On the Evaluation of a Novel Hypoxic 3D Pancreatic Cancer Model as a Tool for Radiotherapy Treatment Screening

SIMPLE SUMMARY: Pancreatic cancer challenges global health with non-specific symptoms, devastatingly low survival rates, and high treatment resistance profiles. Tissue engineering is advancing to facilitate animal free tissue biomimicry, allowing the replication of tumour tissue specific hallmarks of pancreatic cancer that challenge modern treatments. Here, we report the development and characterisation of a low oxygen (hypoxic) 3D polyurethane scaffold system for long-term analysis of radiation responses. This finely tuned platform more accurately recapitulates bio-physical, bio-chemical, and structural-bio-mechanical in vivo tissue niches as well as tumour hypoxia. The latter is a treatment-limiting feature for radiotherapy, allowing the system to streamline the transition of clinical testing from bench to bedside. ABSTRACT: Tissue engineering is evolving to mimic intricate ecosystems of tumour microenvironments (TME) to more readily map realistic in vivo niches of cancerous tissues. Such advanced cancer tissue models enable more accurate preclinical assessment of treatment strategies. Pancreatic cancer is a dangerous disease with high treatment resistance that is directly associated with a highly complex TME. More specifically, the pancreatic cancer TME includes (i) complex structure and complex extracellular matrix (ECM) protein composition; (ii) diverse cell populations (e.g., stellate cells), cancer associated fibroblasts, endothelial cells, which interact with the cancer cells and promote resistance to treatment and metastasis; (iii) accumulation of high amounts of (ECM), which leads to the creation of a fibrotic/desmoplastic reaction around the tumour; and (iv) heterogeneous environmental gradients such as hypoxia, which result from vessel collapse and stiffness increase in the fibrotic/desmoplastic area of the TME. These unique hallmarks are not effectively recapitulated in traditional preclinical research despite radiotherapeutic resistance being largely connected to them. Herein, we investigate, for the first time, the impact of in vitro hypoxia (5% O(2)) on the radiotherapy treatment response of pancreatic cancer cells (PANC-1) in a novel polymer (polyurethane) based highly macroporous scaffold that was surface modified with proteins (fibronectin) for ECM mimicry. More specifically, PANC-1 cells were seeded in fibronectin coated macroporous scaffolds and were cultured for four weeks in in vitro normoxia (21% O(2)), followed by a two day exposure to either in vitro hypoxia (5% O(2)) or maintenance in in vitro normoxia. Thereafter, in situ post-radiation monitoring (one day, three days, seven days post-irradiation) of the 3D cell cultures took place via quantification of (i) live/dead and apoptotic profiles and (ii) ECM (collagen-I) and HIF-1a secretion by the cancer cells. Our results showed increased post-radiation viability, reduced apoptosis, and increased collagen-I and HIF-1a secretion in in vitro hypoxia compared to normoxic cultures, revealing hypoxia-induced radioprotection. Overall, this study employed a low cost, animal free model enabling (i) the possibility of long-term in vitro hypoxic 3D cell culture for pancreatic cancer, and (ii) in vitro hypoxia associated PDAC radio-protection development. Our novel platform for radiation treatment screening can be used for long-term in vitro post-treatment observations as well as for fractionated radiotherapy treatment.