Transcriptome signature of canine osteosarcoma and role of cytokines in the neoplastic growth

Abstract

The broad objectives of the proposed research are (a) to identify the transcriptomic signatures of canine osteosarcoma (OS), their correlation with the biological behaviour and similarities with human OS, and (b) to identify the biological, diagnostic and prognostic role of cytokines in canine OS, with a potential future application in therapeutic settings. OS originates from bone-forming mesenchymal cells and is one of the primary bone tumours in dogs, characterised by a high metastatic ability. More than 80% of OS-affected patients are diagnosed with lung metastasis. Canine OS has an aggressive biological behaviour, and despite therapeutic advancements, survival times have not improved in the past 20 years. This poses a significant challenge for veterinary practitioners and specialists.The field of transcriptomics has undergone rapid growth in the last decade, especially after the publication of the first full canine genome. Transcriptomics has been instrumental in understanding the origin, development and progression of different human and canine cancers. However, many issues still need to be overcome, especially in the veterinary field (i.e., the number of samples, sample storage and preservation, and costs). This part of the study aimed at adding to the growing transcriptomic knowledge for canine OS, examining the gene expression patterns in two datasets of samples with different biological behaviour: canine OS with slow progression (death in over six months) and canine OS with quick progression (death in fewer than six months). The two groups showed a different gene expression pattern profile with upregulation of SOX2, IFG2B1, HAPLN1, LYPD1, DNAJ6 in quickly progressing OS and MMP27, GDF7, IGF2B1, TNN, HAPLN1 upregulated in slowly progressing OS. In addition, canine OS was characterised by the aberrant activation of signalling pathways that are also dysregulated in human OS, such as Ras, PI3K and Hippo pathways. The transcriptomic findings observed in this study highlighted the importance of the mutational profiling of individual patients prior to targeted therapy, given the heterogeneity of the expression pattern and potential different prognostic outcomes.Cytokines and chemokines play essential roles in the tumour microenvironment and stimulate the host response to control cellular stress and reduce cellular damage. In addition, the expression of selected cytokines in human cancers, including OS, has been related to neoplastic growth, viability and progression, and variations in the gene sequence (polymorphism) of different cytokines are associated with increased risk of the human OS. Therefore, targeting the signalling pathway of the cytokine network may represent an attractive therapeutic target. While most of the knowledge of the cytokine expression in OS has been obtained from humans and mouse models, there are no published data on dogs. In this study, bulk ribonucleic acid sequencing (RNA-seq) has been performed to extensively characterise the cellular and cytokines landscape of canine OS, from development to disease. RNA-seq has been conducted on 9 canine bone samples, including 3 OS with slow progression, 3 OS with quick progression and 3 normal bone samples. Our analysis revealed remarkable cytokines and chemokines expression in canine OS. Specifically, IL-2, IL-6, IL-10, IL-15, IL-21, IL-26, IL-37, FLT3LG were highly expressed in OS with slow progression and IL-5, IL-18, IL-19, IL-32, IL-34, TGFβ3, CXCR2 and IL36RN were highly expressed in OS with quick progression by RNA-seq. Further, immunohistochemistry confirmed IL-2, IL-10, IL-15, FLT3, TGF β3 as good candidates as prognostic biomarkers, based on the different immunohistochemical expression in 47 formalin-fixed paraffin-embedded samples of canine OS with different mitotic scores.We conclude that cytokines not only provide a novel target for therapeutic interventions but could also serve as a potential biomarker for the stratification of canine OS patients selected for immunotherapy. Our discovery of cytokine expression in canine OS also opens up avenues for identifying and understanding similar paradigms in human OS.