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

Abstract

While cancer-associated stroma (CAS) in malignant tumours is well described, stromal changes in benign forms of naturally occurring tumours remain poorly characterized. Spontaneous canine mammary carcinomas (mCA) are viewed as excellent models of human mCA. We have recently reported highly conserved stromal reprogramming between canine and human mCA based on transcriptome analysis of laser-capture-microdissected FFPE specimen. To identify stromal changes between benign and malignant mammary tumours, we have analysed matched normal and adenoma-associated stroma (AAS) from 13 canine mammary adenomas and compared them to previous data from 15 canine mCA. Our analyses reveal distinct stromal reprogramming even in small benign tumours. While similarities between AAS and CAS exist, the stromal signature clearly distinguished adenomas from mCA. The distinction between AAS and CAS is further substantiated by differential enrichment in several hallmark signalling pathways as well as differential abundance in cellular composition. Finally, we identify COL11A1, VIT, CD74, HLA-DRA, STRA6, IGFBP4, PIGR, and TNIP1 as strongly discriminatory stromal genes between adenoma and mCA, and demonstrate their prognostic value for human breast cancer. Given the relevance of canine CAS as a model for the human disease, our approach identifies diseasemodulating stromal components with implications for both human and canine breast cancer. It is well accepted that the microenvironment surrounding cancer cells, the so-called cancer-associated stroma (CAS), plays a central role in both cancer initiation as well as its progression 1,2. CAS is composed of various non-cancer cells, among them fibroblasts, immune cells, vascular cells, as well as extracellular matrix. CAS affects tumour cells in several ways: directly by promoting growth and survival of tumour cells, and also by stimulating their invasive and migratory capacity, thereby promoting invasion and metastasis 1,2. Thus, CAS is considered to be a major determinant of tumour malignancy. However, in contrast to malignant tumours, it remains largely unexplored whether and what stromal changes occur in benign forms of naturally occurring tumours, and how stromal changes in benign neoplasms compare to that of malignant tumours in the same tissue. Such knowledge has the potential to help identify disease-promoting and/or suppressive features of the stroma and identify novel prognostic and therapeutic targets therein. Close resemblance with regards to both pathophysiology and clinical aspects have positioned spontaneously occurring tumours in the domestic dog as valuable model to enhance understanding of tumour biology in both canine and human patients 3-5. In particular, canine simple mammary carcinoma (mCA) are regarded as excellent models for human mCA, as they recapitulate the biology of human mCA both histologically and molecularly, and overcome several of the limitations of rodent tumour models 6-8. Canine simple mCA are malignant epithelial neoplasms that infiltrate the surrounding tiss...

Molecular homology between canine spontaneous oral squamous cell carcinomas and human head-and-neck squamous cell carcinomas reveals disease drivers and therapeutic vulnerabilities

“…To analyze gene expression changes in COSCC, we specifically isolated tumor cells and matched normal epithelium from clinical FFPE specimens using LCM coupled with RNAseq as previously established [14] , [15] , [16] , [17] . Representative images of tissue specimens and detailed patient characteristics for all cases are shown in Table 1 and Supplementary Figure 1.…”

“… [13] ). Hence, our group has established a workflow to isolate specific subpopulations of cells from archival formalin-fixed paraffin-embedded (FFPE) tissue sections by laser-capture microdissection (LCM) followed by RNAseq analysis [14] , [15] , [16] , [17] . Using this approach, we set out to specifically isolate tumor cells and matched normal epithelial cells from 10 cases of COSCC in order to analyze the molecular underpinnings of COSCC and its resemblance with human HNSCC.…”

Molecular targets for anticancer therapies in companion animals and humans: what can we learn from each other?

“…Also important is the realization that similar expression pattern does not necessarily translate to identical biological behaviour across species. While most studies in the field rely on immunohistochemistry and PCR techniques to assess expression of a handful of targets, only recently more sophisticated techniques are starting to be introduced that allow for unbiased cross-species analysis, such as wide-genome characterization of the cancer 178 and RNA-sequencing of subpopulations of stroma and neoplastic cells 14 , 179 . Such unbiased analysis enables to investigate similarities between species and to identify new therapeutic targets.…”

Triple-Negative Breast Cancer Comparison With Canine Mammary Tumors From Light Microscopy to Molecular Pathology

“…They revealed strong increases in mesenchymal stem cells, gamma delta T-cells, macrophages, plasmoid dendritic cells, and natural killer T-cells in CAS and demonstrated that commonly deranged pathways between canine and human CAS included angiogenesis, epithelialmesenchymal transition, glycolysis, and immune response pathways. In order to analyze transcriptional reprogramming of adenoma associated stroma (AAS) of 13 canine mammary adenomas compared to previous data from 15 canine mammary carcinomas, Amini et al (95) applied weighted gene coexpression network analysis (WGCNA) and identified six clusters of highly positively correlated genes and subsequently identified four potentially interesting modules including blue, brown, turquoise and yellow modules. They showed that TGFbeta signaling, glycolysis, mitotic spindle, epithelial to mesenchymal transition, mTORC1 signaling, unfolded protein response, apical surface, interferon-gamma response and G2M checkpoint demonstrated greatly increased enrichment only in CAS and pathways involving pancreas beta cells, fatty acid metabolism, spermatogenesis, heme metabolism and IL2-STAT5 signaling showing dramatically reduced enrichment only in CAS.…”

“…They also studied the increased expression of Caveolin-1 (Cav1) and FGF2 in CAS. Potential biomarkers for canine and human mammary carcinoma may be EMT-related genes such as COL11A1, COL8A2, and ADAM12 which are overexpressed in mammary carcinoma (95). Matsumoto et al (140) performed immunohistochemical staining of CD4 and CD8 on tissue microarrays of 164 TNBC cases.…”