Legends
Fig.1. NMF and Seurat analysis verify heterogeneity in PPB. (A) General process of droplet-enabled single cell sequencing. (B). Heatmap of the top five marker genes for each cluster using Seurat. Yellow indicates high expression of a particular gene, and purple indicates low expression. (C) Tumor inferCNV in comparison with normal fibroblasts (fibroN) indicates the CNV changes of tumor cells. A reference group is shown at the top, while the other clusters are below. Brown is chromosome gain, and blue is chromosome loss. (D) Clustering of 10 007 cells and definition of cell types in PPB are displayed in t-SNE plot. (E) Pairwise correlations between the expression modules of the two subtypes (rows, columns) were made to acquire four positive correlated programs. Brown indicates a high Pearson correlation. (F) Top 30 genes in each program were re-clustered and shown in a heatmap. Programs are shown on the y-coordinate and previous clusters of cell types are shown on the x-coordinate.
Fig.2. Subsets of smooth muscle cells in PPB. (A) Feature plots of muscle-related genes revealed the subpopulations of muscle cells. (B) Immunohistochemical staining of Desmin expression (brown) in combination with other muscle proteins, MyoD1 (brown) and MYOG (brown), within PPB tissue sections show striated muscle cells. (C) GO analysis of the selected top 20 GO terms for clusters 5 and 6 is shown in bar plots.
Fig.3. Subsets of chondrocytes in PPB. (A) Feature plots of genes defining various subpopulations of tumor cartilage. (B) GO analysis of the selected top 20 GO terms for clusters 1, 3, 7, and 10 are shown on the bottom.
Fig.4.Two lineage hierarchies of the PPB tumor. (A), (C) Muscle and cartilage cells were respectively divided into three cell subtypes and automatically shown in a Monocle-generated pseudotime trajectory. Dark blue is the start of pseudotime. A proposed model summarizing the lineage hierarchies from relatively stem cells to differentiated cells is shown. PAX7hiMYF5hiMYOD1himyoblast in (A) are defined transitional cell during pseudotime and drawn by a dashed line. (B), (D) Trendgrams of highly expressed genes in the cell subtypes during pseudotime.
Supporting Information Figure
Supporting Information Figure S1Feature and violin plots of genes defining the rough cell subtypes of 10 007 collected single cells.
Supporting Information Figure S2 (A)-(D) GO analysis of the selected top 10 GO terms for programs A-D is shown in bar plots.
Supporting Information Figure S3 Feature plots of genes defining the subpopulations in muscle and cartilage cells of PPB.