Characterisation of Collagen/Chondroitin Sulfate and Collagen/Hyaluronic Acid Scaffolds to Model the Prostate Cancer Microenvironment.
Nezar Kamal1,, Aamir Hameed1, Ciara Murphy1,2,3, Caroline Curtin1,2,3
1Tissue Engineering Research Group (TERG), Department of Anatomy & Regenerative Medicine, Royal College of Surgeons in Ireland (RCSI), Dublin, Ireland. 2Trinity Centre for Bioengineering (TCBE), Trinity College Dublin (TCD), Dublin, Ireland. 3Advanced Materials and Bioengineering Research Centre (AMBER), RCSI and TCD, Dublin, Ireland.
Prostate cancer (PC) is one of the leading causes of cancer-related death in men worldwide. In the early stages of advanced PC, cancer cells undergo epithelial-to-mesenchymal transition (EMT), a process whereby epithelial cells lose their cell-cell adhesion, gain migratory and invasive characteristics, and travel through the blood vessels to invade new tissue, leading to metastasis. Approximately 90.1% of those who die with PC have bone metastases. Studies suggest that combining Docetaxel with hormone therapy helps in reducing PC progression and spread but ultimately only prolongs the length of survival. As a result, there is a major unmet need to develop alternative treatments. The extracellular matrix (ECM) of prostate tissue contains various constituents, including chondroitin sulfate (CS) and hyaluronic acid (HyA), which are significantly increased in metastatic PC and may be of interest for further investigation. This project aims to develop three-dimensional (3D) porous collagen/CS (Col/CS) and collagen/HyA(Col/HyA) scaffolds suitable for studying in vitro PC EMT and as a platform to test and develop treatments for PC.
Three Col/CS scaffold compositions containing 0.5 weight/volume (w/v) collagen and 0.05, 0.55, and 1.05 w/v CS were prepared. Additionally, two Col/HyA scaffold compositions containing 0.5 w/v collagen and 0.05 and 1.05 w/v HyA using freeze-dryer technique. These scaffolds were subjected to rigorous scaffold characterisation, including mechanical testing, SEM, pore size analysis, and porosity measurements. To determine the impact of CS and HyA on PC EMT processes and metastasis growth, osteolytic PC-3 and osteoplastic LNCaP PC cells were cultured on all scaffolds for biochemical evaluations, such as metabolic activity, DNA quantitation, and Live/Dead cell viability assays. Additionally, EMT cytokines expression was examined on the scaffolds using Cytokine Array kit to determine the impact of increasing CS and HyA concentrations on EMT cytokines expression.
Overall, the highly porous scaffolds were successfully developed. The stiffness significantly increased after EDAC cross-linking for all Col/CS, and Col/HyA low but all scaffold compositions were less stiff than PC tumours. All the scaffolds swelled at a high rate and remained stable and free of deformity. All scaffolds supported PC-3 and LNCaP cell growth and proliferation. Moreover, the Col/CS high scaffolds seeded with LNCaP cells showed the highest metabolic activity and proliferation throughout the 14 days cultures. The live/dead images showed only minimal dead cells. All Col/CS scaffolds seeded with PC-3 expressed cytokines associated with EMT processes, and Col/CS high had the highest signal intensity of ENA-78, IL-6, IL-8, Angiogenin, and MCP-1 cytokines among the scaffold types assessed.
Initial findings demonstrate the scaffolds can be used as an in vitro platform for studying PC EMT processes and drug testing.