Effects of Shear Stress Gradients on Ewing Sarcoma Cells Using 3D Printed Scaffolds and Flow Perfusion

Abstract

In this work, we combined three-dimensional (3D) scaffolds with flow perfusion bioreactors to evaluate the gradient effects of scaffold architecture and mechanical stimulation, respectively, on tumor cell phenotype. As cancer biologists elucidate the relevance of 3D in vitro tumor models within the drug discovery pipeline, it has become more compelling to model the tumor microenvironment and its impact on tumor cells. In particular, permeability gradients within solid tumors are inherently complex and difficult to accurately model in vitro. However, 3D printing can be used to design scaffolds with complex architecture, and flow perfusion can simulate mechanical stimulation within the tumor microenvironment. By modeling these gradients in vitro with 3D printed scaffolds and flow perfusion, we can identify potential diffusional limitations of drug delivery within a tumor. Ewing sarcoma (ES), a pediatric bone tumor, is a suitable candidate to study heterogeneous tumor response due to its demonstrated shear stress-dependent secretion of ligands important for ES tumor progression. We cultured ES cells under flow perfusion conditions on poly(propylene fumarate) scaffolds, which were fabricated with a distinct pore size gradient via extrusion-based 3D printing. Computational fluid modeling confirmed the presence of a shear stress gradient within the scaffolds and estimated the average shear stress that ES cells experience within each layer. Subsequently, we observed enhanced cell proliferation under flow perfusion within layers supporting lower permeability and increased surface area. Additionally, the effects of shear stress gradients on ES cell signaling transduction of the insulin-like growth factor-1 pathway elicited a response dependent upon the scaffold gradient orientation and the presence of flow-derived shear stress. Our results highlight how 3D printed scaffolds, in combination with flow perfusion in vitro, can effectively model aspects of solid tumor heterogeneity for future drug testing and customized patient therapies.

Human Organ-Specific 3D Cancer Models Produced by the Stromal Self-Assembly Method of Tissue Engineering for the Study of Solid Tumors

“…Another one consists of the incorporation of the vascular/lymphatic network simulating dynamic blood flow and providing the mechanical signals regulating tumor development and function. An increasing number of studies report the development of such perfusion systems applied to models of various primary and metastatic tumor types [20,[246][247][248][249].…”

Engineering Breast Cancer Microenvironments and 3D Bioprinting

“…Information and research into biomechanical properties of the ECM are limited when synthetic scaffolds are employed for modeling. Biomechanical properties are shown to play a very important part in modulation the ECM and cell progression (Hinton et al, 2015 ; Laronda et al, 2017 ; Trachtenberg et al, 2017 ). Due to the limitations of previous bioprinting methods (Knowlton et al, 2015 ), the architecture of the scaffold is inadvertently neglected because methods such as spheroid formation and inkjet printing have limited capabilities.…”

“…Of the 3DBP methods, EBB shows the most potential for precise architecture control. Factors of fluid shear stress and macro-scale architecture have synergistic effects on tumor phenotype, protein expression, and cell proliferation (Trachtenberg et al, 2017 ). Furthermore, previous studies show that hydrogel architecture including fiber density, size (length and width), and stress relaxation in addition to hypoxic gradients increases sarcoma cell migration (Lewis et al, 2017 ).…”

Tissue engineered models of healthy and malignant human bone marrow

“…Recently, another effort used 3D printing combined with flow perfusion bioreactors to create scaffolds with inverse porosity and shear stress gradients in order to better understand the gradient dependent mechanotransductive regulation of the IGF-1 pathway that plays a major role in ESFT pathogenesis [178]. Our group was the first to investigate the effects of mechanical loading on ES cells [179], in co-cultures of ES cells and MSCs in a collagen type 1 matrix (Fig.…”