Constructing lineage hierarchies of the PPB tumor
To further understand the internal correlation of tumor cells in both subtypes, we constructed differentiation trajectories of cells in pseudotemporal order using Monocle after renaming the clusters of two subtypes to obtain two tightly connected differentiation tumor lineages. In the skeletal muscle lineage, FABP7hisatellite myogenic cells (clusters 6 and 9,FABP7hiPAX7hiMYF5hiMSChi ) were more primordial and at the start of their trajectory. Highly expressed genes, such as PAX7 and MYF5 , were stable in the early and middle stages of the pseudotime and then declined rapidly (Fig.4B). ITM2Ahi satellite myogenic cells (clusters 2 and 4,ITM2AhiPAX7hiMYF5hiMSChi ) emerged slightly later, but were still more primitive thanMYOGhi differentiated myocytes (cluster 5) that dominated on the terminal branch of the trajectory (Fig.4A,4B). The cartilage lineage was similar to the muscle cells. NaïveSOX9hiPAX9hiPAX1hiprechondrocytes (clusters 1, 3, 8, and 11) drove sequential differentiation and generatedMGPhiOGNhi chondrocytes (cluster 7) and DKK2hiTNMDhichondrocytes (cluster 10) (Fig.4C,4D). However, there was no evidence to order the time sequence of these two chondrocyte subpopulations and we could not define the detailed difference between them, although they converged on the same branch in the trajectory. Interestingly, whether in cartilage or muscle subtypes, when primitive cells occupying the trunk of the trajectory developed into differentiated daughter cells, they first produced a small branch filled with daughter cells and then a large branch again from the trunk. The mechanism was not well understood and might be related to cell activation.