et al., 2006; Yang et al., 2010).

Statistical analysis

Chi-square contingency tests were used to test for significant differences in TGCT and metastatic rates between strains. Spearman's correlation test was used to estimate the correlation between the incidence of primary TGCTs and pMETs among strains.

Results

Frequency of males affected with a primary TGCT

We examined a total of 1045 males, ranging in age from 3 to 15 months, from six genetically distinct members of the 129 family of inbred strains. These six groups included the 129S1/SvImJ inbred strain, Dnd1Ter/+ heterozygotes on two backgrounds (129S1 and 129T1), Dnd1Ter/Ter homozygotes on the 129T1 background, KitlSl-J/+ heterozygotes on the 129S1 background, and the 129-Chr19MOLF Chromosome Substitution Strain (CSS). As expected, the frequency of affected males varied widely (Fig. 1a), with rates that were consistent with previous reports (Lam et al., 2004; Matin et al., 1999; Noguchi & Stevens, 1982; Stevens & Hummel, 1957; Youngren et al., 2005).

Figure 1.

Figure 1

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Incidence of TGCTs and pMETs among six TGCT-susceptible strains. (A) Males affected with a primary TGCT. (B) pMETs among males with a TGCT. (C) Anatomical distribution of pMETs in mice and humans.

Frequency of males with a pMET

We found pMETs in each of the six strains surveyed, with the frequency of markers with at least one pMET in TGCT-affected males ranging from 4.5% in the 129/S1 strain to 11.6% in the CSSM19 strain (Fig. 1b). Occur-rence of pMETs did not vary significantly among these strains (χ2 = 2.7, p = 0.7), and there was no correlation between the primary TGCT rate and the metastatic rate (Spearman's r = 0.7, p = 0.23), suggesting that the number of pMETs found in each strain depended only on the absolute number of primary TGCTs found within that strain, and not on the primary TGCT rate in each strain or on the particular genetic attributes of any of the six strains.

Do TGCTs and pMETs arise independently?

An important question concerns whether primary TGCTs and pMETs have independent origins. In particular, if pMETs are derived from primary TGCTs, which would be the case if they are metastases, then pMETs should be found only in males that also have a TGCT. In contrast, independent occurrence would be consistent with separate origins. We tested the hypothesis of independent origins by examining occurrence (numbers) of males with a TGCT or a pMET. Interestingly, pMETs were found only in males that also have a TGCT (χ2 = 61.6, p < 0.0001), suggesting that pMETs are derived from the primary tumour (Table 1).

Table 1.

Test for independent occurrence of TGCTs and pMETs

No pMET pMET present Total
Primary TGCT present 477 56 533
No primary TGCT 556 0 556
Total 1033 56 1089
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Distribution of metastases

We categorized pMETs (n = 63) from our surveys according to secondary tissue sites (Fig. 1c). Regional lymph nodes (renal, inguinal and lumbar lymph nodes) accounted for 38.1% of the sites. Other sub-diaphragmatic sites were peritoneal masses (14.3%), pancreas (12.7%), liver (7.9%) and spleen (6.3%). Super-diaphragmatic metastases in distal lymph nodes (cervical, mediastinal and axillary/brachial) accounted for 17.5% and lung (3.2%) of all metastases. Interestingly, pMETs were not found in adrenal glands or kidneys, which are common sites for metastasis in humans. The wide anatomical distribution of TGCT metastases in our survey is consistent with data for humans (Disibio & French, 2008), suggesting that the source cells for mouse and human TGCT metastases have common biological origins and properties.

Histological composition

To determine whether these pMETs contained cell and tissue types derived from multiple germ layers, which is characteristic of TGCTs and their metastases, we examined their composition histologically. Consistent with the hypothesis that these pMETs originate from germ cells, many contained cells and tissues from multiple germ layers. For example, glandular structures were found in a mediastinal lymph node pMET mixed with less differentiated cells and lined with malignant cells (Fig. 2a,b). A pMET found in the lung showed both epithelioid and papillary cells (Fig. 2c). Although these papillary structures can arise from either ectodermal or endodermal cells, the structures in this pMET suggest an endodermal origin. Epithelioid cells and undifferentiated malignant cells are also found in the pMET from a diaphragmatic nodule (Fig. 2d). Other pMETs display tissues consistent with a mesodermal origin (bone and cartilage, Supplemental Figure S1). Overall, the composition and histological phenotype of these pMETs are consistent with a pluripotent germ-cell origin.

Figure 2.

Figure 2

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Histology of pMETs. (A) Lymph node. At low power, much of the node is effaced with a neoplasm composed of glandular structures intermingled with sheets of poorly differentiated malignant cells. (B) High power view of a portion of the lymph node shown in (A). The glands are irregular in shape and size, and are lined with cytologically malignant cells. A mitotic figure is noted in one of the lining cells. (C) Lung involved with metastatic malignant neoplasm, part of which is composed of densely packed epithelioid cells, and part of which shows papillary architecture. The inset shows the neoplasm at high power. The malignant cells have an appearance suggestive of closely packed papillary structures. (D) Section from a diaphragmatic nodule that consists of undifferentiated cells. The larger cells with more abundant cytoplasm (left) have an epithelioid appearance. A large part of the neoplasm (right) consists of small dark immature-appearing cells that are remarkably proliferative. The arrows indicate abundant mitotic figures.

Immunohistochemistry

To test the germ-cell origin hypothesis for pMETs more rigorously, immunohistochemistry was used to examine OCT4 expression. In humans, OCT4 positive cells in metastases are diagnostic for germ-cell tumours that exhibit pluripotency (Cheng, 2004; Cheng et al., 2007; Looijenga et al., 2003). As a secondary marker for pluripotent cells, we used SSEA-1, which is a specific marker for EC cells, embryonic stem (ES) cells and PGCs in the mouse (Fenderson et al., 1987).

We evaluated pMETs from three different target tissues (lymph node, peritoneum and pancreas) for the presence of OCT4 and SSEA-1 (Fig. 3). In keeping with its role as a transcription factor, OCT4 is normally found in nuclei (Rosner et al., 1990), whereas antibodies to SSEA-1 react with a lactoseries oligosaccharide antigen on the cell surface (Solter & Knowles, 1978). In this analysis, a small number of cells in a pMET from a diaphragmatic nodule were positive for nuclear OCT4 staining in the absence of SSEA-1 (Fig. 3f and h, asterisks), pMETs from a mediastinal lymph node and the diaphragmatic nodule showed discrete cells that stained positively for SSEA-1 (Fig. 3c and g respectively, arrows) without OCT4 (Fig. 3d and h respectively, arrows), and a minority of cells from a pancreatic pMET stained positively for both (Fig. 3l, arrow). Normal lymph nodes were negative for both OCT4 and SSEA-1 staining (data not shown).

Figure 3.

Figure 3

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Immunohistochemistry of OCT4 and SSEA-1 in pMETs. OCT4 positive cells were present in F (asterisks) and J (arrow), but absent in (B). SSEA-1 positive cells (arrows) were observed in each metastasis (C, G, and K) and co-localized with OCT4 in one sample (L, arrow). Nuclei were stained with DAPI (A, E and I). Scale bar: 44 μm.

DNA copy number changes in metastases and primary TGCTs

Finally, we sought genetic evidence that pMETs are derived from TGCTs. This evidence was based on tests for shared genomic alterations between pairs of pMETs and primary TGCTs from individual mice. We expected that pMETs that have a primary TGCT origin should share specific genomic alterations with primary tumour cells in testes from the same mouse. Array-CGH (comparative genomic hybridization) was used to identify DNA copy number changes in metastases, with genomic DNA sequence from the same strain as the wild-type reference. We then used real-time PCR to verify these changes in pMETs as well as test for the same changes in the corresponding primary TGCTs. Although numerous small gains and losses were found in all pMETs compared with normal mouse DNA, no DNA copy number changes were common to all metastases (Table 2). Whenever possible, we examined pMETs from males affected with bilateral TGCTs because DNA copy number changes in metastases originating from a primary TGCT should match changes found in one, but not both, of the primary tumours.

Table 2.

DNA copy number variation in pMETs and corresponding primary TGCTs. Values are given as copy number where n = 2 is normal diploid. Probed genes were chosen based on their central location in the altered DNA segment identified with aCGH. Highlighted values represent shared changes between pMETs and primary TGCTs. pMETs in Samples 1 and 2 originated from males with bilateral primary TGCTs, Sample 3 originated from a male with a fused bilateral left and right TGCT (a), and Sample 4 is from a male with a unilateral left TGCT

Sample Strain and metastasis Gene (Chr) Left TGCT Right TGCT Metastasis
1 CSSM19 Map2k2 (Chr10) 2.44 2.3 1.04
Peritoneal mass Olfr77 (Chr9) 2.42 2.28 1.14
D230025D16Rik (Chr8) 2.14 1.24 1.42
Igf1r (Chr7) 2.28 1.38 0.84
Tmem48 (Chr4) 1.84 1.36 1.42
2 CSSM19 Xiap (ChrX) 2.04 1.16 1.62
Pancreatic mass Map2k2 (Chr10) 2.52 2.74 1.54
Nudt3 (Chr17) 2.94 2.46 2.56
Adam6b (Chr12) 3.86 2.4 3.58
Ywhag (Chr5) 3.24 4.1 3.1
Pou2f2 (Chr7) 2.46 3.56 2.26
3 CSSM19 Lumbar lymph node Slc34a2 (Chr5) 0.97a 1.14
4 129/SvDnd1+/Ter Peritoneal mass Skint3 (Chr4) 6.62 n/a 0.4
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We found consistent evidence with derivation of pMETs from primary TGCTs (Table 2). For example, the peritoneal mass in the CSS-M19 mouse shared 3 of 5 DNA changes (Chrs. 4, 7 and 8) with the right primary TGCT, all of which were deletions (copy number <1.5), which is consistent with derivation of the pMET from the right primary TGCT. The remaining two DNA copy losses, on Chrs. 9 and 10, were found in the pMET, but not in either the right or the left primary TGCT from the same mouse. With the exception of those changes identified in the pMET and the right primary TGCT, the probed genes did not show other detectable copy number variation.

The changes identified in Sample 2 that passed our cut-off threshold were all duplications (copy number >2.5). These DNA changes were most closely shared with the changes found in the left primary TGCT. In particular, the changes on chromosomes 5 and 12 in the pMET were most similar to changes found in the left TGCT because the right TGCT duplications on chromosome 5 were larger, and no copy number changes were found on chromosome 12. The change on chromosome 17 was found in both the pMET and the left TGCT, although a similar change in the right TGCT approached the cut-off threshold. Unlike Sample 1, Sample 2 showed changes in the primary right TGCT that were not found in the contra-lateral TGCT or the pMET itself (Chrs. 7, 10 and X).

Only one DNA copy number change was observed in the pMETs in Samples 3 and 4. Although the bilateral primary TGCTs from the source male in Sample 3 were fused (a rare occurrence in cases of bilateral TGCTs), the DNA copy number loss on chromosome 5 found in the pMET from Sample 3 was shared with the primary TGCT fusion. In contrast, the loss of genomic DNA on chromo-some 4 in Sample 4 found in the pMET was not shared with the unilateral left TGCT, but instead, the left primary TGCT showed a significant gain in this chromosomal region.

Discussion

Metastasis is a prevalent feature of human TGCTs, directly affecting treatment and survival. Although many TGCTs and their metastases respond readily to chemo-therapy, 10–25% of patients with metastases are resistant to treatment (Piulats et al., 2009) and 5–10% of patients relapse after initial treatment (Loehrer et al., 1998). Understanding the mechanisms of metastatic transformation and dissemination is therefore essential to reduce the morbidity and mortality associated with resistant and recurrent TGCT metastases.

Several lines of evidence point to a germ-cell origin for pMETs. pMETs are composed of cell and tissue types derived from all three germ lineages with ectodermal and endodermal derivatives predominating (Fig. 2). In addition, undifferentiated embryonal cells were evident histologically and confirmed with two markers of pluripotency, namely OCT4 and SSEA-1 (Fig. 3), and these cells are highly mitotic (Fig. 2d). These results are consistent with a seminoma or EC component in these metastases (Cheng, 2004; Jones et al., 2004). OCT4 is used to diagnose metastatic germ-cell tumours in humans (Cheng, 2004) and SSEA-1 is a marker for EC cells in mice (Solter & Knowles, 1978). Although OCT4 and SSEA-1 are both markers of EC cells in mice, co-localization of OCT4 and SSEA1 was uncommon in our sample of metastases. This finding of OCT4+/SSEA-1– and OCT4–)/SSEA-1+ cells is not completely unexpected, as EC cell variants lacking OCT4 and SSEA-1 have been described (Mueller et al., 2010; Rosenstraus, 1983).

Assays for DNA copy number changes strongly suggest that pMETs are derived directly from primary TGCTs in the testis rather independently from ectopic stem cells. Among the four pMETs analysed with CGH, seven significant DNA copy number changes were found, the majority of which were deletions (54%). Three of the four samples with validated changes showed shared genomic DNA changes between the primary TGCT and the pMET. Importantly, in the two samples from mice with bilateral primary TGCTs, shared DNA changes were found between the metastasis and one, but not both, of the primary tumours. Together, these results suggest that the pMETs are metastases that originate from a specific primary TGCT and do not represent extragonadal germ-cell tumours (EGCTs).

In humans, extragonadal germ-cell tumours (EGCTs) are rare, occurring in 3–5% of all germ-cell tumours (Richie & Steele, 2007). These tumours have been proposed to originate either from embryonic germ cells that fail to migrate towards the gonads (Dixon & Moore, 1953; Fine et al., 1962; Hailemariam et al., 1997), more primitive totipotent cells (Johnson et al., 1973), or a clinically undetected primary TGCT (Fabre et al., 2004). Recent case reports suggest that most retroperitoneal EGCTs can be considered to be silent or ‘burned out’ primary TGCTs (Fabre et al., 2004). Even in many cases where a primary diagnosis of an EGCT was made, further investigation found that a primary TGCT was present (Angulo et al., 2009). Our data suggest that this is also the case in the mouse model of TGCTs, because metastases were never observed in the absence of a primary TGCT, even in the 129/S1Kitl+/Sl-J line, which has a known PGC migration defect (Runyan et al., 2006).

Management of metastasis plays a pivotal role in treatment of TGCT patients. In addition to the initial effects of chemotherapy and surgical interventions, several late physical effects occur, including an increased risk of cardiovascular disease (Huddart et al., 2003), the development of metabolic syndrome (Huddart & Norman, 2003), secondary cancers (Travis et al., 2005) and reduced fertility (Brydoy et al., 2005). Identification of genetic changes that are associated with TGCT metastasis would enable targeted treatment for at-risk individuals. In our study, however, consistent genomic DNA changes were not found, even in the small number of metastases that were examined. The limited human aCGH data available for TGCT metastases have not, to date, revealed consistent genomic changes (Korkola et al., 2008). The diversity of incidental chromosomal changes in our metastases reflects the variety of changes found in the primary tumours, and is consistent with results from human aCGH surveys (Korkola et al., 2008; Skotheim et al., 2006). These results suggest that the changes driving metastasis may be unrelated to chromosomal rearrangements, and may instead result from epigenetic or miRNA alterations. Recent evidence suggests that DNA methylation patterns distinguish invasive from non-invasive urothelial cancers (Wolff et al., 2010), and that miRNAs play a role in breast cancer [reviewed in (Shi et al., 2010)]. The mouse model reported here may help identify the kinds of genetic and epigenetic changes that are associated with, and perhaps responsible for, TGCT metastasis.

Identification of a mouse model of spontaneous TGCT metastasis presents a unique opportunity to study the mechanisms involved in the acquisition of metastatic potential and the modes of dissemination. The fixed genetic background in this model helps to discriminate between driving and incidental changes in found in metastasis, including miRNA expression and epigenetic states. The nature of this model also allows for genetic manipulation and may serve as a basis for drug discovery in TGCT metastasis treatment. Finding the alterations that lead to metastasis may lead to the identification of metastatic signatures in human TGCTs and the development of treatment strategies based on metastatic risk.

Supplementary Material

Supporting Figure
Supporting Figure Legend
Supporting Tables

Acknowledgements

We thank Sabine Schafer for helpful comments that improved the manuscript. NIH grants NCI CA75056 and NCRR RR12305 supported this work.

Footnotes

Supporting Information

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Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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Supporting Figure Legend
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Supporting Tables
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