Exploring prostate cancer mechanobiology with an in-vitro 3D hydrogel model and computational approaches

Abstract

Prostate cancer (PCa) is the most common cancer and the second leading cause in cancer related mortality in Western men. PCa is an heterogenous and complex disease with insufficient therapies for advanced disease. Current treatments for advanced and metastatic disease revolve around inhibiting the function of the androgen receptor (AR) which is vital for almost all cases of PCa. Though initially effective, eventually virtually all patients develop resistance to therapies through reactivation of AR signalling. The mechanisms in the development of resistance are not well understood and further research is required to recognize what roles the changes in the genomic landscape and the tumour microenvironment (TME) play. Current studies focus on the genomic landscape and how gene expression changes impact the reactivation of AR signalling. The role of TME is far less understood as most current studies on PCa models are done on 2-dimentional (2D) culture platforms. The TME is a dynamic and essential part of the development and progression of PCa. The role it plays in AR signalling reactivation in treatment resistance is unknown. Although several signalling mediators from pathways related to sensing the mechanical microenvironment have been also associated with the AR, suggesting a potential connection with the mechanical TME and AR signalling. The lack of 3-dimensional (3D) cell culture models for PCa with adjustable properties is limiting the possibility of exploring the significance of the TME in PCa. In this thesis, we establish a modifiable 3D hydrogel model based on hyaluronic acid, which can mimic the mechanical and biochemical attributes of the TME of PCa. We employ this model alongside 2D culture RNA sequencing data to investigate the role of ECM stiffness and protein composition on testosterone sensitive vertebral cancer of the prostate (VCaP-T) cells. Additionally, we investigate protein-protein association data to gain understanding of the connection between AR signalling and mechanical signalling provided by the mechanical and cellular TME. Through the network-based exploration of freely available protein-protein association data, it is revealed that in the proteins associated with the AR, there are several enriched pathways relating to cell adhesion and cell-cell contact. This would suggest a possible connection between the mechanical signals provided by the TME and AR signalling. The functionality of the 3D cell culture platform was tested with live/dead viability assays and proliferation assays. These assays of the encapsulated VCaP-T cells were tested utilizing different stiffnesses of hydrogels and protein compositions to explore the impact of TME changes. The initial experiments showed unexpected results which were later linked to unanticipated issues with one of the hydrogel components. Later experiments, with a new batch of material, show good viability of the hydrogel model with the VCaP-T cells alongside clear differences between gel compositions. The cells show a preference to higher stiffness and one common ECM protein. The binding to this protein is additionally supported by the RNA sequencing data which show that the VCaP-T primarily express integrins associated with binding to RGD-peptides and laminin. Overall, this thesis shows functionality of a novel 3D hydrogel cell model for PCa with evidence shown that changes in the mechanical environment of PCa cell changes their proliferation and viability. Additionally, the significance of the TME is shown by the proximity of mechanically active components to the AR, which plays a crucial role in PCa.