Benign prostatic hyperplasia (BPH) is characterized by inflammation, fibrosis of the extracellular matrix and hypercontractility of stromal tissue. In urological diseases with similar symptoms, the ratio of pro nerve growth factor (proNGF) to its mature isoform (NGF) is increased and considered a marker of inflammation. Murine models of diabetic bladder dysfunction display the same imbalance. Chronic treatment with THX-B, an antagonist of the p75NTR receptor of proNGF, restored the neurotrophin ratio and improved bladder contractile parameters. On the other hand, organoids are tridimensional cellular structures which reproduce the interactions between different cell types present in the same organ, removing the problems associated with co-culturing cells in 2 dimensions. They have been widely used to study prostate diseases but mainly in cancer research. We here report a model of organoids from prostate cells to assess the role of neurotrophins in BPH.

Whole prostate tissue of Sprague-Dawley rats was digested with collagenase type II. Epithelial and stromal cells (50/50) were seeded in non-adherent wells. After 48 hours, the structure of organoids was examined by confocal microscopy using smooth muscle myosin heavy chain (SMMHC) and cytokeratin 17 (CK17) to highlight respectively stromal and epithelial cells. In the structure. BPH was induced with hyperglycemic medium (25 mM) combined or not with elevated testosterone concentrations (500 nM). THX-B, the antagonist of p75NTR proinflammatory receptor, was incubated in all conditions. Neurotrophin levels were measured by Elisa. Immunoblottings and RTqPCR were performed on cell extracts to assess inflammatory, contractility and fibrosis markers.

Light microscopy revealed the organisation of cells into spheroids. Confocal microscopy showed epithelial and stromal cells mingled at the surface of a hollow structure, as revealed by CK17 (epithelial) and SMMHC (stromal) markers. Spheroids released NGF and proNGF in the culture medium. NGF secretion was not affected nor by high glucose nor testosterone combined or not, neither by THX-B. ProNGF levels were increased by testosterone and high glucose and reverted to contorl values by THX-B. Inflammatory markers IL1, IL-6 and TNF-alpha expression was unchanged in all conditions. Myosin, smoothelin and actin were unaffected.

Epithelial cells secrete many factors that directly target stromal cells. Co-culture in the form of organoids provide a new tool to examine how different cell type react in culture conditions mimicking a pathological settings, such as hyperglycemia and/or hypertestosteronemia.

Organoid culture of prostate cells was reproducible and constitute a valid model to study prostate cell interaction in BPH in the context of neurotrophin secretions.