A 3D physio-mimetic interpenetrating network-based platform to decode the pro and anti-tumorigenic properties of cancer-associated fibroblasts

Abstract

Three-dimensional (3D) biomaterials with physiologically relevant and experimentally tractable biomechanical features are important platforms to advance our understanding of the influence of tissue mechanics in disease progression. Herein, an interpenetrating network (IPN) of collagen and alginate 3D culture system with tunable extracellular microstructure and mechanics is exploited as a tumor stroma proxy to study phenotypic plasticity of colorectal cancer-associated fibroblasts (CAF). In combination with Next Generation Sequencing (NGS) data analysis, we demonstrated that tuning the storage modulus of the IPN hydrogel between 49 and 419 Pa can trigger a reversible switch between an inflammatory (i-state, α-SMAlowIL-6high) and myofibroblastic (m-state, α-SMAhighIL-6low) state in CAF that is dependent on the polymer network confinement effect and ROS-HIF1-α mechanotransduction signaling axis. Secretome from m-state CAF upregulated several epithelial-mesenchymal-transition (EMT) transcripts and induced robust scattering in DLD-1, HCT116, and SW480 human colorectal adenocarcinoma, while the EMT-inducing capacity is muted in i-state CAF, suggestive of an anti-tumorigenic role. Our findings were further validated through Gene Expression Profiling Interactive Analysis (GEPIA), which showed that cytokines secreted at higher levels by i-state CAF are correlated (p < 0.05) with good overall colorectal cancer patient survival. Therefore, 3D network density and spatial cellular confinement are critical biophysical determinants that can profoundly influence CAF states, paracrine signaling, and EMT-inducing potential.