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).