Feline injection-site sarcoma (FISS), an aggressive iatrogenic subcutaneous malignancy, is challenging to manage clinically and little is known about the molecular basis of its pathogenesis. Tumor transcriptome profiling has proved valuable for gaining insights into the molecular basis of cancers and for identifying new therapeutic targets. Here, we report the first study of the FISS transcriptome and the first cross-species comparison of the FISS transcriptome with those of anatomically similar soft-tissue sarcomas in dogs and humans. Using high-throughput short-read paired-end sequencing, we comparatively profiled FISS tumors vs. normal tissue samples as well as cultured FISS-derived cell lines vs. skin-derived fibroblasts. We analyzed the mRNA-seq data to compare cancer/normal gene expression level, identify biological processes and molecular pathways that are associated with the pathogenesis of FISS, and identify multimegabase genomic regions of potential somatic copy number alteration (SCNA) in FISS. We additionally conducted cross-species analyses to compare the transcriptome of FISS to those of soft-tissue sarcomas in dogs and humans, at the level of cancer/normal gene expression ratios. We found: (1) substantial differential expression biases in feline orthologs of human oncogenes and tumor suppressor genes suggesting conserved functions in FISS; (2) a genomic region with recurrent SCNA in human sarcomas that is syntenic to a feline genomic region of probable SCNA in FISS; and (3) significant overlap of the pattern of transcriptional alterations in FISS with the patterns of transcriptional alterations in soft-tissue sarcomas in humans and in dogs. We demonstrated that a protein, BarH-like homeobox 1 (BARX1), has increased expression in FISS cells at the protein level. We identified 11 drugs and four target proteins as potential new therapies for FISS, and validated that one of them (GSK-1059615) inhibits growth of FISS-derived cells in vitro. (1) Window-based analysis of mRNA-seq data can uncover SCNAs. (2) The transcriptome of FISS-derived cells is highly consistent with that of FISS tumors. (3) FISS is highly similar to soft-tissue sarcomas in dogs and humans, at the level of gene expression. This work underscores the potential utility of comparative oncology in improving understanding and treatment of FISS.

A single domain antibody (SDAb) targeting canine PD-1 was developed as a potential immunotherapeutic for canine cancer. An alpaca was immunized with canine PD-1 protein, and a phage-display library was constructed using mRNA isolated from peripheral lymphocytes. Screening of the library yielded multiple SDAb candidates capable of nanomolar binding to canine PD-1. Among these, clone STX-1b5 demonstrated high expression in a yeast-based recombinant system and was selected for further characterization. Binding and competition assays using ELISA confirmed its ability to bind canine PD-1 and block PDL-1 interaction. In silico structural modeling supported the interaction of STX-1b5 with key PD-1 residues implicated in ligand binding. These findings support the feasibility of using SDAbs and cost-effective yeast expression systems to generate immunotherapeutics for veterinary use, with STX-1b5 representing a promising lead candidate for future clinical development.