Discussion
Pleuropulmonary blastoma (PPB) is a rare malignant mesenchymal tumor in children. It usually presents with sarcoma-like pathological changes18,19. However, there is a gap in the understanding of PPB heterogeneity at the level of single genes. A better understanding of its heterogeneity may facilitate further exploration of the correlation with fetal lung development and the connection between PPB and other diseases such as congenital pulmonary adenomatoid malformation. We used scRNA-seq to roughly define the subtypes of muscle and cartilage in PPB as well as the trajectory of tumor development.
The development of muscle cells in PPB may be related to the differentiation of muscle satellite cells. By clustering, we have easily found striated muscle cells defined by classic genes such as DES . These muscle cells are refined intoMYOGhiMYOD1hi andPAX7hiMYF5hiMYOD1losubpopulations by DEGs. The direction of PPB differentiation is controlled by PAX7hiMYF5hiactive satellite myogenic cells with the capacity for self-renewal20,21. Highly expressed PAX7 is essential for maintenance of satellite stem cells and targets its downstream gene MYF5 for conversion intoPAX7hiMYF5hi satellite myogenic cells without MYOD1 . More precisely, this subpopulation in our sample is at the transition stage from satellite myogenic cells to myoblasts (PAX7hiMYF5hiMYOD1hi ) because of its low expression of MYOD1 . These satellite myogenic cells in PPB express MSC , which generally emerges between embryonic day 10.5 and 16.5 in mice 22 and coordinates with MYF5 gene23 to participate in the proceeding myogenesis21,24. Cells expressing integral membrane protein 2A (ITM2A ) marks a subset that emerges late among satellite myogenic cells and are in the middle of the entire PPB differentiation trajectory. It controls calcium channels to regulate expression of MYOG protein25 and prepares for subsequent mature MYOGhi cells. TheMYOGhi subpopulation comprises mature myocytes (MYOGhiMYL4hiTTNhi ) that exit the cell cycle25. MYOG protein is essential in late myogenesis26 without functional overlap with MYLF or MYOD1 protein27. We have found that theseMYOGhi cells are at the terminal stage of PPB tumor muscle cell development (Fig.4B). The trajectory of skeletal muscle cells in PPB is similar to the differentiation and proliferation of active satellite cells, which is consistent with the fact that the tumor originates from mesoderm.
The cartilage subpopulation is defined by some bone markers emerging in osteogenesis, such as TWIST1 (crucial regulator of the differentiation of skeletal progenitor/stem cell28,29) and HTRA1 (generally expresses in hypertrophic chondrocytes30, but expresses in a large range in PPB). Such a population comprises naïve prechondrocytes and relatively mature chondrocytes. The prechondrocytes with high expression of SOX9 , PAX1 , PAX9 , and SOX5 are at the departure stage of PPB chondrogenesis. Sox9 is a crucial gene in chondrogenesis and initiates the chondrocyte differentiation program31. SOX9 together with SOX5activated by SOX9 gene32 are regulatory markers of mesenchymal cells and prechondrocytes, but disappear after the formation of prehypertrophic chondrocytes33. Paralogous transcription factors PAX1/9 regulate vertebral column development34, activate chondrogenic mesenchymal cells to differentiate into prechondrocytes under the combined action ofSOX9 , and are required for maintenance of BMP4 , a osteogenesis marker35,36. Unfortunately, we currently couldn’t subdivide the large group of prehondrocytes with DEGs further. Located downstream of PPB chondrogenesis areDKK2hi chondrocytes andOGNhiMGPhi chondrocytes, as indicated by the pseudotime trajectory and gene markers. MGP protein is a kind of calcification inhibitor37 synthesized by late, but not telophase, chondrocytes38. DKK2also plays a role in the late stages of osteoblast differentiation to produce mineralized matrices39,40. However, the occurrence time and relationship between the two mature chondrocytes subtypes downstream remain unclear, perhaps because two subtypes have different functions.
The cellular components of pleuropulmonary blastoma and its development trajectory suggest strong intratumoral heterogeneity and the ability to differentiate in multiple directions to skeletal muscle and cartilage. Our atlas of PPB may form the basis for further tumor detection and a more defined molecular mechanism of PPB tumorigenesis. And more detailed cell subtypes and associations between subtypes require our further research.