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 invasion³. 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.