Osteosarcoma (OSA) is a highly aggressive and metastatic neoplasm of both the canine and human patient and is the leading form of osseous neoplasia in both species worldwide. To gain deeper insight into the heterogeneous and genetically chaotic nature of OSA, we applied single-cell transcriptome (scRNA-seq) analysis to 4 canine OSA cell lines. This novel application of scRNA-seq technology to the canine genome required uploading the CanFam3.1 reference genome into an analysis pipeline (10X Genomics Cell Ranger); this methodology has not been reported previously in the canine species, to our knowledge. The scRNA-seq outputs were validated by comparing them to cDNA expression from reverse-transcription PCR (RT-PCR) and Sanger sequencing bulk analysis of 4 canine OSA cell lines (COS31, DOUG, POS, and HMPOS) for 11 genes implicated in the pathogenesis of canine OSA. The scRNA-seq outputs revealed the significant heterogeneity of gene transcription expression patterns within the cell lines investigated (COS31 and DOUG). The scRNA-seq data showed 10 distinct clusters of similarly shared transcriptomic expression patterns in COS31; 12 clusters were identified in DOUG. In addition, cRNA-seq analysis provided data for integration into the Qiagen Ingenuity Pathway Analysis software for canonical pathway analysis. Of the 81 distinct pathways identified within the clusters, 33 had been implicated in the pathogenesis of OSA, of which 18 had not been reported previously in canine OSA.

骨肉瘤（OSA）是犬类和人类患者的高度侵袭性和转移性肿瘤，并且是全世界这两个物种中骨性肿瘤形成的主要形式。为了更深入地了解OSA的异质性和遗传混沌性质，我们将单细胞转录组（scRNA-seq）分析应用于4种犬OSA细胞系。scRNA-seq技术在犬基因组中的这种新颖应用需要将CanFam3.1参考基因组上载到分析管道中（10X Genomics Cell Ranger）；据我们所知，以前在犬种中尚未报道这种方法。通过将scRNA-seq输出与逆转录PCR（RT-PCR）的cDNA表达和Sanger测序对4种犬OSA细胞系（COS31，DOUG，POS，和HMPOS）的11个基因与犬OSA的发病机制有关。scRNA-seq输出揭示了所研究的细胞系（COS31和DOUG）内基因转录表达模式的显着异质性。scRNA-seq数据显示了COS31中10个不同的簇，它们具有相似的转录组表达模式。在DOUG中鉴定出12个簇。另外，cRNA-seq分析提供了数据，可以整合到Qiagen Ingenuity Pathway Analysis软件中，以进行规范的途径分析。在集群中确定的81种不同途径中，有33种与OSA的发病机制有关，其中18种以前在犬OSA中未见报道。scRNA-seq输出揭示了所研究的细胞系（COS31和DOUG）内基因转录表达模式的显着异质性。scRNA-seq数据显示了COS31中10个不同的簇，它们具有相似的转录组表达模式。在DOUG中鉴定出12个簇。另外，cRNA-seq分析提供了数据，可以整合到Qiagen Ingenuity Pathway Analysis软件中，以进行规范的途径分析。在集群中确定的81种不同途径中，有33种与OSA的发病机制有关，其中18种以前在犬OSA中未见报道。scRNA-seq输出揭示了所研究的细胞系（COS31和DOUG）内基因转录表达模式的显着异质性。scRNA-seq数据显示了COS31中10个不同的簇，它们具有相似的转录组表达模式。在DOUG中鉴定出12个簇。另外，cRNA-seq分析提供了数据，可以整合到Qiagen Ingenuity Pathway Analysis软件中，以进行规范的途径分析。在集群中确定的81种不同途径中，有33种与OSA的发病机理有关，其中18种以前在犬OSA中未见报道。cRNA-seq分析提供了用于整合到Qiagen Ingenuity Pathway Analysis软件中以进行规范路径分析的数据。在集群中确定的81种不同途径中，有33种与OSA的发病机理有关，其中18种以前在犬OSA中未见报道。cRNA-seq分析提供了用于整合到Qiagen Ingenuity Pathway Analysis软件中以进行规范路径分析的数据。在集群中确定的81种不同途径中，有33种与OSA的发病机理有关，其中18种以前在犬OSA中未见报道。