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Single-cell analysis and functional characterization uncover the stem cell hierarchies and developmental origins of rhabdomyosarcoma

Nature Cancer volume 3, pages 961–975 (2022)Cite this article

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Abstract

Rhabdomyosarcoma (RMS) is a common childhood cancer that shares features with developing skeletal muscle. Yet, the conservation of cellular hierarchy with human muscle development and the identification of molecularly defined tumor-propagating cells has not been reported. Using single-cell RNA-sequencing, DNA-barcode cell fate mapping and functional stem cell assays, we uncovered shared tumor cell hierarchies in RMS and human muscle development. We also identified common developmental stages at which tumor cells become arrested. Fusion-negative RMS cells resemble early myogenic cells found in embryonic and fetal development, while fusion-positive RMS cells express a highly specific gene program found in muscle cells transiting from embryonic to fetal development at 7–7.75 weeks of age. Fusion-positive RMS cells also have neural pathway-enriched states, suggesting less-rigid adherence to muscle-lineage hierarchies. Finally, we identified a molecularly defined tumor-propagating subpopulation in fusion-negative RMS that shares remarkable similarity to bi-potent, muscle mesenchyme progenitors that can make both muscle and osteogenic cells.

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Fig. 1: scRNA-seq reveals distinct cell states and intertumoral heterogeneity in human RMS.
Fig. 2: Single RMS cells can remake all tumor cell heterogeneity within the cancer.
Fig. 3: LARRY barcoding of human FN-RMS RD cells reveals that the mesenchymal-enriched cell subfraction is capable of driving tumor growth following culture in low-serum, stress conditions.
Fig. 4: Tumor-propagating potential is found within the mesenchymal-enriched cell fraction of FN-RMS.
Fig. 5: Limiting dilution cell transplantation confirms that the FN-RMS mesenchymal-enriched subfraction has tumor-propagating potential in vivo.
Fig. 6: RMS subtypes share common gene expression patterns and are arrested at distinct stages of fetal and embryonic muscle development.
Fig. 7: Mesenchymal-enriched FN-RMS TPCs share transcriptional and functional similarities with the bi-potent SkM. Mesen.

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Data availability

scRNA-seq and snRNA-seq data are available at the Gene Expression Omnibus (GEO) under accession GSE195709. Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

Code availability

All of the analysis scripts have been deposited at GitHub and can be accessed using the links: https://github.com/qinqian/sc_normal_muscle, and https://github.com/qinqian/rms_analysis.

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Acknowledgements

This work was supported by NIH grants no. R01CA154923 (D.M.L.), no. R01CA215118 (D.M.L.), no. R01CA211734 (D.M.L.), no. U54CA231630 (D.M.L.), no. R00HG008399 (L.P.), no. R35HG010717 (L.P.) and no. R01AR064327 (A.D.P.). Additional funding included the Liddy Shriver Sarcoma Initiative (D.M.L.), the MGH Research Scholars Program (D.M.L.), the Infinite Love for Kids Fighting Cancer Grant (D.M.L., F.D.C), the Rally Foundation (D.M.L.), The Truth 365 (D.M.L.), the Summer’s Way/Friends of TJ Young Investigator Award (Y.W.), the Tosteson & Fund for Medical Discovery Fellowship from MGH (C.Y.), the Alex’s Lemonade Stand Foundation Young Investigator Award (C.Y.), CIRM Quest DISC2-10696 (A.D.P.), UCLA BSCRC (A.D.P.), the Ayoub Centennial Chair (A.D.P.), the Paulie Strong Foundation (F.D.C.), The Grayson Fund (F.D.C.), Willens Family Fund (F.D.C.) and the Pediatric Cancer Foundation (F.D.C.). We thank the MGH Department of Pathology Flow and Image Cytometry Research Core which has been supported by NIH grants no. 1S10OD012027-01A1, no. 1S10OD016372-01, no. 1S10RR020936-01 and no. 1S10RR023440-01A1. We thank M. Dyer and E. Stewart from the Childhood Solid Tumor Network (CSTN) at St. Jude for a subset of PDX models used in this work. We thank A. Friedmann and D. Ebb from MGH Pediatric Hematology/Oncology department. We also thank D.C. O’Neill and L. Millet for helpful and stimulating discussions.

Author information

Author notes

  1. These authors contributed equally: Yun Wei, Qian Qin.

  2. These authors jointly supervised this work: Luca Pinello, David M. Langenau.

Authors and Affiliations

  1. Molecular Pathology Unit, Massachusetts General Research Institute, Charlestown, MA, USA

    Yun Wei, Qian Qin, Chuan Yan, Madeline N. Hayes, Sara P. Garcia, Daniel Do, Alexander H. Jin, Tiffany C. Eng, Karin M. McCarthy, Abhinav Adhikari, Maristela L. Onozato, A. John Iafrate, Mario L. Suvà, Luca Pinello & David M. Langenau

  2. Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA, USA

    Yun Wei, Qian Qin, Chuan Yan, Madeline N. Hayes, Daniel Do, Alexander H. Jin, Tiffany C. Eng, Karin M. McCarthy, Abhinav Adhikari, Maristela L. Onozato, A. John Iafrate, Mario L. Suvà, Luca Pinello & David M. Langenau

  3. Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA, USA

    Yun Wei, Qian Qin, Chuan Yan, Madeline N. Hayes, Daniel Do, Alexander H. Jin, Tiffany C. Eng, Karin M. McCarthy, Abhinav Adhikari & David M. Langenau

  4. Harvard Stem Cell Institute, Cambridge, MA, USA

    Yun Wei, Qian Qin, Chuan Yan, Madeline N. Hayes, Daniel Do, Alexander H. Jin, Tiffany C. Eng, Karin M. McCarthy, Abhinav Adhikari, Mario L. Suvà & David M. Langenau

  5. Department of Microbiology, Immunology, and Molecular Genetics, University of California Los Angeles, Los Angeles, CA, USA

    Haibin Xi & April D. Pyle

  6. Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California Los Angeles, Los Angeles, CA, USA

    Haibin Xi & April D. Pyle

  7. Center for Sarcoma and Connective Tissue Oncology, Department of Orthopedic Surgery, Massachusetts General Hospital, Boston, MA, USA

    Dimitrios Spentzos & Gunnlaugur P. Neilsen

  8. Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, USA

    Leonard H. Wexler & Filemon Dela Cruz

  9. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Mario L. Suvà

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Contributions

Y.W., D.M.L. and M.N.H. designed the project. Q.Q., S.P.G. and L.P. performed computational modeling and data analysis. Y.W., C.Y., D.D., A.H.J., A.A., K.M.M. and T.C.E. performed sequencing, mouse work and sample processing. H.X. and A.D.P. shared single-cell sequencing data of normal muscle development before publication. M.L.O. and A.J.I. validated fusion status by FOXO1-FISH break-apart assay. D.S., G.P.N., L.H.W. and F.D.C. contributed PDX models and patient samples. M.L.S. aided in high-throughput sequencing and computational approaches for single-cell sequencing data.

Corresponding authors

Correspondence to Luca Pinello or David M. Langenau.

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Competing interests

A.J.I. receives royalties from ArcherDx and consults for Paige.AI, Repare Therapeutics, Oncoclinicas Brasil and Kinnate Biopharma. M.L.S. is an equity holder, scientific co-founder and advisory board member of Immunitas Therapeutics. L.P. has financial interests in Edilytics and SeQure Dx, Inc. All potential competing interests are reviewed and managed by Massachusetts General Hospital and Mass General Brigham HealthCare in accordance with their conflict-of-interest policies. D.M.L. receives sponsored research funds from NextCure for an unrelated project.

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Nature Cancer thanks Junyue Cao, Max Wicha and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Frozen RMS patient samples have similar cell states as those in PDX models.

a,b, UMAP showing all cells sequenced from representative FN-RMS 20696. Non-tumor cells were assigned using Cellassign and clusterprofiler enricher analysis (a, n = 1,726 cells) and tumor cells analyzed for expression of diagnostic markers for rhabdomyosarcoma (b, n = 1,676 cells). In total, 3,402 cells were analyzed in this experiment. c, UMAP visualization of tumor cells from primary FN-RMS 20696 (n = 1,676 cells). d, Heatmap showing single cells (x axis) and genes enriched for specific transcription modules (y axis, FN-RMS 20696). Cells are arranged by UMAP clusters, combined based on expression similarity, and then assigned a specific cell state as noted. Cell numbers same as panel c. e, UMAP renderings for primary RMS samples juxtaposed with graphical analysis showing detected genes/tumor cells when analyzed across different cell states (n = 1,676 cells for sample 20696; n = 9,675 cells for 29806; n = 11,902 cells for 21202; and n = 6,188 cells for 20082).

Extended Data Fig. 2 Cell states are identified by scRNA sequencing in all RMS PDXs and similar numbers of detected genes per cell are found across cell states.

a, UMAP renderings of all PDXs, with exception of MAST111, MAST139, MAST85-r2 and MSK72117, which were shown in Figs. 1a and 2d. Number of tumor cells analyzed are denoted within the figure panels. b, Representative examples of FN-RMS (left) and FP-RMS (right). UMAP showing genes detected per cell (left). Violin plots showing genes detected within each cell for a given RMS subpopulation (right). c All PDX models assessed by violin plots denoting the number of detected genes per cell across RMS subpopulations.

Extended Data Fig. 3 A subset of fusion-positive RMS contain unique and tumor-specific cell clusters that express neural genes.

a, Top enriched molecular signatures from MSigDB are shown for each unique cell cluster identified from individual FP-RMS PDX models. False Discovery Rate (FDR) Q values noted. Tumor and cluster number are noted (that is, MSK74711-8). b, Venn diagram showing little overlap in gene expression across unique transcription clusters identified from different tumors. c, Upset plot quantifying the gene set enrichment of unique clusters with the GO_NEUROGENESIS gene set (P values defined by Fisher’s exact test).

Extended Data Fig. 4 RMS cells ubiquitously express a subset of muscle lineage and cancer-specific genes.

a,b, UMAP visualizations showing cell states (left panels) and compared with gene expression for MYOD1, DESMIN and MYC. Representative examples shown for fusion-negative (MAST39, n = 10,577 cells) and fusion-positive RMS (MAST95, n = 4,771 cells). c,d, Histological analysis of PDXs grown in NSG mice. Representative sections of three tumors shown with similar results. Hematoxylin and Eosin (left) and immunohistochemistry for MYOD1 and DESMIN (right). Fusion-negative (FN, c) and fusion-positive RMS (FP, d). Scale bar = 50 μm. n = 3 independently engrafted tumors were stained and imaged.

Extended Data Fig. 5 Immunofluorescence antibody staining reveals intermingling of cell states in PDX tumors.

a, Immunofluorescence staining within the central tumor mass. Dashed lines indicate clustered cell populations. Arrows denote rare cells detected by IF staining. Scale bar = 50 μm. b,c Heterogeneity identified by single-cell RNA sequencing (b) and compared with immunofluorescence staining of the central tumor mass (IF-C, c) or at the invasive edge (IF-E, c). Color coding denotes that immunofluorescence was detected in tumor cells within the sections analyzed. ND, not detected; NA, not applicable. Evenly distributed through tumor (ED) or clustered (C) based on immunofluorescence staining. d Quantitation of cell state percentages assessed by scRNA-sequencing or immunofluorescence (center of mass, C compared with tumor edge, E). Error bar equals s.e.m. (n = 4 image fields analyzed per condition, range 207–643 cells/field).

Extended Data Fig. 6 Cell state heterogeneity in primary patient samples, PDX models, single cell engrafted tumors and RD cells grown in mouse xenografts.

a–c, 3D renderings of gene expression for muscle (x axis), proliferation (z axis), mesenchymal-like (y axis) gene modules identified in RMS samples (a, FN-PDXs; b, FP-PDXs; c, primary patient samples). Individual cells are noted by dots and color coded based on cell assignments shown in Fig. 1d. Not detected (ND) denotes lack of a given cell state both in the initial UMAP cell cluster annotations and in 3D gene expression space. d, Combined UMAP visualization for all parental PDXs profiled (n = 10, and includes both replicates of MAST85) and single cell derived PDXs (n = 3). See Supplementary Table 1 for detailed information and cell numbers. e,f, Single cell RNA sequencing of RD xenograft (n = 2,619 tumor cells profiled). Heatmap showing single cells (x axis) and genes enriched for specific transcription modules (y axis, e). Cells are arranged by UMAP clusters, combined based on expression similarity, and then assigned a specific cell state as noted. f UMAP rendering of xenografted RD cells following single cell sequencing (left) and quantification of cell state composition of all 2,619 RD cells profiled (right). Similar cell states are observed in RD cells raised in 2D cell culture (see Fig. 3).

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Extended Data Fig. 7 Tumor-propagating potential is enriched in the mesenchymal-enriched tumor cell fraction in FN-RMS.

a,e,i,m, Flow cytometry analysis of FN-MSK74711 cells harvested directly from PDX tumors grown in NSG mouse (a) or cell line models (e,i,m) prior to (left) and after FACS enrichment (right two panels). b,f,j,n, Quantitative PCR confirming cell state enrichment following FACS (n = 2 independently engrafted tumors (MSK74711) and analyzed in triplicate from independent sorts, 6 datum points shown). Three replicates were analyzed from independent sorting of RD, 381 T and SMS-CTR cells. Two-way ANOVA followed by two-sided Student’s t-test comparison (*P < 0.05, **P < 0.01; ***P < 0.001 and ****P < 0.0001). c,g,k,o, Representative images of sphere size following FACS enrichment and plating for two weeks (scale bar = 20 μm). Shown is a representative micrograph of tumorspheres (left) with similar results seen across replicate wells. Combined quantification from two independent experiments (c,g,k) and three independent experiments in panel o. Three wells/experiment were completed and the combined analysis shown (o, +/− SEM noted). d,h,l,p, Quantification of tumor sphere numbers from representative limiting dilution experiments (datum points represent number of spheres found within individual wells from a single experiment). Experiments replicated twice (d,h,l) or three times (p) with similar results. Mean + /− SEM noted for SMS-CTR, two-way ANOVA followed by two-sided Student’s t-test (*P < 0.05, **P < 0.01, ***P < 0.001). Mesenchymal-enriched (Mesen, Mes or Me, muscle (Musc, Mu), interferon (INF), proliferative (Prolif).

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Extended Data Fig. 8 Limiting dilution cell transplantation confirms that mesenchymal-enriched cells from FN-RMS PDX 74711 are enriched for tumor propagating potential in vivo.

a Representative images of NSG mice engrafted with CHODL + /CD90 + mesenchymal-enriched or CHODL-/CD90- MSK74711 PDX RMS cells (all three mice from 10,000 cells/mouse group are shown). Mice were imaged at days post-transplantation as noted. Dashed lines delineate tumor. b, Latency of tumor regrowth following engraftment into NSG mice. TPC frequency + /− 95% confidence interval noted per condition in parenthesis. Quantification by ELDA *P < 0.05, **P < 0.01. n = 3 animals were transplanted for each limiting dilution across each of the four sorted cell populations analyzed. c, Flow analysis of tumors generated from sorted cell populations, mean + /− SEM noted, n = 3 independent tumors, *P < 0.05, **P < 0.01, ***P < 0.001 by two-sided Student’s t-test comparing the Mesen+ vs. Mesen− populations. d, Immunostaining of Ki67 proliferation and MF20 differentiation muscle markers in animals engrafted with FACs sorted cells. n = 3 independent tumors. For each tumor, four random fields were selected for quantification. mean + /− SEM., *P < 0.05, **P < 0.01, ***P < 0.001, by two-sided Student’s t-test.

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Extended Data Fig. 9 Subtype-specific RMS core signatures are expressed at specific muscle development stages.

a,b Dot plot renderings showing the expression of ten representative genes that comprise the fusion-negative or fusion-positive core signature across all PDXs and their identified cell states (a) and across normal muscle cells stratified by age (b). c UMAP rendering of scRNA sequencing data from embryonic (n = 5 samples), fetal (n = 4 samples), and adult skeletal muscle (n = 4 samples) showing expression of representative subtype-specific core signature genes. Week or year of life is noted (Wk and Yr, respectively). n = 3,251 cells analyzed across all stages of muscle development.

Extended Data Fig. 10 Osteogenic markers are expressed in the mesenchymal-enriched FN-RMS tumor propagating cells.

a, TSNE renderings denoting cell state (left) and compared with OGN and MGP expression in representative FN-RMS PDXs, MAST111 (n = 2,199 cells) and MAST39 (n = 10,577 cells). b, FACS sorting of RD and 381 T FN-RMS cells followed by Q-PCR validates the enrichment of osteogenic markers OGN and MGP within the mesenchymal-enriched subpopulation. FACs plots are representative across three independent experiments completed on different days (left). Q-PCR samples are the same as those rendered in Fig. 4 and Extended Data Fig. 7, Mean+/−SEM (n = 3 samples isolated from independent sorts completed on different days). ***P < 0.001, ****P < 0.0001 by ANOVA followed by two-sided Student’s t-test. c, Representative flow cytometric analysis confirming cell surface expression of OGN and MGP in mesenchymal-enriched subfractions of RD and 381 T RMS cells (completed in three independent biological replicates).

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Supplementary information

Supplementary Information

Representative example of sorting strategy used for FACS experiments.

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Supplementary Table 1

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Wei, Y., Qin, Q., Yan, C. et al. Single-cell analysis and functional characterization uncover the stem cell hierarchies and developmental origins of rhabdomyosarcoma. Nat Cancer 3, 961–975 (2022). https://doi.org/10.1038/s43018-022-00414-w

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