| Objectives Osteosarcoma occur most frequently in adolescents and are associated with a high degree of malignancy and poor prognosis.At present,neoadjuvant chemotherapy and surgical treatment are the main treatments for osteosarcoma.Over the past 30 years,there has been no change in overall survival for osteosarcoma,and immunotherapy has not achieved desirable clinical outcomes.The reasons are the large individual differences,strong heterogeneity and complex tumor microenvironment.Drug resistance,recurrence,and metastasis of osteosarcoma often lead to poor prognosis.In order to develop targeted therapies for osteosarcoma,a better understanding of its complex tumor microenvironment is needed.The purpose of this study was to analyze the prior treatment osteosarcoma map using single-cell transcriptome technology,identify the heterogeneity of various cells in the osteosarcoma microenvironment,and calculate the interactions between cells to elucidate the important cell characteristics of osteosarcoma angiogenesis and pathological fracture.Single cell sequencing data of osteosarcoma post treatment were combined to identify tumor cell populations insensitive to current neoadjuvant chemotherapy,and the features of endothelial cell dysfunction,immune infiltration and osteoclast osteolysis in osteosarcoma post treatment were calculated,providing theoretical support for clinical drug research and development.Methods Single cell data of 6 osteosarcoma patients prior treatment and7 osteosarcoma patients post treatment were included in this study.We analyzed the profiles of myeloid cells,osteosarcoma cells,NK/T cells,osteoclasts,cancer-associated fibroblasts,and B cells in terms of gene expression,cell function analysis,transcription factor activity analysis,pseudotime analysis,and RNA velocity analysis by downdimension clustering of single cell data from 6osteosarcoma patients prior treatment.Osteosarcoma cells were identified from the perspective of copy number variation,and the osteosarcoma cell groups of interest were identified by deconvolution algorithm combined with bulk RNA sequencing data,and the survival significance was determined.Macrophage and osteosarcoma cell typing were validated by immunofluorescence.The interaction between cells was verified by cell communication analysis,co-culture experiment and ELISA experiment.We analyzed the profiles of myeloid cells,NK/T cells,and osteoclasts in terms of gene expression,cell wind energy analysis,transcription factor activity,pseudotime analysis,and RNA rate analysis by downdimension clustering of single cell data of 13 osteosarcoma patients prior treatment and post treatment.The insensitive group of osteosarcoma to chemotherapeutic drugs was calculated by drug sensitivity analysis.The role of osteosarcoma cells in promoting endothelial dysfunction was calculated by spatial analysis,logistic regression and cell communication analysis.Results We obtained tissue from 6 patients with prior treatment osteosarcoma and sequenced them using the 10×Genomics platform.A total of29278 cells were obtained after quality control,and 9 main cell types were identified.The identified osteosarcoma cells were divided into 5 subpopulations and the deconvolution algorithm was used to determine the osteosarcoma cell subpopulations with significant prognostic correlation.We then reported on different transcription patterns in osteosarcoma cell subtypes and identified key transcription factors associated with survival prognosis.Furthermore,we revealed that C1＿osteoblastic OS cells promote osteoclast differentiation and bone resorption by TNFSF11＿TNFRSF11A,and regulate DEPLETION of CD8~+T cells by PVR＿TIGIT and FAM3C＿PDCD1.CD8~+T cell depletion is regulated by C3＿TXNIP~+macrophages and C5＿IFIT1~+macrophages through LGALS9＿HAVCR2.C3＿TXNIP~+macrophages,C5＿IFIT1~+macrophages,cancer-associated fibroblasts,and osteosarcoma cells regulate endothelial angiogenesis through VEGFA＿KDR and VEGFA＿FLT1.A total of 108176 cells were collected from 13 osteosarcoma patients with prior or post treatment,and 10 main cell types were identified.In osteosarcoma cells,220 genes were up-regulated post treatment compared with those prior treatment,25 genes were associated with better prognosis,306 genes were down-regulated post treatment compared with those prior treatment,and 21genes were associated with worse prognosis.The specific target of tumor necrosis rate on survival was analyzed from the perspective of tumor necrosis rate post treatment.We identified Treatment cluster 1(TC1)that is insensitive to neoadjuvant chemotherapy in osteosarcoma cells and constructed a high-quality survival prognosis model.We also identified five genes that are extremely regulated by drug-resistant transcription factors and identified the heterogeneity of their metabolism-related pathways.TC1 distinguishes TC0 and the tumor necrosis rate>90%cluster metabolic pathways are mainly:vitamin B2metabolism of riboflavin,angiotensin original to angiotensin metabolism,amine source sex hormone metabolism,Foxo mediated gene transcription,oxidative stress,metabolism and neurons metabolism of creatine,metabolism of bile acid and bile salt,aspartic acid and asparagine metabolism.The C5＿endothelial cells associated with metastasis was identified and the spatial distance between TC1and C5＿endothelial cells was proved to be near at the spatial projection level.We revealed TC1 via CD44＿SELE mediated C5＿Endothelial cells dysfunction.At the same time,we also identified the key gene CD63 in TC1 that affects the infiltration of CD8~+T cells.CD63 knockdown of TC1 can increase the infiltration of CD8~+T cells,and CD63 overexpression of TC1 can reduce the infiltration of CD8~+T cells.In pathological osteolysis of osteosarcoma,we identified that current neoadjuvant chemotherapy drugs can inhibit the migration,differentiation,and fusion of osteoclasts in osteosarcoma tissues,but cannot affect the proliferation of osteoclasts.Conclusion1.Prior treatment,osteosarcoma cells with inflammatory(C1＿osteoblastic OS cells)and osteoblastic(C5＿osteoblastic OS cells)functions were associated with poorer survival.2.Prior treatment,osteosarcoma inflammatory cell population C1＿osteoblastic OS cells promoted osteoclast differentiation and bone resorption function through TNFSF11＿TNFRSF11A,and regulated exhaust of CD8~+T cells through PVR＿TIGIT and FAM3C＿PDCD1,and regulated angiogenesis of Endothelial through VEGFA＿KDR.3.Post treatment,the activity of TC1 transcription factors FOXF1,SETDB1,DLX2,EN1 and ZIC5 in osteosarcoma cell population increased and regulated its drug resistance phenotype.4.Overexpression of CD63 can reduce the infiltration of CD8~+T cells into osteosarcoma TC1 cells,while knockdown of CD63 can increase the infiltration of CD8~+T cells into osteosarcoma TC1 cells.5.C5＿endothelial cells are associated with metastasis of osteosarcoma,and the specific mechanism is that they are spatially close to osteosarcoma TC1 cells,which has endothelial cell dysfunction mediated by CD44＿SELE.6.Post treatment,neoadjuvant chemotherapy drugs can inhibit the migration,differentiation and fusion of osteoclasts,thus inhibiting the pathological osteolysis process. |
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