Distinct and overlapping sarcoma subtypes initiated from muscle stem and progenitor cells

Abstract

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, whereas undifferentiated pleomorphic sarcoma (UPS) is one of the most common soft tissue sarcomas diagnosed in adults. To investigate the myogenic cell(s) of origin of these sarcomas, we used Pax7-CreER and MyoD-CreER mice to transform Pax7(+) and MyoD(+) myogenic progenitors by expressing oncogenic Kras(G12D) and deleting Trp53 in vivo. Pax7-CreER mice developed RMS and UPS, whereas MyoD-CreER mice developed UPS. Using gene set enrichment analysis, RMS and UPS each clustered specifically within their human counterparts. These results suggest that RMS and UPS have distinct and overlapping cells of origin within the muscle lineage. Taking them together, we have established mouse models of soft tissue sarcoma from muscle stem and progenitor cells.

Figure 1. Transforming Pax7⁺ myogenic progenitors in vivo generates RMS and UPS

(A) P7KP mice (n=17) were injected with systemic tamoxifen (IP TMX) and generated tumors (n=67) at clinically relevant anatomic locations, such as the orbit. Median tumor free survival was 44 days. (B) eRMS displayed small round, blue cell histology with rhabdomyoblasts (arrowheads) by H&E, and were positive for the myogenic marker MyoD (C). (D) pRMS displayed characteristic large rhabdomyoblasts (arrowheads) with spindle cell histology by H&E, and were positive for the myogenic marker MyoD (E). (F) Non-myogenic UPS displayed characteristic spindle cell histology with giant cells (arrowheads) by H&E, but lacked MyoD expression (G). (H) In contrast, myogenic UPS displayed spindle cell histology, but were positive for MyoD (I) (40X, scale bar=50μm). (J) Anatomic distribution of sarcomas generated in P7KP mice injected with IP tamoxifen. (K) Histological distribution of sarcoma subtypes in P7KP mice injected with IP tamoxifen. (L) Myogenic distribution in the P7KP derived UPS sarcoma subset. See also Figure S1.

Figure 2. Characterization of MyoD^CE/+ mice

(A) Schematic of the MyoD^CE/+ allele. CreER was targeted to the endogenous MyoD locus. In addition, the avian tumor virus receptor A (tva) and MyoD are expressed from dual Internal Ribosome Entry Sites (IRES). (B) Pax7^CE/+;R26^mTmG/+ (n=2) and MyoD^CE/+;R26^mTmG/+ (n=2) mice were injected (IP) with tamoxifen (TMX) for five consecutive days and sacrificed ten days later. Pax7^CE/+;R26^mTmG/+ mice contained a GFP⁺ satellite cell population while MyoD^CE/+;R26^mTmG/+ mice had no identifiable GFP⁺ population. (C) Following injury with intramuscular cardiotoxin (CTX) and five daily TMX IP injections, both Pax7^CE/+;R26^mTmG/+ and MyoD^CE/+;R26^mTmG/mTmG mice (n=3) exhibited GFP⁺ regenerating muscle fibers (40X, scale bar=50μm). See also Figure S2.

Figure 3. Transforming MyoD⁺ myogenic progenitors in vivo generates UPS

(A) MDKP mice injected with IP tamoxifen (TMX) generated single tumors at clinically relevant anatomic locations, such as the proximal thigh. Median tumor-free survival was 153 days (n=12 mice). (B) MDKP derived myogenic UPS had spindle cell histology by H&E and expressed the myogenic marker MyoD (C). In contrast, non-myogenic UPS maintained the classic spindle cell and pleomorphic histology (D), but lacked immunoreactivity for MyoD (E) (40X, scale bar=50μm), Pax7, and Myogenin (Figure S3). (F) Anatomic distribution of sarcomas generated in MDKP mice injected with IP tamoxifen. (G) Histological distribution of sarcomas generated in MDKP mice injected with IP tamoxifen. (H) Distribution of myogenic staining in MDKP derived UPS. See also Figure S3.

Figure 4. Sarcomas cluster by histologic subtype using unbiased genomic analysis

(A) Principal component analysis clustered P7KP derived eRMS separately from P7KP derived UPS and MDKP derived tumors along the first principal component (PC1). The second principal component (PC2) separated the UPS P7KP sarcomas from the MDKP sarcomas. (B) Unsupervised hierarchical clustering separated the sarcomas into an RMS-like clade that includes most P7KP derived eRMS and pRMS and a UPS-like clade that includes most P7KP derived UPS and MDKP derived tumors. (C) GSEA demonstrates that a gene set derived from P7KP RMS (eRMS and pRMS) enrich in human RMS, while a gene set from P7KP derived UPS and MDKP sarcomas enrich in human UPS. See also Figure S4.