Abstract
ABSTRACTSpontaneous canine simple mammary carcinomas (mCA) are often viewed as models of human mCA. Cancer-associated stroma (CAS) is central for initiation and progression of human cancer, and is likely to play a key role in canine tumours as well. However, canine CAS lacks characterisation and it remains unclear how canine and human CAS compare. Formalin-fixed paraffin embedded (FFPE) tissue constitutes a valuable resource of patient material, but chemical crosslinking has largely precluded its analysis by next-generation RNA sequencing (RNAseq). We have recently established a protocol to isolate CAS and normal stroma from archival FFPE tumours using laser-capture microdissection followed by RNAseq. Using this approach, we have analysed stroma from 15 canine mCA. Our data reveal strong reprogramming of canine CAS. We demonstrate a high-grade molecular homology between canine and human CAS, and show that enrichment of upregulated canine CAS genes strongly correlates with the enrichment of an independently derived human stromal signature in the TCGA breast tumour dataset. Relationships between different gene signatures observed in human breast cancer are largely maintained in the canine model, suggesting a close interspecies similarity in the network of cancer signalling circuitries. Finally, we establish the prognostic potential of the canine CAS signature in human samples, emphasising the relevance of studying canine CAS as a model of the human disease. In conclusion, we provide a proof-of-principle to analyse specific subsections of FFPE tissue by RNAseq, and compare stromal gene expression between human and canine mCA to reveal molecular drivers in CAS supporting tumour growth and malignancy.
Highlights
We provide the proof-of-principle for this novel protocol by analysing Cancer-associated stroma (CAS) and matched normal stroma isolated by laser-capture microdissection (LCM) from Formalin-fixed paraffin embedded (FFPE) tissue from 15 clinical cases of canine simple mammary carcinomas (mCA)
Differential expression analysis with a false discovery rate (FDR) cut-off of 0.01 and fold change threshold of 2 revealed 884 genes to be significantly deregulated in CAS compared to normal stroma, with 446 genes significantly upregulated and 438 genes significantly downregulated in CAS (Fig. 1B and Table S1)
Over-representation analysis of Gene Ontology (GO) terms associated with biological processes, cellular components and molecular functions for up- and downregulated genes suggested the strongest changes in the following categories: immune system process, biological adhesion, cell differentiation, proliferation, growth, extra cellular matrix and collagen organisation (Fig. 1C-H)
Summary
The microenvironment surrounding cancer cells is pivotal for growth and survival of many different tumours The role of CAS in tumour biology has been widely documented (Bissell and Hines, 2011). CAS directly promotes the growth of tumour cells by secreting and/or activating molecules such as growth factors, nutrients and cytokines, among others (e.g. reviewed by Bissell and Hines, 2011; Hanahan and Coussens, 2012). The mechanisms underlying the formation of CAS and the molecular dialogue between CAS and cancer cells remain poorly understood
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