EMT Transition Alters Interstitial Fluid Flow-Induced Signaling in ERBB2-Positive Breast Cancer Cells.

UNLABELLED: A variety of biophysical forces are altered in the tumor microenvironment (TME) and these forces can influence cancer progression. One such force is interstitial fluid flow (IFF)-the movement of fluid through the tissue matrix. IFF was previously shown to induce invasion of cancer cells, but the activated signaling cascades remain poorly understood. Here, it is demonstrated that IFF induces invasion of ERBB2/HER2-expressing breast cancer cells via activation of phosphoinositide-3-kinase (PI3K). In constitutively activate ERBB2-expressing cells that have undergone epithelial-to-mesenchymal transition (EMT), IFF-mediated invasion requires the chemokine receptor CXCR4, a gradient of its ligand CXCL12, and activity of the PI3K catalytic subunits p110α and ß. In wild-type ERBB2-expressing cells, IFF-mediated invasion is chemokine receptor-independent and requires only p110α activation. To test whether cells undergoing EMT alter their signaling response to IFF, TGFß1 was used to induce EMT in wild-type ERBB2-expressing cells, resulting in IFF-induced invasion dependent on CXCR4 and p110ß. IMPLICATIONS: This study identifies a novel signaling mechanism for interstitial flow-induced invasion of ERBB2-expressing breast cancer cells, one that depends on EMT and acts through a CXCR4-PI3K pathway. These findings suggest that the response of cancer cells to interstitial flow depends on EMT status and malignancy.

Three-dimensional cell culture model for measuring the effects of interstitial fluid flow on tumor cell invasion.

The growth and progression of most solid tumors depend on the initial transformation of the cancer cells and their response to stroma-associated signaling in the tumor microenvironment (1). Previously, research on the tumor microenvironment has focused primarily on tumor-stromal interactions (1-2). However, the tumor microenvironment also includes a variety of biophysical forces, whose effects remain poorly understood. These forces are biomechanical consequences of tumor growth that lead to changes in gene expression, cell division, differentiation and invasion(3). Matrix density (4), stiffness (5-6), and structure (6-7), interstitial fluid pressure (8), and interstitial fluid flow (8) are all altered during cancer progression. Interstitial fluid flow in particular is higher in tumors compared to normal tissues (8-10). The estimated interstitial fluid flow velocities were measured and found to be in the range of 0.1-3 µm s(-1), depending on tumor size and differentiation (9, 11). This is due to elevated interstitial fluid pressure caused by tumor-induced angiogenesis and increased vascular permeability (12). Interstitial fluid flow has been shown to increase invasion of cancer cells (13-14), vascular fibroblasts and smooth muscle cells (15). This invasion may be due to autologous chemotactic gradients created around cells in 3-D (16) or increased matrix metalloproteinase (MMP) expression (15), chemokine secretion and cell adhesion molecule expression (17). However, the mechanism by which cells sense fluid flow is not well understood. In addition to altering tumor cell behavior, interstitial fluid flow modulates the activity of other cells in the tumor microenvironment. It is associated with (a) driving differentiation of fibroblasts into tumor-promoting myofibroblasts (18), (b) transporting of antigens and other soluble factors to lymph nodes (19), and (c) modulating lymphatic endothelial cell morphogenesis (20). The technique presented here imposes interstitial fluid flow on cells in vitro and quantifies its effects on invasion (Figure 1). This method has been published in multiple studies to measure the effects of fluid flow on stromal and cancer cell invasion (13-15, 17). By changing the matrix composition, cell type, and cell concentration, this method can be applied to other diseases and physiological systems to study the effects of interstitial flow on cellular processes such as invasion, differentiation, proliferation, and gene expression.

Proteomic profiling of infiltrating ductal carcinoma reveals increased cellular interactions with tissue microenvironment.

Progression of invasive carcinoma involves the deregulation of molecular signaling pathways that results in the acquisition of oncogenic phenotypes. Functional enrichment analysis allows for the identification of deregulated pathways from omics scale expression data. Given the importance of post-transcriptional regulatory mechanisms on protein expression and function, identification of deregulated pathways on the basis of protein expression data is likely to provide new insights. In this study, we have developed methods for label-based mass spectrometry in a large number of samples and applied these methods toward identification and quantification of protein expression in samples of infiltrating ductal carcinoma, benign breast growths, and normal adjacent tissue. We identified 265 proteins with differential expression patterns in infiltrating ductal carcinoma relative to benign growths or normal breast tissue. Analysis of the differentially expressed proteins indicated the deregulation of signaling pathways related to proliferation, invasion and metastasis, and immune response. Our approach provides complementary information to gene expression microarray data and identifies a number of deregulated molecular signaling pathways indicative of breast cancer progression that may enable more accurate, biologically relevant diagnoses and provide a stepping stone to personalized treatment.