Rhabdomyosarcoma (RMS) is a pediatric malignancy that shares common features with skeletal muscle arrested in embryonic development (Xia et al., 2002
). The two main subtypes of pediatric rhabdomyosarcoma, embryonal RMS (ERMS) and alveolar RMS (ARMS), differ in their clinical, biological, and molecular characteristics. For example, ERMS and ARMS can be distinguished based on histology and have different long-term prognosis with ERMS patients having better overall outcome than ARMS. These divergent clinical features likely reflect the use of different molecular programs that lead to transformation. For example, we have identified that the RAS pathway is active in a majority of human ERMS (Hettmer et al., 2011
; Langenau et al., 2007
). By contrast, 85% of ARMS have recurrent chromosomal translocations that juxtapose PAX3 or PAX7 with the forkhead transcription factor (FKHR) (Xia et al., 2002
). Finally, it is likely that ERMS and translocation-positive ARMS arise in different cell types that eventually undergo transformation. Keller et al. found that PAX3-FKHR+ ARMS can arise from Myf6 expressing myoblast cells but not dermamyotome or satellite cells that express Pax7 (Keller et al., 2004
). By contrast, ERMS can arise from either satellite cells or myoblasts that eventually reinitiate molecular programs found in satellite cells (Rubin et al., 2011
). Despite elegant studies defining possible cells of origin in RMS, identification of an ERMS-propagating cell that is required for continued tumor growth in vivo
has not been described in mice or humans.
Tumor-propagating cells have been characterized in many malignances, and in some tumors, this potential is confined to a molecularly definable cell population that can be enriched by cell surface markers. For example, in AML a rare CD34+
cell enriches for leukemia-propagating potential while in breast cancer CD44+
expression is associated with tumor-propagating potential (reviewed in Dalerba et al., 2007
). Molecularly defined, rare CD133+
tumor-propagating cells have also been identified in subset of gliomas and exhibit striking differences in response to nitric oxide and hypoxia inducible factor (HIF) signaling when compared to more differentiated tumors cells (Eyler et al., 2011
; Li et al., 2009
). Thus, it is likely that many tumors contain hierarchically organized cell subpopulations that retain the capacity to remake tumor and yet give rise to differentiated tumor cell progeny. One might expect that selection would favor the evolution of tumors with high numbers of tumor-propagating cells at a cost of differentiated cell types. Yet paradoxically, in most malignancies, tumor-propagating cells are far less abundant than differentiated tumor cells that are incapable of remaking tumor. These data suggest that differentiated tumor cells may provide important supportive roles in overall growth and maintenance. To date, a role for differentiated, non-tumor-propagating ERMS cells has yet to be fully explored.
Stem cells often reside in distinct niches in normal tissue and their functions are exquisitely controlled by local factors secreted by supporting cells. For example, hematopoietic stem cells (HSCs) have been shown to home to niches within the calvarium that are tightly associated with osteoblasts (Lo Celso et al., 2009
). These and other niche-associated cells presumably provide paracrine-signaling factors to recruit and maintain these cells in a specific niche. Unlike other tissues, the muscle stem cell niche is defined by juxtaposition of satellite cells next to differentiated muscle fibers, and their numbers and differentiation capacity are controlled by complex signaling pathways regulated by mature muscle cells (reviewed in Bentzinger et al., 2012
). Despite a large body of data defining stem cell niches in normal tissue, few studies have identified tumor-specific niches and/or regions of compartmentalized tumor cell function and less have used microscopic imaging to directly visualize tumor-propagating cells within live animals. In one example, Sipkins et al used a combination of multiphoton and confocal microscopy to image the HSC niche in the calvarium of mice and demonstrated that these sites can attract multiple tumor cell types (Sipkins et al., 2005
); however, it is unknown if these malignant cells are capable of reinitiating tumors. In ERMS, as with most solid tumors, it is unknown if tumor-propagating cells reside in distinct regions within the tumor mass and if the more differentiated cells play a role in promoting tumor progression.
Here, we utilize a transgenic zebrafish model of embryonal rhabdomyosarcoma to identify the tumor-propagating cell in this disease and to define the functional consequences of tumor cell heterogeneity within live animals. Because ERMS cell subpopulations can be fluorescent-labeled based on myogenic factor expression, ERMS cell subtypes can be visualized in live animals and the processes of cell growth, division, and local dissemination can be visualized as dynamic processes in live animals. Our data provide an explanation for the large number of non-tumor propagating cells in established cancers and reveals an important supportive role for differentiated tumor cell types in local dissemination and metastasis.