Introduction

Osteosarcoma (OSA) is a highly aggressive neoplasia of the canine and human patient. Genome sequencing has revealed OSA has a high mutational load and appears highly heterogenous. However, while bulk genome sequencing identified heterogeneity between OSA tumors, intra-tumoral heterogeneity has not been established in OSA. By performing Single-Cell Transcriptomics (SC-T), a novel, high-resolution analysis of individual cells can be generated to reveal intra-tumoral populations. This technology has not previously been applied to the canine genome. From SC-T analysis, information on tumoral subpopulations that are otherwise masked by bulk sequencing is evident.

Methods

Established cOSA cell lines were utilized for a SC-T run and the results were validated against a genomic profile generated via Sanger sequencing. The data generated from the SCT analysis were run against the canine genome via the use of HiperGator super computers. The data was then formatted and input into Ingenuity Pathway Analysis to identify pathways up or down regulated within the clusters and into Cell Loupe to generate cellular clusters.

Results

Within the ∼10,000 cells analyzed from each sample, unique cellular clusters were identified. 81 pathways of relevance were identified and of those 33 had been implicated in OSA tumorigenesis-18 which had been investigated in cOSA and human OSA (hOSA) and 15 which had only been investigated in hOSA.

Conclusion

SC-T can be successfully applied to the canine genome and to cOSA. From the data, we were able to identify the expression of new pathways implicated in cOSA and gain further insight into the heterogeneity of OSA.