The development of nanoparticle-based targeted therapeutics for the treatment of cancer requires a well-defined understanding of the tumor microenvironment, which is challenging due to tumor complexity and heterogeneity. Recent advancements in three-dimensional (3D) cell models such as tumor-on-a-chip devices can overcome some of these challenges by providing coculture in vitro systems (tumor surrounded by tubular endothelial cells) that mimic native cellular environments to accurately study the enhanced permeability and retention (EPR) potential of drug delivery systems under flow conditions. However, inducing “leaky” vasculature in endothelial cells surrounding solid tumors in microfluidic devices is not readily controllable and highly dependent on tumor cell identity. Utilizing a microfluidic tumor model (MTM) consisting of a tumor region surrounded by a 3D microvascular network, we have simulated the EPR effect by activating a known regulator of endothelial junction formation and edema: the transient receptor potential vanilloid 4 (TRPV4) ion channel, to rapidly assess extravasation and tumor accumulation of nanoparticles of different sizes and surface chemistries. Treatment with a selective TRPV4 agonist stimulated reorganization of the actin cytoskeleton and disruption of adherens junctions to provide a concentration-dependent or “tunable” leakiness, confirmed by increased tumor uptake of fluorescent dextran macromolecular tracers from the vascular channels. Although this controlled 3D in vitro vascular-edema system may not exemplify all of the complexities of edema mechanisms in vivo, it provides a rapid, materials-focused screening method to assess the extravasation and tumor uptake potential of nanoparticles with distinct properties. We show that the passage of nanoparticles through leaky vasculature is not solely governed by particle size but also by surface chemistry, where surface tertiary amines limit tumor cell association due to unwanted endothelial interactions.