3D bioengineered scaffold-based model to investigate the role of the tumour microenvironment in localised prostate cancer progression

摘要

Introduction: Prostate cancer (PCa) is the second most frequent malignancy in men, with 1.1 million cases diagnosed annually. Fortunately, most men are diagnosed when the malignancy is confined to the gland (localised disease). However, approximately 10-33% of patients will go on to progress to advanced, metastatic disease, which is ultimately lethal. It is widely acknowledged that the tumour microenvironment (TME; stroma, extracellular matrix (ECM), vasculature, immune cells and hormones) plays a fundamental role in this disease progression. Despite this, most in vitro PCa models are simple, 2D monocultures of epithelial tumour cell lines. There is an immediate need to develop multi-cellular in vitro constructs to investigate the effect of the TME on tumourigenicity. To this end, we have developed a novel 3D bioengineered in vitro scaffold-based model, which aims to recapitulate specific stages of early prostate tumourigenesis, namely fibroblast-epithelial and immune-epithelial interactions. Materials and Methods: Melt-electrospun scaffolds were formatted from poly(E-caprolactone) (PCL) polymer spun in a 0/90° pattern, forming flexible 15×15mm~2 meshes with a pore size of -100μm. Scaffolds were plasma treated to enhance cell attachment. Validated patient-derived cancer-associated fibroblasts (CAF) or non-malignant prostatic fibroblasts (NPF) were incorporated into the treated scaffolds. Once confluent, benign (initiated) epithelial cells (BPH-1) were co-cultured on the fibroblast constructs +/- mast cell (MC) conditioned media. At day 1, scaffolds were fixed and tumourigenicity was assessed by analysing the morphological transformation of BPH-1 cells (sphericity, volume, orientation, migration) using confocal microscopy and Imaris software. Results: CAFs and NPFs proliferate and deposit ECM to form a 3D stromal network within the scaffolds. Cell penetrance is ～90μm (6-8 cell layers). CAF, but not NPF, induce an invasive morphology in the benign epithelium, recapitulating the in vivo biology. Significantly, when an immune component was incorporated - MC conditioned media -the CAF-induced transformation of BPH-1 cells was further potentiated. Ongoing work includes investigating the transcriptome of the BPH-1 cells when co-cultured with CAF/NPF +/- mast cells and proteomic analysis of CAF and NPF grown in both 2D and scaffold (3D) environments. Conclusion: This 3D scaffold-based model provide a physiologically relevant in vitro platform to study the prostate TME, with data highlighting the fundamental contribution of fibroblast-epithelial and immune-epithelial interactions on early Pca progression.