Intratumoral heterogeneity of tumor cells
We divided the tumor cluster into two cell subtypes of muscle and cartilage based on pathological characteristics of the sample and DEGs of each cell clustered by Seurat (mentioned more specifically in the next parts). To further explore its intratumoral heterogeneity, we used nonnegative matrix factorization (NMF) to rebuild 20 modules of each subtype and scored each cell for these expression modules using AddModuleScore in Seurat. There were four programs (program A–D) with a relatively high Pearson correlation (Fig.1E) associated with skeletal system development (such as SFRP1 and HTRA1 ), DNA packaging (such as MKi67 and PTTG1 ), RNA transport (such as MALAT1 and NK1R ), and muscle tissue development (such as MYLPF and ACTC1 ), respectively (Supporting Information Figure S2). In the scope definition of program D, we proactively ignored some ribosomal gene-related modules (such as Mus_v5 and Carti_v14) to obtain a clear function of cell subsets (Fig.1E). The classification of tumor cells was validated again by NMF clustering. The top 30 highly expressed genes in bone- and muscle-related programs were separately and highly distributed in both subtypes. Other cell cycle programs were expressed in several clusters of both subtypes, which may be the only similarity between cell functions of the two subtypes (Fig.1F).
Skeletal muscle cells in PPB
Clusters 2, 4, 5, 6, and 9 (4 038 cells) were kinds of skeletal muscle tumor cells. Using t-SNE projection, we found that classic muscle cell genes DES , NEB , TNNT1 , MYOD1 , TNNI1 ,ACTC1 , and RYR1 were highly expressed in these clusters (Fig.2A, Supporting Information Figure S3). Immunohistochemical analysis of DES and MyoD1 protein expression confirmed their presence in these cells (Fig.2B). We continued to subdivide this group and found that it was roughly divided into two subpopulations including mature differentiated myocytes (cluster 5,MYOGhiTTNhiENO3hiMYL4hi , 726 cells) and proliferating satellite myogenic cells (clusters 2, 4, 6, and 9,PAX7hiMYF5hiMSChiSTC1hi , 3 312 cells). We found positivity for anti-MYOG antibody staining in these minor differentiated myocytes, showing scattered brown nuclear muscle cells (Fig.2C). Furthermore, these satellite myogenic cells, which were perhaps cells in transition to myoblasts, were namedPAX7hiMYF5hi satellite myogenic cells, and clusters 6, 9 (1 149 cells) among these satellite cells clusters expressed metabolic regulator FABP7 , while the other clusters (2 and 4, 2 163 cells) had high expression ofITM2A , a gene expressed in a muscle cell line and associated with myogenesis16. GO analysis verified the potential biological processes of these clusters. Cluster 5 had more precise muscle characteristics such as participating in the muscle system process. The other clusters contained more cell cycle genes at G2/M phase with the biological process of chromosome segregation (Fig.2C).