Identification Of PDPN-Positive Sarcoma Stem-Like Cells In Soft Tissue Sarcoma And The Potential Mechanism Of Formation

Sarcoma is a rare malignant stromal tumor,which is divided into osteosarcoma and soft tissue sarcoma（STS）.Regarding STS,their rarity,ubiquity,and diversity of clinical manifestations at the onset of the disease make it difficult to diagnose.As a result,32 to70 percent of STS patients are not treated according to international guidelines.However,correctly locating patients suspected of STS for appropriate diagnosis and treatment management maximizes the chances of a favorable outcome and prognosis.However,currently,due to the complex classification of STS and the large molecular mutant spectrum,the treatment methods are relatively simple.Surgery and chemoradiotherapy are the main clinical treatments for STS.Therefore,the treatment and management of STS are still quite difficult,and the prognosis is poor.It is particularly important to identify the specific pathogenesis of STS.Cancer stem cells（CSCs）are subtypes of tumor cells that are associated with key steps in cancer,including tumor progression,treatment resistance,relapse,and in some pathological cases metastasis.Sarcomas,including STS,also have a class of tumor stem cells that are sarcoma stem cells during their development.Sarcoma stem cells are very heterogeneous.The heterogeneity of sarcoma stem cells may depend on the type of sarcoma mutation.The formation of sarcoma stem cells is an important factor in the occurrence,development,and drug resistance of sarcoma.It is important to understand the formation mechanism of sarcoma stem cells.In addition,finding new markers of sarcoma stem cells is of great benefit to the study of the formation mechanism of tumor stem cells and the development of targeted drugs.Mesenchymal stem cells（MSCS）are a kind of mesenchymal stem cells with multidirectional differentiation potential.The classical tumor stem cell theory suggests that mesenchymal stem cells are the cellular origin of sarcomas.Starting with mesenchymal stem cells and following a given differentiation path through progenitor cells to eventually differentiated cells,it is natural to expect that a portion of this hierarchy will survive in sarcomas.Thus,we can hypothesize the existence of sarcoma stem cells capable of self-renewal and differentiation,as in cancer and leukemia.Of course,only certain types of sarcomas,such as osteosarcoma,chondrosarcoma,liposarcoma,leiomyosarcoma,or rhabdomyosarcoma,are allowed to follow certain differentiation pathways reminiscent of physiological differentiation.Other types of sarcomas,diagnosed as undifferentiated pleomorphic sarcoma or merely spindle cell sarcoma,do not show any easily recognized pattern of differentiation.Indeed,even in high-grade tumors of the former sarcoma type,the remaining differentiation capacity is not easily recognized.In this case,tumor differentiation may be diverse in terms of function,such as clonicity,tumorigenicity,therapeutic resistance,motility,and invasiveness.But how mesenchymal stem cells turn into sarcoma stem cells has not been clear.Protein tyrosine phosphatase non-receptor type 11（PTPN11）encodes Src homologous region 2（SH2）containing protein tyrosine phosphatase 2（SHP2）.Under physiological conditions,SHP2 is self-inhibitory and has almost no catalytic activity.However,activation of the mutant SHP2 structurally exposes the protein tyrosine phosphatase（PTP）domain,enabling it to dephosphorylate downstream proteins or act as a signal adapter protein,thereby triggering carcinogenic signaling cascades such as RAS/ERK,JNK,m TOR,PI3K/AKT,and TP53.SHP2 mutations are closely associated with a number of diseases,such as Noonan syndrome and LEOPARD syndrome,where Gain-of-function（GOF）mutations in SHP2 are found in Noonan syndrome and related cancers,Loss-of-function（LOF）mutations in SHP2 are common in LEOPARD syndrome.Although GOF mutations in SHP2 have been found in cancer,the underlying mechanism is unknown.In addition,previous laboratory studies have shown that Sh P2^E76K activating mutations can induce malignant transformation of mesenchymal stem cells to form soft tissue sarcomas.However,whether the mutated mesenchymal stem cells activated by SHP2^E76K transform into sarcoma stem cells and the mechanism of its formation remain to be clarified.In this work,we found that mouse mesenchymal stem cells with SHP2^E76K mutations underwent malignant transformation into sarcoma stem-like cells.Further proteomic study on WT and SHP2^E76K mutant mouse mesenchymal stem cells showed that compared with WT mouse mesenchymal stem cells,SHP2^E76K mouse mesenchymal stem cells expressed higher level of Podoplanin（PDPN）.PDPN-positive SHP2^E76Kmouse mesenchymal stem cells showed the characteristics of sarcoma stem-like cells.In addition,the results of clinical samples suggest that PDPN-positive liposarcoma cells exhibit the potential of sarcoma stem-like cells compared with PDPN-negative liposarcoma cells.Proteomic KEGG pathway analysis and cholesterol assay showed that cholesterol synthesis was involved in the formation of PDPN-positive sarcoma stem-like cells.Inhibition of cholesterol synthesis can reduce the malignancy of PDPN-positive sarcoma stem-like cells in mice.Further analysis showed that N-acetylglucosamino-6-phosphate deacetylase（Naga）,which regulates lipid synthesis,was underexpressed in SHP2^E76K mouse mesenchymal stem cells.Higher expression of Naga inhibited the malignancy of SHP2^E76K mouse mesenchymal stem cells and decreased the proportion of PDPN-positive sarcoma stem-like cells.Therefore,this study identified a novel sarcoma stem-like cell subpopulation and suggested that Naga may provide a new therapeutic target for the treatment of soft tissue sarcomas.Objective:To investigate whether PDPN is a new marker of soft tissue sarcoma stem cells and explore its formation mechanism.Methods:WT and SHP2^E76K mouse mesenchymal stem cells were first isolated and potential tumor stem cell surface molecular markers were screened by proteomics.Then,PDPN-positive and PDPN-negative SHP2^E76K mouse mesenchymal stem cells were separated,and malignant biological experiments such as tumor spheroid formation experiment and continuous transplantation experiment were conducted to examine whether PDPN-positive SHP2^E76K mouse mesenchymal stem cells had sarcoma stem cell characteristics.Next,in order to verify the presence of PDPN-positive sarcoma stem cell-like cells,6 clinical liposarcoma samples and 6 normal adipose tissues were collected.The proportion of PDPN-positive cells in liposarcoma was determined by flow cytometry.In addition,PDPN-positive and PDPN-negative liposarcoma cells were further isolated,and biological experiments such as tumor spheroid formation experiment and continuous transplantation experiment were also used to verify whether PDPN-positive sarcoma cells has significantly increased malignant biological potential of proliferation,metastasis,invasion and so on compared with PDPN-negative liposarcoma cells.Next,KEGG pathway analysis was used to identify potential signaling pathways that may regulate the formation of sarcoma stem-like cells from SHP2^E76K mouse mesenchymal stem cells.PDPN-positive and PDPN-negative SHP2^E76K mouse mesenchymal stem cells were separated from human liposarcoma cells.The role of lipid rafts in sarcoma stem cell-like cells was determined by immunofluorescence staining and cholesterol content detection.To verify the role of lipid rafts in sarcoma stem cells,we also added a lipid raft specific inhibitor,MβCD,to detect PDPN expression and lipid raft formation.In addition,cholesterol content and the ability of PDPN-positive cells to form tumor sphere,tumor,metastasis and cloning were also tested,so as to further verify the role of cholesterol-mediated lipid raft formation in the stemness maintenance of PDPN-positive cells.Finally,we used proteomics to identify potential molecules that may regulate lipid synthesis dysfunction in SHP2^E76K mouse mesenchymal stem cells.The role of this molecule in PDPN-positive sarcoma stemlike cell formation was further verified by lentivirus transfection and overexpression.Results:1.PDPN-positive SHP2^E76K mouse mesenchymal stem cells could be sarcoma stem-like cells.1.1 The expression of PDPN in SHP2^E76K mouse mesenchymal stem cells was significantly higher than that in wild-type mouse mesenchymal stem cells.1.2 Compared with PDPN negative SHP2^E76K mouse mesenchymal stem cells,PDPN positive SHP2^E76K mouse mesenchymal stem cells have stronger soft agar cloning ability.1.3 Compared with PDPN-negative SHP2^E76K mouse mesenchymal stem cells,PDPN-positive SHP2^E76K mouse mesenchymal stem cells have stronger ability to form microspheres.1.4 Compared with PDPN-negative SHP2^E76K mouse mesenchymal stem cells,PDPN-positive SHP2^E76K mouse mesenchymal stem cells have stronger tumor formation ability.2.PDPN positive human liposarcoma primary cells are sarcoma stem cell-like cells.2.1 The percentage of PDPN positive cells in human liposarcoma was significantly higher than that in adjacent tissues.2.2 Compared with PDPN-negative human liposarcoma primary cells,PDPN-positive human liposarcoma primary cells have stronger plate clone formation ability.2.3 PDPN-positive human liposarcoma primary cells have stronger soft agar clone formation ability than PDPN-negative human liposarcoma primary cells.2.4 PDPN-positive human liposarcoma primary cells have stronger metastasis and invasion ability than PDPN-negative human liposarcoma primary cells.2.5 PDPN-positive human liposarcoma primary cells have stronger microsphere formation ability than PDPN-negative human liposarcoma primary cells.2.6 PDPN-positive human liposarcoma primary cells have stronger tumor formation ability than PDPN-negative human liposarcoma primary cells.3.Cholesterol-mediated lipid raft is essential for maintaining the stemness of PDPN-positive sarcoma stem cell-like cells in mice.3.1 Proteomic KEGG analysis showed that SHP2^E76K mouse mesenchymal stem cells were rich in cholesterol biosynthesis related genes compared with wild-type mouse mesenchymal stem cells.3.2 The cholesterol level of SHP2^E76K mouse mesenchymal stem cells is higher than that of wild-type mouse mesenchymal stem cells.3.3 The cholesterol level of mesenchymal stem cells in PDPN positive SHP2^E76Kmice was higher than that in PDPN negative SHP2^E76K mice.3.4 MβCD inhibits the expression of lipid raft marker Caveolin-1 in SHP2^E76Kmesenchymal stem cells.3.5 MβCD inhibits the formation of SHP2^E76K mesenchymal stem cell plate clone formation.3.6 MβCD inhibits the formation of soft agar clone of SHP2^E76K mesenchymal stem cells3.7 Inhibitory effect of mβCD on microspheres formation of SHP2^E76Kmesenchymal stem cells.3.8 MβCD inhibited PDPN-positive SHP2^E76K sarcoma stem-like cells.4.Cholesterol-mediated lipid raft formation promotes stemness of PDPN-positive human liposarcoma stem-like cells4.1 The cholesterol level of human liposarcoma cells is higher than that of normal cells.4.2 The cholesterol level of PDPN positive human liposarcoma cells was higher than that of PDPN negative human liposarcoma cells.4.3 Co-localization of PDPN and lipid raft marker Caveolin-1 in human liposarcoma cells.4.4 MβCD inhibits the formation of human liposarcoma cell plate clone.4.5 MβCD inhibits the formation of soft agar clone of human liposarcoma cells.4.6 Inhibitory effect of mβCD on microspheres formation in human liposarcoma cells.4.7 Inhibitory effect of mβCD on PDPN positive cells in human liposarcoma cells.5.N-acetylglucosamin-6-phosphate deacetylase（Naga）is a key molecule that regulates lipid raft formation in PDPN-positive sarcoma stem cells.5.1 The expression of Naga in SHP2^E76K mouse mesenchymal stem cells was significantly higher than that in wild-type mouse mesenchymal stem cells.5.2 The expression of Naga in SHP2^E76K mouse mesenchymal stem cells after lentivirus transfection was significantly higher than that in no-loaded group.5.3 Overexpression of Naga rescues plate cloning ability of SHP2^E76K mouse mesenchymal stem cells.5.4 Overexpression of Naga saves metastatic ability of SHP2^E76K mouse mesenchymal stem cells.5.5 Overexpression of Naga rescues the ability of SHP2^E76K mouse mesenchymal stem cells to form microspheres.5.6 Overexpression of Naga saves tumor formation ability of SHP2^E76K mouse mesenchymal stem cells.5.7 Ratio of sarcoma stem cell-like cells saved by overexpression of Naga in PDPN positive SHP2^E76K mice.Conclusion:1.PDPN may be a novel marker of soft tissue sarcoma stem-like cells.2.Cholesterol-mediated lipid rafts are essential for maintaining the stemness of PDPN-positive sarcoma stem-like cells.3.Naga is a key molecule that regulates the abnormal formation of lipid rafts in PDPN-positive sarcoma stem-like cells.