Interstitial Fluid Flow Increases Hepatocellular Carcinoma Cell Invasion through CXCR4/CXCL12 and MEK/ERK Signaling.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer (~80%), and it is one of the few cancer types with rising incidence in the United States. This highly invasive cancer is very difficult to detect until its later stages, resulting in limited treatment options and low survival rates. There is a dearth of knowledge regarding the mechanisms associated with the effects of biomechanical forces such as interstitial fluid flow (IFF) on hepatocellular carcinoma invasion. We hypothesized that interstitial fluid flow enhanced hepatocellular carcinoma cell invasion through chemokine-mediated autologous chemotaxis. Utilizing a 3D in vitro invasion assay, we demonstrated that interstitial fluid flow promoted invasion of hepatocellular carcinoma derived cell lines. Furthermore, we showed that autologous chemotaxis influences this interstitial fluid flow-induced invasion of hepatocellular carcinoma derived cell lines via the C-X-C chemokine receptor type 4 (CXCR4)/C-X-C motif chemokine 12 (CXCL12) signaling axis. We also demonstrated that mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling affects interstitial fluid flow-induced invasion; however, this pathway was separate from CXCR4/CXCL12 signaling. This study demonstrates, for the first time, the potential role of interstitial fluid flow in hepatocellular carcinoma invasion. Uncovering the mechanisms that control hepatocellular carcinoma invasion will aid in enhancing current liver cancer therapies and provide better treatment options for patients.

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.