The tumour microenvironment (TME) plays a fundamental role in prostate carcinogenesis. Classical tumour recombination experiments have shown that cancer‐associated stroma directs tumour formation in benign epithelia. Despite this, stromal components are often overlooked in human PCa models. Currently, there are no models of human PCa which adequately examines the combined contribution of cancer‐associated fibroblasts (CAF), their aberrant extracellular matrix (ECM) and a key resident immune population, mast cells (MC). Here we describe a three‐dimensional (3D) bioengineered TME microtissue in vitro model which interrogates the interaction and contribution of these components in early prostate carcinogenesis.

Melt‐electrospun scaffolds were formatted from medical grade poly(ε‐caprolactone). Patient‐derived primary CAFs or non‐malignant prostatic fibroblasts (NPFs) were incorporated into the scaffolds, forming a 3D microtissue. Once confluent, tagged benign epithelial cells (BPH‐1 or RWPE‐1) were co‐cultured on the microtissues ± MC (HMC‐1 or LAD2), MC‐conditioned media (CM) or recombinant tryptase. Subsequently, microtissues were fixed and tumourigenicity was assessed by analysing the 3D morphological transformation of epithelial cells. Additionally, live cell migration assays were performed to further quantify invasive potential.

Our data show that CAF and NPF proliferate and each deposit distinct ECM to form a 3D stromal network within the scaffolds. CAF, but not NPF, microtissues induce an invasive morphology in the benign epithelium. MCs cooperate with CAFs, potentiating CAF‐induced tumourigenic effects. These effects are mediated by MC secreted factors, specifically tryptase (a serine protease). Our data indicate that MC‐derived tryptase likely acts by remodelling the native aberrant ECM deposited by primary CAFs, conferring tumourigenicity on the benign epithelia.

Overall, this model demonstrates the cascade of interactions between CAFs, ECM and MCs, mediated by tryptase, to drive early epithelial transformation in the human prostate. Our data also highlight tryptase as a key mediator of these effects, which may be a novel therapeutic target to slow carcinogenesis.