Analysis of Gene Expression Signatures in Cancer-Associated Stroma from Canine Mammary Tumours Reveals Molecular Homology to Human Breast Carcinomas

Abstract

Cancer-associated stroma (CAS) plays a key role in cancer initiation and progression. Spontaneously occurring canine mammary carcinomas are viewed as excellent models of human breast carcinomas. Considering the importance of CAS for human cancer, it likely plays a central role in canine tumours as well. So far, however, canine CAS lacks characterisation, and it remains unclear whether the biology between CAS from canine and human tumours is comparable. In this proof-of-principle study, using laser-capture microdissection, we isolated CAS and normal stroma from 13 formalin-fixed paraffin embedded canine simple mammary carcinomas and analysed the expression of seven known human CAS markers by RT-qPCR (Reverse Transcription quantitative PCR) and validated some targets by immunohistochemistry. We found that Col1a1 (Collagen1α1), αSMA (alpha Smooth Muscle Actin), FAP (Fibroblast activation protein), PDGFRβ (Platelet-derived growth factor receptor beta), and Caveolin-1 were significantly upregulated in canine CAS, and the expression of CXCL12 (Stromal cell derived factor 1) significantly decreased, whereas MMP2 (Matrix Metalloproteinase 1) and IL6 (Interleukin 6) did not change. Our results suggest strong similarities in CAS biology in canine and human mammary carcinomas but also reveal some differences. To the best of our knowledge, this is the first report to provide a comprehensive expression analysis of the most important CAS markers in canine simple mammary carcinomas and further supports the validity of the dog as model for human cancer.

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).…”

“…Such knowledge has the potential to help identify both disease--promoting and/or suppressing features of CAS and identify novel prognostic and therapeutic targets therein. Given that canine mammary tumours are regarded as valuable model for human breast cancer, and the central role of CAS in human cancer in mCA, we have recently analysed stromal reprogramming in canine mCA, and demonstrated the presence of strong molecular homology in stromal reprogramming between canine and human mCA, emphasizing the relevance of the canine model for the human disease Ettlin et al, 2017). To understand whether stromal reprogramming also occurs in benign mammary tumours, and to compare stromal reprogramming between benign and malignant mammary tumours, we have now analysed stromal reprogramming in 13 cases of canine mammary adenoma and compared it to that in canine mCA.…”

“…Table 1 provides clinical details, such as age and breed of each patient, sample age and tumour type, for all cases included in the study. Laser--capture microdissection Laser capture microdissection (LCM) for selective isolation of matched CAS and normal stroma was performed as previously described Ettlin et al, 2017)). Areas for dissection were reviewed by a veterinary pathologist.…”

Know Thy Model: Charting Molecular Homology in Stromal Reprogramming Between Canine and Human Mammary Tumors

“…Driven by the lack of detailed characterization of stromal reprogramming in CMTs caused by technical limitations described above, we established a workflow to isolate subsections of formalin-fixed paraffin-embedded clinical tumor samples by laser-capture microdissection, and analyze gene-expression changes therein. In a first study, we isolated CAS and matched "normal" stroma (i.e., stroma isolated adjacent to unaltered mammary glands) from FFPE specimen of 13 cases of canine simple mCA, and analyzed the expression of seven well-described CAS-markers in human mCA (PDGFRβ, MMP2, Col1α1, FAP, ACTA2/αSMA, CXCL12/SDF1, and IL6) by RT-qPCR (Ettlin et al, 2017). Our results demonstrated that ACTA2, COL1A1, and FAP were upregulated in canine CAS, while PDGFRB, MMP2, and IL6 expression did not significantly change between normal stroma and CAS.…”

Safety and immunogenicity of a potential checkpoint blockade vaccine for canine melanoma

“…FAP is highly conserved between different species. It is expressed on fibroblasts within human or canine cancers [39] and was, therefore, selected to target the tumor stroma. To increase responses to FAP, the vaccine expressed the mouse sequence, which shows 87% homology with the corresponding canine protein.…”