Next-generation RNA sequencing of FFPE subsections reveals highly conserved stromal reprogramming between canine and human mammary carcinoma

Abstract

Spontaneous 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.

Differential stromal reprogramming in benign and malignant naturally occurring canine mammary tumours identifies disease-promoting stromal components

“…These changes are consistent with fibroblast--and immune--cell driven remodelling of the tumour microenvironment, which are known to be heavily involved in tumour biology of mCA (Bissell and Hines, 2011;Hanahan and Coussens, 2012). Indeed, we find clear signs of fibroblast activation and reprogramming, as evidenced by the increase in αSMA and the decrease in vimentin by IF (Figure 2), which is also seen in malignant canine mCA (Amini et al, 2019;Ettlin et al, 2017;Yoshimura et al, 2011). It is interesting that the stroma surrounding adenomas shows such a clear reprogramming, as these non--infiltrative benign mammary tumours are generally associated with little fibrovascular supporting stroma (Goldschmidt et al, 2011).…”

“…To understand how stromal reprogramming in benign canine mammary adenomas compares to malignant mCA, we juxtaposed our dataset for CAS in adenoma to a dataset of matched CAS and normal stroma from 15 canine mCA that we had obtained using the same methodology as previously reported (Amini et al, 2019). As the histopathological appearance of normal, uninvolved stroma showed no difference between adenomas and mCA as expected, we merged the two data sets while adjusting for potential batch effects (see methods for details).…”

“…Immunofluorescence was performed as outlined in (Amini et al, 2019). Antibodies and conditions used for immunofluorescence are detailed in Supplementary table 6.…”

“…Antibodies and conditions used for immunofluorescence are detailed in Supplementary table 6. RNA sequencing library preparation 10 ng of RNA from Elution 1 (E1) diluted to a concentration of 0.33 ng/μl in a total volume of 30 μl was submitted for next--generation RNA sequencing and analysed as outlined in (Amini et al, 2019).…”

“…To understand stromal reprogramming in canine mCA and how it compares to human mCA, we have previously analysed CAS reprogramming in formalin--fixed paraffin embedded (FFPE) breast cancer tissue using by laser--capture--microdissection (LCM) and quantitative PCR (RT--qPCR), and further advanced the approach to analyse LCM subsections by next--generation sequencing (RNAseq) . Using this powerful RNAseq--driven approach, we have very recently assessed stromal reprogramming in a set of 15 canine mCA, and demonstrated strong molecular homology in stromal reprogramming between canine and human mCA, emphasizing the relevance of the canine model for the human disease also with regards to CAS reprogramming (Amini et al, 2019).…”