Effects of interstitial flow and fluid shear stress on tumor cell invasion and metastasis

Introduction: Tumor cells are exposed to fluid flow during the onset of angiogenesis, at the tumor periphery while invading the stroma, metastasis through blood and lymphatic vessels, and during vascular normalization therapy. Studies have shown that forces generated by fluid flow, specifically fluid shear stress, generate a cascade of processes that affect cell function. Tumor cell lines that have been exposed to fluid flow forces in vivo have invasive and metastatic potentials distinct from in vitro cell motility assays without flow. Brain tumor cell lines exposed to fluid flow forces in vivo have lower invasive potentials than in vitro cell motility assays without flow indicate. On the other hand, metastatic cell lines exposed to fluid flow forces in vivo display augmented invasive potentials. This study hypothesized that interstitial flow and fluid shear stress are un-recognized factors that dramatically influence cell motility and play a role in suppression or augmentation of invasion - phenomena involving mechanotransduction.;Methods: Several non-invasive/non-metastatic and invasive/metastatic cell lines were investigated with emphasis placed on non-invasive (U87, CNS-1) and invasive (U251) gliomas, and highly metastatic (SN12L1) and low metastatic potential (SN12C) renal cell carcinomas. Modified Boyden chambers, modeling 2D (rotating disk) and 3D (Darcy flow through collagen/cell gel suspensions) fluid shear stress, were designed to mimic the fluid dynamic microenvironments in blood circulation and interstitial flow in the tumor interstitium. Emphasis was placed on the 3D suspensions - modeling flows up to 24 hours in length. Novel methods addressed gel compaction, flushing effects of flow, and isolation of chemotactic migration from flow stimulation. Matrix Metalloproteinase (MMP) levels were manipulated via MMP shRNA constructs, broad-spectrum and specific MMP inhibitors, and were quantified by MMP assays, zymography, and RT-PCR (gel electrophoresis and quantitative). Involvement of cell adhesion molecules (CD44, α3 integrin) and glycocalyx components (heparan sulfate and hyaluronans) were also investigated.;Principal Findings: Physiologic levels of fluid shear stress suppressed the migratory activity of non-invasive U87 and CNS-1 glioma cells while the motility of the invasive U251 glioma cell line remained unaltered within the 3D interstitial flow model. MMP inhibition experiments and assays demonstrated that the glioma cells depended on MMP activity to invade, and suppression in motility correlated with downregulation of MMP-1 and MMP-2 levels - confirmed by RT-PCR and with the aid of MMP-1 and MMP-2 shRNA constructs. On the other hand, interstitial flow and fluid shear stress upregulated MMP levels and enhanced the motility of the SN12L1 highly metastatic cells while the migratory activity of low metastatic potential SN12C cells remained unaffected. The augmented migration rates of the metastatic cell lines correlated with the flow-induced upregulation of adhesion molecules (CD44 and integrins), and MMP gene expressions. Blocking the enhanced expression of adhesion molecules and MMP activity resulted in diminished migratory activity near baseline control levels. The presence of a glycocalyx-like layer of glycosaminoglycans was confirmed to be present around tumor cells in 3D. Degradation of this layer by hyaluronidase and heparinase blocked the flow enhanced motility effects on the metastatic cells.;Significance/Conclusions: Exposure to flow induced migration trends that were consistent with reported invasive potentials of implanted tumors. Interstitial flow and fluid shear stress in the tumor microenvironment suppresses the migratory activity of non-invasive cells while enhancing the motility of metastatic cells. This study emphasizes that flow effects involve mechanotransduction of fluid shear stress via glycocalyx components that regulate MMP expression. The models developed for this study should be useful for further study of the fluid mechanical processes that affect tumor progression.