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.