Cre-LoxP based conditional systems are considered superior to simple transgenic models due to the ability to target `tumor-inducing' genetic events in specific tissue type. Nonetheless, using standard tissue-specific Cre drivers, a large number of cells within the targeted tissue are affected by the oncogene expression thereby creating a highly un-physiological environment for tumorigenesis. This is a significant limitation of this strategy. Some studies have tried to address this issue such as spontaneous recombination dependent random activation of K-ras
leading to lung cancer (24
). In this study, we have tried to address this issue by intentionally making Cre activity (and hence SYT-SSX2 oncogene expression) sporadic and randomly distributed within a new mouse model of synovial sarcoma. Although this strategy does not permit tracking of the cell of origin, it does recapitulate the natural pathogenesis more closely by restricting oncogene expression within a small subset of cells, each of which is surrounded by a `normal physiological' microenvironment. In this strategy, random induction of oncogene expression takes place throughout the lifetime of the mouse and across most cell types within a single mouse significantly enhancing the chance of recapitulating the necessary cell and the microenvironment of origin to generate a tumor. Neither a prior knowledge of the cell of origin is required, nor will it be revealed, by this strategy. Although knowledge of the tissue source of a tumor is important from a biological point of view, the ability to rapidly generate mouse models is highly desirable for preclinical applications. Furthermore, the strategy described here is conditional and allows investigators to combine multiple genetic anomalies to attempt at generating a tumor models or investigate the roles of downstream genetic hits within a model. Therefore, with the exception of our inability to trace the origin, the strategy of random induction incorporates the unique advantages of a conditional system minus the disadvantage of `collateral damage' induced by widespread oncogene expression within the target tissue.
Comparing tumors within the new R-CreER+/−/SSM2+/− mouse model with our previous model established by expressing SYT-SSX2 in myoblasts of Myf5-Cre+/−/SSM2+/− mice is instructive. Despite similarity in histology, expression of markers, and transcriptional profile, there are certain differences between these two models. To begin, the age of onset is different in these two models. While Myf5-Cre+/−/SSM2+/− mice died by 3–5 months of age (), R-CreER+/−/SSM2+/− mice live much longer, beyond 1 year, and showed massive tumor load prior to death (). The tumors within Myf5-Cre+/−/SSM2+/− mice, in most cases, did not appear to be large enough nor numerous enough (based on fluorescence-based visual detection at necropsy) to be the primary cause of death. This suggested either early widespread microscopic metastasis or lethality from a non-tumor related phenotype of widespread SYT-SSX2 expression within Myf5 lineage. In R-CreER+/−/SSM2+/− mice, however, the tumor load was uniformly large enough to be the primary cause of death. This highlights an important limitation of widespread tissue specific oncogene expression. `Collateral damage' induced by oncogene expression in a particular cell lineage may confound phenotype analysis. Of particular concern is pre-clinical application where a candidate drug may have a potent effect on the tumor phenotype, but not on the other `tumor-unrelated phenotype' induced by widespread oncogene expression. In such cases, the mouse model may fail to show a survival benefit despite an effect of the drug on the tumor itself. This is particularly relevant in the case of the synovial sarcoma model since we demonstrate that SYT-SSX2 has two dramatically distinct effects; tumorigenesis and cell death.
Anatomical distribution of tumors in ROSA-CreER+/−/SSM2+/− mice was distinct from Myf5-Cre+/−/SSM2+/− mice, suggesting additional non-myoblast cell of origin in the new model (). Tumors within Myf5-Cre+/−/SSM2+/− mice were observed most frequently in the intercostal region while tumors in the R-CreER+/−/SSM2+/− mice were mostly noted in the para-spinal region (back and tail) and face but not in the intercostal region. Both models, however, often harbored tumors in the limbs. CT scanning and histology revealed significant calcification in some tumors generated in the new model. This fits the range of synovial sarcoma phenotypes in humans where calcification is a relatively synovial sarcoma-specific radiological finding amongst soft tissue sarcomas. Tamoxifen-induced Cre mediated expression of β-galactosidase (LacZ) in R-CreER+/−/R-LacZ+/− mice revealed surprising paucity of LacZ expressing skeletal muscle fibers (). The differences in age of onset and anatomic location when compared to the myoblast derived synovial sarcomas, makes a non-myogenic origin a possibility in this new model. However, a muscle origin cannot be ruled out in this strategy of random induction. In the light of these findings, the possibility exists that synovial sarcomas have more than one cell-of-origin. Furthermore, the predilection of these tumors to occur in proximity to skeletal elements in both models also raises the issue of whether the cell type or the micro-environment has a more crucial role in the induction of synovial sarcomas. We are currently investigating other potential sources of this lethal disease and address whether origin may have any clinical or prognostic influence.
SYT-SSX2 expression has a prominent cytotoxic effect based on in vivo and in vitro data we show here. It will be very important to investigate the mechanism of cell death and, in particular, how some cells escape this and progress towards neoplasia. From a therapeutic point of view, reactivating these SYT-SSX2 induced pro-apoptotic pathways that were silenced in the tumor cells may be an effective strategy towards designing targeted therapy for this lethal disease. The R-CreER+/−/SSM2+/− random sporadic model in conjunction to the TAT-Cre based in vitro approach we describe here will be very useful in the investigation of cytotoxic pathways activated by SYT-SSX2.
In summary, we describe here a variation on the conditional approach in tumor modeling by taking advantage of the CreER inducible system. We describe a new mouse model of synovial sarcoma utilizing this system and compare it to our previously reported mouse model to highlight the various advantages of this alternate strategy.