Cultured PGCs (EGCs) were derived from E12.5 C57BL/6 mice. Karyotype analysis of the primary EGC line determined that this line was 40,XY. Transplantation of 0.5–1 × 106 EGCs into the testes of SCID mice resulted in tumor formation in 25 of 26 transplants at 4–6 weeks following surgery (). EGCs transplanted directly into the testes result in locally invasive testicular tumors with trilineage embryonic differentiation that included large amounts of primitive neuroepithelium () and endodermally derived gut epithelium (). In the majority of cases, the testis architecture was completely destroyed by tumor cells.
Figure 1 Testicular transplantation of EGCs. (A): Solid tumor generated by transplantation of 5 × 105 EGCs directly into the testis (arrow shows testis). (B): Primitive neural tissue (arrowhead) and neural epithelium (arrow) (magnification, ×100). (more ...)
To identify whether the testicular tumors had a stem cell subpopulation capable of self-renewal, we evaluated the proportion of tumor cells that expressed a unique cell surface marker called SSEA1, which is present on the cancer-initiating EGCs used to make the primary tumor. SSEA1 is not expressed on adult testicular cells (; supporting information Fig. 1A, 1B
). We reasoned that tumor stem cells would maintain SSEA1 expression, whereas the majority of tumor cells would lose expression of SSEA1 in the process of differentiation. Indeed, we determined that SSEA1-positive cells derived from the testicular tumors averaged 12.5% of the total cell population compared with the tumor-initiating EGCs cultured on mouse embryonic fibroblasts, which are >90% positive for SSEA1 prior to transplantation (). Therefore, the majority of cells within the testicular tumor are SSEA1-negative.
To determine whether stem cell-like cells could also be identified in tumors derived from ESCs, we performed transplantations with mouse ESCs; diploid human ESCs (HSF-6); karyotypically abnormal hESCs (H9) with a karyotype of 46, XX, add(7); and NTERA-2 cl.D1 EC cells with a hypotriploid karyotype (supporting information Table 2
). Our results show that 4–6 weeks after transplantation into the testis, teratomas derived from mouse ESCs had a detectable SSEA1-positive population at 12.17%, which is very similar to the proportion of SSEA1-positive cells in EGC-derived tumors (12.5%). However, in contrast to the murine EGC line, transplantations of murine ESCs into the testis of SCID mice resulted in metastasis in two-thirds of tumors, suggesting that pluripotent ESCs are more tumorigenic than EGCs in vivo. With regard to human pluripotent cells, the pluripotent embryonal carcinoma line (called NTERA2), which was derived from a metastatic teratocarcinoma contains a 6.89% SSEA4 positive population following transplantation and tumor formation, whereas tumors from karyotypically normal hESCs result in a 1% positive SSEA4-positive population. Interestingly, analysis of a karyotypically abnormal hESC line results in a population of SSEA4-positive cells equivalent to NTERA2 at 5.52%. These results suggest that similar to the EGCs in the current study, murine ESCs transplanted into the testis generate teratocarcinomas with a clearly reproducible stem cell population, whereas human pluripotent cells with aneuploidies are generated teratocarcinomas with a clearly distinguishable stem cell population.
Next we used immunofluorescence and confocal microscopy to determine the location of SSEA1-positive cells within the EGC-derived tumors. We found that SSEA1-positive cells within testicular tumors were arranged in small clusters (). Costaining with anti-Oct4 demonstrated that all SSEA1-positive tumor cell clusters expressed this pluripotent transcription factor in the nucleus (). Given the abundance of immature neural structures in the primary tumors, Nestin and Oct4 costaining was performed and showed that Nestin and Oct4 did not colocalize, suggesting that the SSEA1-positive cells in tumors were not related to neural precursor or neural stem cells (). Two additional germ cell markers, Dazl and Mvh, were also evaluated because these markers label the spermatogonia and spermatocytes in adult testis (supporting information Fig. 2A, 2B
). These germ line markers did not colocalize with SSEA1 or Oct4 staining in the testicular tumors (). In sum, the lack of germ line or neural identity, as indicated by the absence of Dazl, Mvh, and Nestin costaining, coupled with the expression of SSEA1 and Oct4, strongly suggests an immature stem-like tumor cell population rather than a primitive germ or neural cell population within these small tumor clusters.
Figure 2 Immunofluorescent staining of testicular tumors at 6 weeks (A–C). (A): Double immunofluorescence for SSEA1 (green) and Oct4 (red). (B): Double immunofluorescence for Nestin (green) and Oct4 (red). (C): Double immunofluorescence for Mvh (green) (more ...)
To determine whether the SSEA1-positive population was capable of self-renewal and differentiation, SSEA1-positive cells at various numbers were serially transplanted into recipient testes following MACS or FACS sorting without intervening culture (). We found that transplantation of just 2.5 × 105 positive cells resulted in not only a testicular tumor but also metastasis throughout the peritoneum and also to the kidney (). This was observed in 100% of secondary tumors at this cell number (). In contrast, metastatic activity was only observed 1 of 26 transplants of primary EGCs at numbers of 5 × 105 cells and greater. As controls, transplantation of STOs did not result in tumors, and SSEA1-negative tumor cells isolated by FACS did not self-renew when plated on STOs in the presence of leukemia inhibitory factor. Together, these observations strongly suggest that the SSEA1-negative population does not contain a stem cell component capable of self-renewal.
Comparison of tumor potential between EGC transplants to generate primary tumors and SSEA1-positive serial transplants without intervening culture to generate secondary tumors
Figure 3 Secondary tumors derived following re-transplantation of stage-specific embryonic antigen 1 (SSEA1)-positive cells without intervening culture. (A): Testicular tumor (white arrow) and metastasis from the primary tumor into the kidney (black arrow) and (more ...)
Given that SSEA1-positive cells had an undifferentiated identity in the primary tumors, we performed histology on both the testicular tumors and metastases to evaluate the degree of differentiation. Similar to the results shown in , the secondary tumors were highly differentiated and contained cells from all three embryonic layers, including primitive neuronal tissue (), bone (), and muscle (not shown). To determine whether stem cell populations can be isolated from the secondary tumors, we performed MACS sorting before culturing on STOs (). This resulted in the generation of cell lines capable of robust self-renewal (). Retransplantation of tumor-derived SSEA1-positive cells that had been cultured for >10 passages resulted in highly metastatic tumors 6 weeks following surgery. Together, these results demonstrate that the SSEA1-positive cells within a primary tumor can differentiate and regenerate a new teratoma with a new stem cell population upon transplantation into a new host testis. Furthermore, these tumors are more aggressive than the primary tumors. This suggests that genetic, epigenetic, and/or transcriptional differences between primary EGCs and the subsequent cancer stem cell population are responsible for this malignant potential.
We first addressed genetic stability between the EGCs and a first-generation cancer stem cell line by performing G-banding karyotype analysis. The EGC line was 40,XY; however, G-banding identified two stable structural changes in 100% of cells involving a duplication of B2-C7 on chromosome 4 and a deletion of C3-F1 on chromosome 6. In contrast, the SSEA1-positive cells derived from primary tumors displayed a number of genomic rearrangements, including a major clone with three nonclonal sublines, and a minor clone with two nonclonal sublines. The gross genomic changes included isochromosomes iso(8) and iso(15), translocations (t) t(6;8) and t(11;14), as well as loss of the Y chromosome del(Y) and in one case a hypotriploid karyotype (supporting information Fig. 3
). This result indicates that generation of cancer stem cells in vivo are not the result of clonal selection from a single karyotypically abnormal cell in the original EGC population and instead occurs via more generalized genetic instability during tumorigenesis.
Given that teratomas are generated through a process of differentiation from self-renewing cells, our next aim was to determine whether disruption of self-renewal prior to transplantation was sufficient to block the formation of tumors and the generation of SSEA1-positive cancer stem cells. To achieve this we pretreated the EGCs with all-trans retinoic acid (RA) prior to transplantation [41
]. Tumor growth was quantified in vivo using live-animal imaging of EGC lines that stably expressed a trifusion-imaging vector (supporting information Fig. 4A
). Imaging was performed to quantify tumor growth and also to determine whether metastases arose from RA-treated primary EGCs. Treatment of EGCs with RA for 4 days disrupted self-renewal, as monitored by changes in colony morphology, and significantly reduced mRNA expression of pluripotent transcription factors (supporting information Fig. 5
). Furthermore, RA-treated EGCs displayed increased expression of genes associated with differentiation, as well as increased numbers of apoptotic cells and a decreased proliferation rate (supporting information Fig. 5
). Western blot was also performed to confirm loss of NANOG protein and to confirm that RA treatments resulted in reduced Rb phosphorylation. We did not identify appreciable differences in P53 expression between control and RA treated cells (supporting information Fig. 5
). Next we transplanted 5 × 105
control (untreated) and RA-treated EGCs into the testis of SCID mice, and tumor growth was quantified by bioluminescent imaging from 5 to 41 days post-transplantation. Bioluminescent images of mice are shown at day 41 following transplantation (). Tumors derived from control cells were localized to the testis (no metastasis) and were detected in two of eight cases beginning at 5 days following transplantation. By day 10, six of eight transplants had a detectable signal above background, and by day 14, eight of eight transplants had a detectable signal. In contrast, tumors derived from RA-treated EGCs were first detectable in two of four transplants at day 14. By day 21, two of four transplants had a signal above background, and one transplant had lost the signal (). By day 41 only one of four transplants had retained a bioluminescent signal above background. In the cohort treated with RA, one of four transplants never developed a detectable signal, and the testis containing the transferred cells was of normal size at necropsy except for a small tuft of tubules at the site of injection, suggesting that tumor formation never progressed.
Figure 4 Analysis of tumor growth following treatment of embryonic germ cells (EGCs) with RA. Shown is live-animal imaging of tumor growth. (A): Dynamics of tumor growth following transplantation of EGCs into the testis as monitored by live-animal imaging. Each (more ...)
To determine whether SSEA1-positive cells could be identified in tumors derived from RA-treated cells, flow cytometry was performed (). SSEA1-positive cells derived from control testicular tumors averaged 9.2% (+ SD) of the total population. In contrast, testes from transplants of RA-treated EGCs contained, on average, 1.8% SSEA1-positive cells (+ SD). Cell lines were attempted from the SSEA1-positive cells derived from RA-treated tumors; however, no cells survived after the first split, whereas SSEA1-positive cell lines were derived from the control tumors. Transplants were performed using the SSEA1-positive cells derived from RA-treated tumors, and no tumors formed. This result indicates that the small population of SSEA1-positive cells in teratomas derived from RA-treated EGCs are not capable of self-renewal and are therefore not cancer stem cells. Histology of the primary tumors derived from RA-treated EGCs revealed small foci of stromal cells (, arrow) without differentiated structures. Furthermore, SSEA1 and Oct4 did not stain the tumor cells (). Although we did detect isolated Vasa-positive cells intermingling with the negatively staining stroma (), we hypothesize that these correspond to isolated germ cells migrating from the tubules, which have degenerated as a consequence of the initial transplant. Taken together, these results suggest the generation of genetically unstable SSEA1-positive cells in teratomas is dependent upon an ability to self-renew via RA-sensitive pathways.
Finally, we assessed transcriptional differences between SSEA1-positive cells derived from primary and secondary tumors and EGCs using microarray analysis (). For this experiment, EGCs were used to generate a primary tumor, and 6 weeks later the SSEA1-positive cells (11.83%) were isolated by MACS and used to generate secondary tumors (n = 2 mice) without intervening culture, as well as an SSEA1-positive cancer stem cell line (CSC66). The secondary tumors were harvested at 6 weeks, and the SSEA1-positive cells (21.5%) were collected by MACS and used to generate tertiary tumors (n = 2 mice) without intervening culture, as well as an SSEA1-positive cancer stem cell line (CSC84). The history of this tumor series is shown in . Serial transplants are still continuing; however, at the quaternary tumor stage, one of the two mice died at 5 weeks, and in septenary tumors one of two mice died at 4 weeks due to tumor burden and increased metastasis now including the liver. This suggests that the cancer stem cells are becoming more functionally aggressive with serial transplantation, which we hypothesize will be reflected by significant changes in the transcriptional profile.
Figure 5 Microarray comparing primary EGCs with the stage-specific embryonic antigen 1 (SSEA1)-positive cells derived from primary tumors and secondary tumors. (A): First- and second-generation tumors were generated by serial transplantation without intervening (more ...)
Grouping of the transcriptional profiles was first performed using cluster analysis (supporting information Fig. 6
). This analysis revealed that the transcriptome of the SSEA1-positive cancer stem cell lines clustered independently from the primary EGCs. The top 10 differentially expressed genes are shown in supporting information Table 3
. The most upregulated gene in second-generation cancer stem cells was Igf2
, which has previously been associated with human testicular cancers [42
]. Although the EGCs CSC66 and CSC84 were highly similar, we could identify 1,000 differentially expressed genes on the basis of a t
value of .00001 to account for multiple testing corrections (). K
-Means clustering () of these 1,000 most differentially expressed genes revealed that the transcriptional change from EGC to CSC84 was associated with repression of fibroblast growth factor signaling pathways (FGF-4, FGF-17
) and repression of inhibin, nodal
, and lefty1
in CSC84 compared with CSC66 and EGCs (cluster 1). The second-generation tumor CSC84 exhibited reduced expression of tumor suppressors Pten
and, interestingly, some genes associated with pluripotency and germ cell development, including Deleted in azoospermia like, Developmental pluripotency associated
) 2, Dppa3, Dppa4, Sox2, Utf1
(cluster 3). However, there was no change in Oct4
. The CSC84 line was particularly enriched in oncogenes, including JunD, Rad23a
, and Rab8b
as well as exhibiting increased expression of the Wnt receptor Frizzled2
(cluster 4). CSC84 was also enriched in genes associated with motility and migration, including SDF-1, SCF, lasp1, lpp
, and Insig2
, which may account for the more aggressive behavior of these cells with serial transplantation. Cluster two contained enriched transcripts in both of the cancer stem cells lines, CSC84 and CSC66, relative to EGCs. This cluster contains genes associated with developmental processes and metabolism, as well imprinted genes such as Insulin growth factor 2
, as well as Igfbp4
. The list of differentially expressed genes and the Gene Ontology analysis from K
-mean clustering are given in supporting information Chart 1
. Therefore, our results show that the cancer stem cell populations derived from the pluri-potent tumors are not identical at the transcriptional level to EGCs used to generate the primary tumors, and with sequential transplantation they become progressively more distinct and tumorigenic.