Novel application of single-cell next-generation sequencing for determination of intratumoral heterogeneity of canine osteosarcoma cell lines

Abstract

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.

Immune pathways and TP53 missense mutations are associated with longer survival in canine osteosarcoma

“…Among the gene changes they confirmed using RT-qPCR and IHC analysis, we also saw elevated expression of MMP3, SLC2A1, DKK3, POSTN, and ASPN in OSA tumors compared to normal bone samples. A recent article reported loss of PTEN expression in canine OSA cell lines (POS/HMPOS) but continued expression of RB1 in all four tested cell lines 30 . WES analysis of 8 canine OSA cell lines coupled with prior microarray analysis identified TP53 missense mutations in 3 cell lines (Supplementary Fig.…”

Immunohistochemical Characterisation of GLUT1, MMP3 and NRF2 in Osteosarcoma

“…Canine OSA remains challenging to treat and carries a poor prognosis due to the very aggressive and metastatic nature of the tumors. Given how common OSA is in dogs and likely genetic contribution to OSA in large breeds, we (16) and others (25,(41)(42)(43) have sought to extend understanding of the molecular determinants of OSA in dogs and to compare these results to those obtain from OSA from people.…”

Deep targeted sequencing of cytological tumor cells using whole genome amplification

“…22 In previous research, a comprehensive single-nucleotide polymorphism array and analysis of intratumoral heterogeneity was conducted using whole genome-amplified DNA retrieved from culture cells or frozen tissues isolated by laser capture microdissection (LCM) or cell sorting. [23][24][25][26] The application of WGA to cytological specimens was first reported by Beltinger et al in 1997. 27 Subsequently, Murphy et al reported genome structural variant analysis and single nucleotide variant (SNV) analysis using a large number of tumor cells.…”

“…The MDA method amplifies genomic DNA with a lower error rate than other methods 21 ; therefore, it is applied in the detection of somatic variants 22 . In previous research, a comprehensive single‐nucleotide polymorphism array and analysis of intratumoral heterogeneity was conducted using whole genome‐amplified DNA retrieved from culture cells or frozen tissues isolated by laser capture microdissection (LCM) or cell sorting 23–26 …”