Project description:Cancer-associated stroma (CAS) profoundly influences development and progression of tumours including mammary carcinoma (mCA). Spontaneous canine simple mCA represent relevant models of human mCA, notably also with respect to CAS reprogramming. While transcriptomic changes in CAS of mCA are well described, it remains unclear to what extent these differences translate to the protein level. Therefore, we sought to gain insight into the proteomic changes in CAS and compare them with transcriptomic changes in the same tissue. To this end, we analysed CAS and matched normal stroma isolated by laser-capture microdissection (LCM) by LC-MS/MS in a cohort of 14 Formalin-fixed paraffin embedded (FFPE) canine mCAs that we had previously characterized using LCM-RNAseq. Our results reveal clear differences in protein abundance between CAS and normal stroma, which are characterised by changes in the extracellular matrix, the cytoskeleton, and cytokines such as TNF. While the proteomics-based analysis of LCM-FFPE tissue detects fewer targets than RNAseq, the protein and RNA levels show a decent degree of correlation, especially for the most deregulated targets. Moreover, the results from both approaches reveal a comparable picture with regards to pathway activation. Finally, we validate transcriptomic upregulation of LTBP2, IGFBP2, COL6A5, POSTN, FN1, COL4A1, COL12A1, PLOD2, COL4A2, and IGFBP7 in CAS on the protein level and demonstrate their adverse prognostic value for human breast cancer. Given the relevance of canine mCA as model for the human disease, our analysis substantiates these targets as disease-promoting stromal components with implications for breast cancer in both humans and dogs.
Project description:Meningiomas are the most commonly reported primary intracranial tumor in dogs and humans. Canine meningiomas closely resemble human meningiomas in histological appearance as well as biological behavior. Because of the similarities between canine and human meningiomas, dogs have been proposed as models and surrogates for studying and treating human cancers including meningioma. However, little is known about specific pathways and individual genes that are involved in the development and progression of canine meningioma. In addition, there is a paucity of suitable fresh material available for studies of differential expression (DE) of genes in cases of veterinary neoplasia. We report here the use of formalin-fixed paraffin embedded (FFPE) specimens as much as a decade old, to provide RNA suitable for transcriptome analysis using next-generation sequencing (NGS). RNA was extracted from 13 canine meningiomas – 12 from (FFPE) and one flash-frozen. These represented 6 grade I and 7 grade II/III. RNA was also collected from flash-frozen meningeal tissue (arachnoid) from 3 control dogs. RNA from FFPE was of sufficient quality to successfully identify 125 significantly DE genes, the majority of which were related to oncogenic processes. 12 genes (AQP1, BMPER, FBLN2, FRZB, MEDAG, MYC, PAMR1, PDGFRL, PDPN, PECAM1, PERP, ZC2HC1Cc) were validated using qPCR. Among the DE genes were oncogenes, tumor suppressors, transcription factors, VEGF-related genes, and members of the WNT pathway. Our work demonstrates that RNA of sufficient quality can be extracted from FFPE samples to provide biologically relevant transcriptome analyses using a NGS technique, such as RNA-seq.
