Development of Three-dimensional Scaffold Models Suitable for the Study of Prostate Cancer Progression

Abstract

Prostate cancer (PC) is one of the leading causes of cancer-related death in men worldwide. In advanced PC, cancer cells undergo an epithelial-to-mesenchymal transition (EMT) process, characterised by increasing mesenchymal factors, leading to metastasis. At the metastatic sites, cells undergo a mesenchymal-to-epithelial (MET) process, characterised by increasing epithelial and angiogenic factors for more stable colonisation. In recent years, the extracellular matrix (ECM) of the PC tumour microenvironment (TME) has been recognised as a critical player in cancer development and the EMT process. The ECM of prostate tissue consists of collagen and various constituents, including chondroitin sulfate (CS) and hyaluronic acid (HyA), which significantly increase during PC progression and warrant further investigation. Due to their inherent limitations, 2D cultures cannot entirely mimic the in vivo ECM of PC TME, displaying significant differences in cell shape, proliferation, and differentiation. Therefore, this project aims to develop physiologically relevant 3D collagen-based scaffolds containing different concentrations of CS and HyA to mimic the ECM of PC and be suitable for studying the EMT process, evaluating anti-cancer therapeutics, including chemotherapy and gene therapy, and studying the PC bone metastasis in bone-mimicking scaffolds. In Chapter 2 of this thesis, four collagen-based scaffolds were successfully developed and characterised. The scaffolds were named according to CS and HyA concentration (Col/CS low, Col/CS high, Col/HyA low and Col/HyA high). The scaffolds revealed excellent biocompatibility and facilitated the attachment and survival of two PC cell lines, LNCaP and PC-3. Additionally, the scaffolds maintained the typical phenotype of PC cells, confirming their suitability for PC research. Chapter 3 focused on examining the influence of ECM components, including CS and HyA, on the EMT process of PC using the scaffolds developed in Chapter 2. The findings indicated that CS promotes EMT and aggressiveness in PC cells by stimulating the expression of mesenchymal markers, metastatic markers, and pro-inflammatory, angiogenic, and proliferation-related cytokines, particularly in PC-3 cells. On the other hand, HyA was found to suppress EMT in LNCaP cells by enhancing epithelial marker expression and reducing angiogenic cytokines. This highlighted the crucial functions of ECM components in regulating EMT and PC advancement. In Chapter 4, the scaffolds created in Chapter 2 were used to assess the chemotherapy response generated by PC in a 3D environment compared to the 2D culture. Moreover, the potential therapeutic effect of miRNA-23a (miR-23a) was evaluated using these scaffolds as novel gene therapy. The results underscored the role of CS in promoting chemoresistance in PC-3 cells, making it the perfect target to assess the effect of miR- 23a overexpression in PC progression. The study showed that miR-23a can inhibit cell migration, invasiveness, metastatic behaviour, and aggressive phenotype. This indicates that miR23-a is a promising gene therapy in PC and highlights the efficiency of collagen- based scaffolds. Finally, a bone-mimicking collagen-nanohydroxyapatite scaffold was used as a platform to mimic PC bone metastasis in Chapter 5 and study the cellular interactions within the TME. The study was designed by co-culturing different osteoblast maturities with PC cells (LNCaP and PC-3) to examine their relationships more deeply and simulate the TME. The co-culture systems revealed that the scaffolds mimic the TME of PC bone metastasis, highlighting the role that osteoblasts play in shaping the morphology, proliferation, and migration of PC cells. The results also showed that co-culture of PC cells with osteoblasts supported bone remodelling and activated MET processes for more stable colonisation. These investigations confirmed the ability of the scaffolds to simulate the interactions between tumours and stroma found in PC bone metastases. In conclusion, this thesis successfully introduced collagen-based scaffolds incorporating native ECM components (CS and HyA) as physiologically relevant 3D models for PC research. These models provided valuable insight into mechanisms driving PC progression, EMT, and therapeutic resistance while offering a platform for evaluating novel treatments, including gene therapy. The co-culture systems further expand their application to studying tumour-stroma and cell-cell interactions, particularly in bone metastasis, making it a valuable tool for in vitro studies for PC research.