Three-Dimensional Microfluidic Collagen Hydrogels for Investigating Flow-Mediated Tumor-Endothelial Signaling and Vascular Organization

Abstract

Hyperpermeable tumor vessels are responsible for elevated interstitial fluid pressure and altered flow patterns within the tumor microenvironment. These aberrant hydrodynamic stresses may enhance tumor development by stimulating the angiogenic activity of endothelial cells lining the tumor vasculature. However, it is currently not known to what extent shear forces affect endothelial organization or paracrine signaling during tumor angiogenesis. The objective of this study was to develop a three-dimensional (3D), in vitro microfluidic tumor vascular model for coculture of tumor and endothelial cells under varying flow shear stress conditions. A central microchannel embedded within a collagen hydrogel functions as a single neovessel through which tumor-relevant hydrodynamic stresses are introduced and quantified using microparticle image velocimetry (μ-PIV). This is the first use of μ-PIV in a tumor representative, 3D collagen matrix comprised of cylindrical microchannels, rather than planar geometries, to experimentally measure flow velocity and shear stress. Results demonstrate that endothelial cells develop a confluent endothelium on the microchannel lumen that maintains integrity under physiological flow shear stresses. Furthermore, this system provides downstream molecular analysis capability, as demonstrated by quantitative RT-PCR, in which, tumor cells significantly increase expression of proangiogenic genes in response to coculture with endothelial cells under low flow conditions. This work demonstrates that the microfluidic in vitro cell culture model can withstand a range of physiological flow rates and permit quantitative measurement of wall shear stress at the fluid-collagen interface using μ-PIV optical flow diagnostics, ultimately serving as a versatile platform for elucidating the role of fluid forces on tumor-endothelial cross talk.

Flow shear stress regulates endothelial barrier function and expression of angiogenic factors in a 3D microfluidic tumor vascular model

“…38 Briefly, 1 £ 10 6 MDA-MB-231 cells were resuspended in 8 mg/ml neutralized collagen, poured into FEP tubing fit concentrically with a 22G needle (711 mm), and capped with PDMS sleeves (Fig. 1).…”

“…A graded increase in flow that subjected the endothelium to a low shear stress of t D 0.01 dynes/cm 2 up to t D 0.1 dyne/cm 2 over 72 hours was utilized to encourage the establishment of a viable and functional endothelialized channel. 38 Following this initial preconditioning time period, the flow was increased to induce one 2 ) for 6 hours, after which analysis of angiogenic activity (expression of angiogenic factors and vascular permeability) was conducted. The 6h time point was selected to probe the initial and transient endothelial response to flow as well as the subsequent changes in tumor expressed angiogenic genes.…”

“…Flow rates, Q, were initially estimated based on an assumption of Poiseuille flow, t w D 4Qm=pr 3 , using the measured viscosity of EBM-2 culture media at 37 C (m D 0.78 § 0.0057 cP) and then validated using micro-particle image velocimetry as described previously. 38 Mono-cultures of MDA-MB-231 cells or TIME cells alone were used as controls.…”

“…This decreased permeability is likely due to increased endothelial alignment and spreading in the direction of flow, 38 in which high WSS (t W D 10 dyn/cm 2 ) significantly decreased P d relative to the preconditioned endothelium. These findings are consistent with others showing shear stress-mediated endothelial morphological adaptations 22,38,[49][50][51][52] and permeability. 14,17,19,21,23,53 The presence of tumor cells also increased P d relative to endothelial mono-cultures, with a significantly greater P d under high WSS conditions.…”

“…Using a previously established 3D, in vitro microfluidic tumor vascular model, 38 tumor and endothelial cells were co-cultured under a range of tumor-relevant hydrodynamic stresses. It was hypothesized that high flow shear stress will decrease vascular permeability and tumor cell expression of angiogenic growth factors.…”