 4). In the periphery of the tumor (Figure 3D, stage 1) proliferating (Ki67-positive) satellite cells, surrounding old fibers, were mixed with more mature MyoD- and Myogenin-positive cells. These cells were intermingled with areas of neomyogenesis, including small fibers with central nuclei, positive for embryonal MHC (eMHC). Immunofluorescence confirmed the presence of Desmin-positive centro-nucleated fibers contiguous to old fibers (Figure 3E, left panel). At this early stage, Pax7-positive cells were detectable beneath the basal lamina of the newly formed fibers, suggesting that satellite cells retained the ability to return to quiescence (Figure 3E, right panel). Closer to the tumor mass the expanded population of proliferating satellite cells appeared to take over, while the number of newly formed fibers was reduced (Figure 3D, stages 2 and 3). The central area of the tumor consisted of tissue with high cellularity and no further evidence of terminal myogenic differentiation (Figure 3D, stage 4). Accordingly, the tumor bulk was eGFP-negative indicating that the cells were undifferentiated (Figure 3B, Figure 3—figure supplement 1B). Morphologically, small round cells were associated with elongated polygonal cells or with cells displaying pleomorphic nuclei (Figure 3D, stage 4). The histological markers (Pax7, MyoD and Myogenin, Figure 3D, stage 4) and the anatomical localization (trunk, neck and occasionally the orbit) (Figure 3—figure supplement 1B), were compatible with the ERMS subtype. MHI-null mice developed tumors with a short latency (3.95 months), while mice heterozygous for the Cdkn2a locus (MHI-het) showed delayed tumor formation (6 months) (Figure 3—figure supplement 1C). PCR analysis revealed that the majority of the tumors in MHI-het mice had lost the wild type allele (Figure 3—figure supplement 1D). To exclude the involvement of fetal myoblasts in ERMS development, MHI-null mice were maintained under Dox treatment until P10. Given the persistence of Dox in the tissues, transgenic Hgf and eGFP expression became detectable from day 40 onward (Figure 3—figure supplement 2A,B), when only resident muscle stem cells are present. In this cohort of mice, tumor development occurred with the previously described latency and incidence (Figure 3—figure supplement 2C).
SC niche perturbation in Cdkn2a-null mice results in a multi-step model of ERMS development.
(A) Representative image of a control (M) and a hyperplastic mouse (MHI-null). (B) Representative fluorescent images of MHI-null trunk muscles at different stages of progression: normal muscle, hyperplasia and ERMS. (C) Distribution of MHI-null mice developing ERMS with or without hyperplasia. (D) H&E staining and representative immunohistochemical analysis of MHI-null specimens collected at different stages of tumor progression (from hyperplasia to ERMS, stage 1–4). (Scale bar = 100 µm). (E) Representative immunofluorescence on MHI-null hyperplastic muscle sections. Pax7 (red), Laminin (green), Desmin (red) and DAPI (blue). (Scale bar = 100 µm).
It has been shown that ERMS and UPS are part of a tumor continuum in terms of histological presentation and expression profiling (Rubin et al., 2011). Intriguingly, the SC niche, in addition to muscle stem cells, also contains resident muscle fibroblasts that are responsible for extracellular matrix production within skeletal muscle fibers (Thomas et al., 2015). To assess the contribution of these two cell types to sarcomagenesis we moved the MHI-null mice in a Pax7 null genetic background. Pax7-deficient C57/B6 mice are viable at birth, but progressively die in 2–3 weeks (Mansouri et al., 1996; Seale et al., 2000). In these mice the number of satellite cells is reduced at birth, and further declines during postnatal development. However, in a mixed genetic background, between 5 and 10% of mutant animals survive into adulthood (Oustanina et al., 2004). Thereby, we generated a cohort of MHI-null Pax7-null mice (herein referred as MHl-null P-null) (Figure 4A) sufficient to determine the effect of the Pax7 null background on sarcoma development. The mixed background had no effect on sarcoma incidence and latency (Figure 4B). However MHI-null P-null mice showed a drastic reduction in ERMS incidence (Figure 4C). The MHI-null P-null tumors were characterized by cells displaying a variegate morphology, that occasionally included the presence of visible cross-striations. Notably, immunohistochemistry revealed the positivity for MyoD and Myogenin in less than 30% of cases, which were classified as ERMS (Figure 4C,D). Interestingly, the majority of tumors (more than 70%) presented extensive cellular heterogeneity from round to spindle-shape cells, with nuclear appearance ranging from hyperchromatic to pleomorphic. The complete absence of tissue-specific markers of differentiation categorized these tumors as UPS (Figure 4C,D). The switch in sarcoma subtype is in line with the expansion of myofibroblasts observed in Pax7-null muscles upon exposure to a regenerative microenvironment (Maltzahn et al., 2013). Accordingly, while both UPS and ERMS tumors expressed Met, only UPS were positive for the pan-fibroblast marker PDGFRα (Figure 4D,E), and FACS analysis on freshly isolated tumors revealed that they were Sca1-positive and Integrin-α7-negative (Figure 4F), indicating a mesenchymal-like phenotype (Driskell et al., 2013; Guarnerio et al., 2015; Joe et al., 2010). In contrast, the ERMS were Sca1-negative and Integrin-α7-positive, confirming the myogenic identity of this subtype. Altogether our data clearly indicate that perturbation of the SC niche microenvironment can give rise to distinct sarcoma subtypes in a Pax7 lineage-dependent manner, and provide evidence for a fibroblast cell origin of UPS.
Genetic ablation of Pax7 results mainly in UPS development.
(A) Schematic representation of the system used to generate Ckm Hgf Cdkn2a-null Pax7-null (MHI-null P-null) mice. (B) Kaplan-Meier curve showing the relative sarcoma-free survival in the indicated genotypes. (C) Penetrance (pie charts) and histological distribution (bar graphs) of tumors. (D) Representative immunohistochemical analysis of MHI-null tumors in Pax7 heterozygous or null genetic background. UPS: undifferentiated pleomorphic sarcoma. (Scale bar = 250 µm). (E) Western blot analysis of murine UPS and ERMS probed for the indicate proteins. (F) Sca1 and Int-α7 expression analysis in primary murine UPS and ERMS (left panel, single staining; center and right panels, double staining).
Genetically engineered animal models represent a robust preclinical platform to investigate the efficacy of novel treatments, among which targeted therapies and combination therapies are the most attractive (Day et al., 2015). In our model transgenic Hgf comes from differentiated muscle fibers. Thus the tumor mass is no longer exposed to transgenic HGF for lack of differentiated muscle cells, as shown by the absence of expression of the eGFP reporter in the tumors (see Figure 3B, Figure 3—figure supplement 1B). Accordingly, Dox-mediated Hgf downmodulation did not impair tumor growth (Figure 5—figure supplement 1A,B) indicating that transgenic HGF production was not essential for tumor maintenance. Although all primary murine tumors retained basal Met expression, in a subset of ERMS (which later turned out to be Met-amplified), Met was present at much higher level and overexpression was accompanied by receptor phosphorylation (Figure 5A,B, and Figure 5—figure supplement 2B). On the other hand, at earlier stages, paracrine HGF stimulation promoted receptor downregulation (Figure 5—figure supplement 2A) and this resulted in a difficult detection of Met and phospho-Met (Figure 5—figure supplement 2B).
Only a minor fraction of ERMS harbors MET amplification.
(A) Western blot analysis in a panel of primary murine MHI-null ERMS probed for the indicate proteins. (B) Immunohistochemical analysis of primary murine MHI-null ERMS shown in A, probed for Met and phosphorylated Met. (Scale bar = 100 µm). (C) Waterfall plot showing the MET Z-score expression from a panel of previously characterized human RMS tumors (Shern et al., 2014) (http://pob.abcc.ncifcrf.gov/cgi-bin/JK). The exceptionally high MET expression in Patient 2083 (highlighted) is associated with focal amplification of chromosome 7q31.2 (Shern et al., 2014). (D) FISH for MET in a human ERMS sample (INT291) characterized by small cluster signals (with an average of 9 signals per cell; MET is labeled in green and centromeres in red). (E) CGH analysis of murine MHI-null ERMS. Red indicates copy number gain, green indicates copy number loss. Arrowheads mark the Met (red) and Kras (blue) loci.
Despite extensive attempts to stabilize cell lines from both sarcoma subtypes, we obtained a significant number of cell lines only from ERMS. To identify signaling pathways involved in tumor maintenance, we tested a panel of 18 ERMS lines with receptor tyrosine kinase (RTK) phosphoarrays. Met activation was detected in three cell lines (#187R, #1640 and #1796, 17% of the total) (Figure 5—figure supplement