Basal-like breast cancers have several well-characterized distinguishing molecular features, but most of these are features of the cancer cells themselves. The unique stromal-epithelial interactions, and more generally, microenvironmental features of basal-like breast cancers have not been well characterized. To identify characteristic microenvironment features of basal-like breast cancer, we performed cocultures of several basal-like breast cancer cell lines with fibroblasts and compared these with cocultures of luminal breast cancer cell lines with fibroblasts. Interactions between basal-like cancer cells and fibroblasts induced expression of numerous interleukins and chemokines, including IL-6, IL-8, CXCL1, CXCL3, and TGFb. Under the influence of fibroblasts, basal-like breast cancer cell lines also showed increased migration in vitro. Migration was less pronounced for luminal lines; but, these lines were more likely to have altered proliferation. These differences were relevant to tumor biology in vivo, as the gene set that distinguished luminal and basal-like stromal interactions in coculture also distinguishes basal-like from luminal tumors with 98% accuracy in 10-fold cross-validation and 100% accuracy in an independent test set. However, comparisons between cocultures where cells were in direct contact and cocultures where interaction was solely through soluble factors suggest that there is an important impact of direct cell-to-cell contact. The phenotypes and gene expression changes invoked by cancer cell interactions with fibroblasts support the microenvironment and cell-cell interactions as intrinsic features of breast cancer subtypes. Mol Cancer Res; 9(1); 3-13. Ó2010 AACR.

IntroductionA gene expression signature indicative of activated wound responses is common to more than 90% of non-neoplastic tissues adjacent to breast cancer, but these tissues also exhibit substantial heterogeneity. We hypothesized that gene expression subtypes of breast cancer microenvironment can be defined and that these microenvironment subtypes have clinical relevance.MethodsGene expression was evaluated in 72 patient-derived breast tissue samples adjacent to invasive breast cancer or ductal carcinoma in situ. Unsupervised clustering identified two distinct gene expression subgroups that differed in expression of genes involved in activation of fibrosis, cellular movement, cell adhesion and cell-cell contact. We evaluated the prognostic relevance of extratumoral subtype (comparing the Active group, defined by high expression of fibrosis and cellular movement genes, to the Inactive group, defined by high expression of claudins and other cellular adhesion and cell-cell contact genes) using clinical data. To establish the biological characteristics of these subtypes, gene expression profiles were compared against published and novel tumor and tumor stroma-derived signatures (Twist-related protein 1 (TWIST1) overexpression, transforming growth factor beta (TGF-β)-induced fibroblast activation, breast fibrosis, claudin-low tumor subtype and estrogen response). Histological and immunohistochemical analyses of tissues representing each microenvironment subtype were performed to evaluate protein expression and compositional differences between microenvironment subtypes.ResultsExtratumoral Active versus Inactive subtypes were not significantly associated with overall survival among all patients (hazard ratio (HR) = 1.4, 95% CI 0.6 to 2.8, P = 0.337), but there was a strong association with overall survival among estrogen receptor (ER) positive patients (HR = 2.5, 95% CI 0.9 to 6.7, P = 0.062) and hormone-treated patients (HR = 2.6, 95% CI 1.0 to 7.0, P = 0.045). The Active subtype of breast microenvironment is correlated with TWIST-overexpression signatures and shares features of claudin-low breast cancers. The Active subtype was also associated with expression of TGF-β induced fibroblast activation signatures, but there was no significant association between Active/Inactive microenvironment and desmoid type fibrosis or estrogen response gene expression signatures. Consistent with the RNA expression profiles, Active cancer-adjacent tissues exhibited higher density of TWIST nuclear staining, predominantly in epithelium, and no evidence of increased fibrosis.ConclusionsThese results document the presence of two distinct subtypes of microenvironment, with Active versus Inactive cancer-adjacent extratumoral microenvironment influencing the aggressiveness and outcome of ER-positive human breast cancers.

Purpose Cancer cells have altered metabolism, with increased glucose uptake, glycolysis, and biomass production. This study performed genomic and metabolomic analyses to elucidate how tumor and stromal genomic characteristics influence tumor metabolism. Experimental Design Thirty-three breast tumors and six normal breast tissues were analyzed by gene expression microarray and by mass spectrometry for metabolites. Gene expression data and clinical characteristics were evaluated in association with metabolic phenotype. To evaluate the role of stromal interactions in altered metabolism, cocultures were performed using breast cancer cells and primary cancer-associated fibroblasts (CAFs). Results Across all metabolites, unsupervised clustering resulted in two main sample clusters. Normal breast tissue and a subset of tumors with less aggressive clinical characteristics had lower levels of nucleic and amino acids and glycolysis byproducts, while more aggressive tumors had higher levels of these Warburg-associated metabolites. While tumor intrinsic subtype did not predict metabolic phenotype, metabolic cluster was significantly associated with expression of a wound response signature. In cocultures, CAFs from basal-like breast cancers increased glucose up take and basal-like epithelial cells increased glucose oxidation and glycogen synthesis, suggesting interplay of stromal and epithelial phenotypes on metabolism. Cytokine arrays identified hepatocyte growth factor (HGF) as a potential mediator of stromal-epithelial interaction, and antibody neutralization of HGF resulted in reduced expression of glucose transporter 1 (GLUT1) and decreased glucose uptake by epithelium. Conclusions Both tumor/epithelial and stromal characteristics play important roles in metabolism. Warburg-like metabolism is influenced by changes in stromal-epithelial interactions, including altered expression of HGF/Met pathway and GLUT1 expression.

Introduction Overall survival of early-stage breast cancer (BC) patients is similar for those who undergo breast conserving therapy (BCT) and mastectomy, however, 10-15% of women undergoing BCT suffer ipsilateral breast tumor recurrence. The risk of recurrence may vary with BC subtype. Understanding the gene expression of the cancer-adjacent tissue and the stromal response to specific tumor subtypes is important for developing clinical strategies to reduce recurrence risk. Methods We utilized two independent data sets to study gene expression data in cancer-adjacent tissue from invasive BC patients. Complementary in vitro cocultures were used to study cell-cell communication between fibroblasts and specific BC subtypes. Results Our results suggest that intrinsic tumor subtypes are reflected in histologically-normal cancer-adjacent tissue. Gene expression of cancer-adjacent tissues shows that triple negative (Claudin-low or Basal-like) tumors exhibit increased expression of genes involved in inflammation and immune response. While such changes could reflect distinct immune populations present in the microenvironment, altered immune response gene expression was also observed in cocultures in the absence of immune cell infiltrates, emphasizing that these inflammatory mediators are secreted by breast-specific cells. In addition, while triple negative BCs are associated with upregulated immune response genes, Luminal breast cancers are more commonly associated with estrogen-response pathways in adjacent tissues. Conclusions Specific characteristics of BCs are reflected in the surrounding histologically-normal tissue. This commonality between tumor and cancer-adjacent tissue may underlie second primaries and local recurrences. Impact Biomarkers derived from cancer-adjacent tissue may be helpful in defining personalized surgical strategies or in predicting recurrence risk.