Characterisation of stromal gene expression in soft tissue sarcoma

Background: Soft Tissue Sarcomas (STS) are a biologically rare, heterogeneous and complex population of solid tumours of mesenchymal origin. Recent studies have emphasised the important role of mesenchymal cells like fibroblasts in cancer biology. Very little is known about the role of Sarcoma Associated Fibroblasts (SAF) as most studies have focused on Carcinoma Associated Fibroblasts (CAF). The study of SAF is challenging for two reasons; firstly, there are few STS cell lines available that are matched to their original histotype. Secondly, as STS cells and SAF are both mesenchymal, no selective markers have been identified to facilitate their study. This study will focus on characterising SAF in the poorly differentiated, genomically complex and mesenchymal-like STS including Undifferentiated Pleomorphic Sarcoma (UPS) and Myxofibrosarcoma (MFS). Objectives: To identify STS and SAF specific gene expression profiles to improve our fundamental understanding of STS biology. Methodology: Eight STS cell lines (UPS, MFS, and LMS), recently described by Salawu et al (Salawu et al., 2016) or donated by Professor Heymann (Nantes, France) were used in this project. Expression of mesenchymal proteins (transgelin, αSMA, vimentin, fibronectin, N-cadherin, FSP-1, FAPα and SOX2) was compared between the STS cell lines and normal human mesenchymal cells (dermal, lung, uterine fibroblasts and mesenchymal stem cells) using western blot and Immunocytochemistry. Tumours were then grown from the UPS cell lines in NSG mice and immunohistochemistry and immunofluorescence staining were applied to tumour sections to further characterise mesenchymal protein expression. Finally, RNAseq was used to characterise differences in stromal gene expression between the STS cells and the mouse stroma by aligning reads against the human and mouse reference genomes (Bradford et al. 2013). Conclusions: Of all the mesenchymal proteins examined so far, Transgelin showed the most promise for the identification of SAF versus STS cells. Analysis of the RNAseq data shows it was possible to identify mouse stromal genes from UPS xenografts. Differential gene expression between the three UPS tumours was used to identify a novel stromal gene signature and subsequently led to the identification of a possible “SAF gene panel” for use in future STS studies.