(P 01) A PILOT STUDY ON SINGLE-NUCLEUS RNA SEQUENCING IN CANINE SOFT TISSUE SARCOMAS

Objective: Soft tissue sarcomas (STS) are common cancers with limited therapeutic options. Dogs develop STS spontaneously but with higher frequency than humans, making them an excellent model for studying the disease. Notably, canine STS share significant clinical, biological, genomic, and transcriptomic similarities with human STS and the shorter lifespan of dogs allows for faster observation of disease progression and treatment outcomes. To uncover the transcriptomic profiles at single-cell resolution and potential therapeutic targets of STS, we conducted a pilot study of single-nucleus RNA sequencing (snRNA-seq) on canine STS samples.

Methods: We performed snRNA-seq on four snap-frozen grade I-III STS samples. Diagnosis and subtyping were confirmed through histopathology and immunohistochemistry. Nuclei were isolated and libraries were prepared using Chromium kits from 10x Genomics, and indexed cDNA libraries were sequenced on a NextSeq 550 with an aimed sequencing depth of 20,000 paired end reads per cell. Data processing and visualization were performed using the canine reference genome CanFam6, Cell Ranger (v8.0), and Seurat (v5.0.2) in R (v4.3.3).

Results: Tumor cells were the dominant population in all samples, with immune cell infiltrates comprising 3-19% of the cells. Transcriptomic analysis at single-cell resolution revealed clusters corresponding with histopathological findings. Notably, a distinct cluster characterized by significant up-regulation of mitotic genes predominantly consisting of the neoplastic cells from grade III STS was identified. Among the immune cells, we identified subsets of tumor-infiltrating T cells and actively proliferating immune cells. These patterns were consistent across all samples despite the heterogeneous cancer cell clustering.

Conclusion: Our pilot study highlights the potential of snRNA-seq to generate comprehensive transcriptomic profiles and pinpoint therapeutic targets in canine STS. This approach not only holds promise for advancing research and developing new therapeutics for canine STS but also provides valuable insights that are directly translatable to human STS. Further studies with larger sample sizes and diverse STS subtypes are warranted to validate and expand upon our findings, fostering the development of novel therapeutic strategies for both canine and human patients.