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.

Keywords: canine; neoplasia; osteosarcoma; single-cell transcriptomics; tumor heterogeneity.

Figure 1.

RT-PCR agarose gels from 4 canine osteosarcoma cell lines showing cDNA expression of MYC, ERBB2, IGF1, P21, RB1, MET, STAT3, PTEN, P53, EGFR, and P16 genes. Primers were selected to cover 350–700 bp of the cDNA sequence of the selected gene (Suppl. Table 1). A. HMPOS expressed 9 of 11 genes but did not express PTEN or IGF1. B. POS expressed 10 of 11 genes but did not express PTEN. C. DOUG expressed 10 of 11 genes but did not express P16. D. COS31 expressed 10 of 11 genes but did not express P16. Both DOUG and COS31 cell lines showed lower IGF1 band intensity relative to other genes, and a lower expression was later confirmed by scRNA-seq (Suppl. Fig. 4B).

Figure 2.

Loupe Browser output displayed as a t-SNE plot for the COS31 canine osteosarcoma cell line, which is based on globally distinguishing gene clusters from 10,319 barcoded cells. Globally distinguishing genes are those genes that are highly expressed within a cluster relative to the entire dataset. Ten clusters were identified: cluster 1 (1,929 cells); 2 (1,527 cells); 3 (1,454 cells); 4 (1,378 cells); 5 (1,355 cells); 6 (1,117 cells); 7 (576 cells); 8 (459 cells); 9 (387 cells); 10 (137 cells) (see online version for colors).

Figure 3.

Loupe Browser output displayed as a t-SNE plot for the DOUG canine osteosarcoma cell line which is based on globally distinguishing gene clusters from 9,791 barcoded cells. Globally distinguishing genes are those genes that are highly expressed within a cluster relative to the entire dataset. Twelve clusters were identified: cluster 1 (1,470 cells); 2 (1,202 cells); 3 (1,142 cells); 4 (1,078 cells); 5 (1,018 cells); 6 (977 cells); 7 (788 cells); 8 (691 cells); 9 (648 cells) 10 (294 cells); 11 (281 cells); 12 (202 cells) (see online version for colors).

Figure 4.

Loupe Browser output as a t-SNE plot for COS31 and DOUG in the active feature search browser. The log₂-expression level is reflected by the color scale and the lower numerical value to the right of the scale. The numerical value reflects if a gene is present or absent; the color scale indicates the intensity of expression in barcoded cells across all clusters. Both COS31 and DOUG lacked P16 expression (no barcoded cells and 0 log₂-expression) but had intense MET expression (positive barcoded cells [color scale] and 0–3.9 [COS31], 0–3.0 [DOUG] log₂-expression) (see online version for colors).