Interactions between breast cancer cells and their stromal component: An analysis of alterations in gene expression Christos Sotiriou, Frank El Ouriaghli, Samira Majjaj, Benjamin Haibe-Kains, Christine Desmedt, Denis Larsimont, Martine Piccart. Translational Research and Medical Oncology Unit, Université Libre de Bruxelles (U.L.B.), Institut Jules Bordet, 121 Boulevard de Waterloo, 1000 Brussels, Belgium Methods: Myo-fibroblast cells (CD10) were isolated and purified from breast tumor (N= 28) and normal (N=4) tissues. Gene expression analysis was performed using the Affymetrix GeneChip® Human Genome U133 Plus 2.0 arrays. Survival analysis was carried out using 12 publicly available microarray datasets including more than 1200 systemically untreated breast cancer patients. 100 101 102 103 0 10 Leukocytes 1 10 2 10 3 10 102 103 104 Leukocytes Clinical Relevance? Molecular Subtypes CD10+ Tumor vs CD10+ Normal ER-/HER2- Tumor CD10+ vs ER+/HER2- 4 10 100 101 102 103 104 CD10 (F ibroblasts) 101 Epcam (cancer cells) Epcam (cancer cells) 100 Tumor Fibroblast Signature Leukocytes Epcam+ (Tumor cells) fibroblasts Conclusions: Our results highlight the importance of tumor epithelial-stroma cell interactions in breast carcinogenesis and breast cancer sub-typing. Moreover, it shows the role of stroma cells in tumor dissemination particularly within the HER2+ subtype and provide basis for the development of novel therapeutic strategies. 100 Leukocytes 101 102 Leukocytes 103 104 CD10+ (Fibroblasts) 103 probe sets Filtre: Fold Chance >= 4 & FDR < 0.01 Differences in Gene Expression of CD10 (fibroblasts) According to Molecular Subtypes HER2+/ER- 89 mapped Known function N=51 genes 51 Tumor samples Uknown function N=38 genes HR=3.076 CI 1.544-6.131 P=0.001 Epcam+ (Tumor cells) Experimental Protocol 104 of CD10+ and Epcam+ cells Normal CD10+ CD10 (Fibroblasts) Epcam (c ancer cells) Background: Epithelial-stromal interactions are known to be important in normal mammary gland development and to play a role in breast carcinogenesis. The aim of this study was to explore the influence of breast tumor microenvironment on primary tumor growth, breast cancer sub-typing and prognosis. Results: Breast tumor myo-fibroblast stroma cells showed an altered gene expression patterns to the ones isolated from normal breast tissues. While some of the differentially expressed genes are found to be associated with extracellular matrix formation/degradation and angiogenesis, the function of several other genes remains largely unknown. Unsupervised hierarchical clustering analysis clustered breast tumor myo-fibroblast cells into subgroups recapitulating the molecular portraits of breast cancer based on ER, HER2 status and tumor differentiation. A stroma gene expression signature developed from myo-fibroblast cells isolated from normal versus BC tissues showed a statistically significant association with clinical outcome. Breast tumors with high expression levels of the stroma signature were significantly associated with worse prognosis (HR 1.55; CI 1.20-1.99; p=5.57 10- 4). This association was only observed within the clinically high risk HER2+ subtypes. Interestingly, HER2+ tumors with high and low expression levels of the stroma signature showed 45% and 85% distant metastasis free survival at 5-year follow-up respectively (HR 2.53; CI 1.31-4.90; p=5.29 10-3). Unsupervised Analysis Purification of CD10 (fibroblasts) and Epcam+ (tumor) cells Abstract N=21 CD10+ Tumor Fibroblast Signature Response to Therapy (fibroblasts) N=28 Type 1: ER-/HER2- (basal-like) Type 2: HER2+ (HER2-like) Type 3: ER+/HER2- (luminal-like) L:grade 1; I:grade 2; H:grade 3 Primary tumor Mechanic dissection Single cell suspenssion Enzymatic digestion Dissection Digestion Epcam CD45 CD10 Amplification protocol Module in silico approach Clinical Relevance? All patients N!1400 Systemically Untreated Proliferation Immune Response Isolation/Separation Ingenuity Pathway Analysis Network 1 Cancer cells (Epcam+) Leukocytes (CD45+) Fibroblasts (CD10+) “Stromal cells” Tumor Invasion (Plau) Neoadjuvant Epirubicine (ER- pts only, N=81) Potential predictive value of the tumor invasion module and fibroblast signature HER-2 signaling ER signaling High correlation with stroma signature Tumor Invasion Cancer Cellular movement Connective tissue disorder HR=1.169 CI 0.913-1.497 P=0.213 Acknowledgments FNRS, MEDIC Foundation, Breast Cancer Research Foundation (BCRF, Evelyne Lauder)