Interactions with fibroblasts are distinct in Basal-like and luminal breast cancers

Abstract

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 TGFβ. 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.

Figure 1. Basal-like and Luminal Cocultures Show Distinct Gene Expression Profiles

Hierarchical clustering shows 369 genes that were significantly and consistently differentially expressed between Basal and Luminal breast cancer, when interacting with fibroblasts. The heatmap shows the log2(R/G) of the Buess interaction coefficient, so the color represents the fold change relative to monoculture. A complete list of genes and their interaction coefficients across these samples are available in Supplemental Table 1.

Figure 2. Basal-like vs. Luminal Differences in Proliferation and Migration in Response to Coculture

Interaction cocultures were used to measure proliferation using an MTT assay (A), migration using a Transwell migration assay (B) and wound healing using a scratch assay (C). Relative to monoculture, cocultures induced significant proliferation changes for all three Luminal lines and for one of the three Basal-like lines. Migration and wound healing were more pronounced both at baseline and following coculture for the Basal-like breast cancer cell lines.

Figure 3. Gene Expression Differences Observed in Direct Coculture are Not Fully Recapitulated in Interaction (Transwell) Cocultures

This 1-dimensional (genes clustered only) cluster of the 369 genes from coculture included direct (A) and interaction (B) cocultures where either fibroblasts alone (but under the influence of Luminal or Basal-like breast cancer cell lines) or breast cancer cell lines alone (but under the influence of fibroblasts) were used to harvest RNA for microarrays. The color bars along the top indicate which cell type was represented in the microarray: orange indicates fibroblasts, blue indicates Luminal cells, and red indicates Basal-like cells. A small percentage of genes (C) show strong overlap between direct and interaction cocultures.