(466c) Biomimicry of the Hypoxic Tumour Microenvironment of Pancreatic Cancer in 3D – on the Design of Novel Studies of Radiotherapy Combined with a Static Magnetic Field

The interdisciplinary field of biomimetics is emerging to bridge the gap between simplistic 2D cell culture models and complex xenografts, more readily emulating tumour architecture, porosity, cell-cell and cell-matrix interactions, as well as hypoxic regions^[1]. More specifically, tissue engineering allows mimicry of bio-physical-chemical and mechanical properties of tumour microenvironments in a finely tuned in vitro setting, facilitating an animal free and cost effective platform as a representative tool for treatment screening and resistance profiling for cancer research ^[2].

Pancreatic ductal adenocarcinoma (PDAC) is a notoriously tragic disease, non-specific symptoms and high metastatic occurrence results in devastatingly low patient prognosis^[1-4]. More specifically, PDAC has a 5-year relative survival rate of just 9% ^[4]. This figure has not significantly improved for over 50 years ^[6] and predictions that this disease will rise to one of the most lethal cancers in coming decades ^[7] outline that this is a cancer of unmet needs. The PDAC tumour microenvironment (TME) is extremely complex and hypoxic^[8]. Unique disposition of PDAC extracellular matrix results in histological and behavioural hallmarks such as dense desmoplasiaand stromal formation that aid tumour survival and impair treatment delivery^[9]. Chaotic blood vessel formation and collapse paired with this desmoplasia contribute to hypoxia, which is directly related to radio-resistance and treatment failure ^[9]. Thus, this unique and heterogeneous TME requires biomimicry to predict the clinical relevance of evolving treatments.

Radiotherapy for PDAC is traditionally controversial with contradictory clinical trial outcomes, such as the LAP02 2016 trail^[10] and the ESPACI study^[11] in which no improved overall survival and damage to organs at risk were observed, highlighting a need for further understanding and optimisation. Advances in radiotherapy such as image guided and proton therapy, aim to enhance treatment and reduce damage to healthy tissue in neighbouring organs, facilitating specific tumour targeting and higher dose disposition. Such emerging modalities are associated with varying biological effectiveness compared to conventional therapies, thus there is a need for a reliable and representative system to test delivery strategies. Moreover, it is evident that realistic 3D models as well as improved and applied therapies are required for PDAC.

Image guided radiotherapy has inspired a small number of pre-clinical studies investigating the presence of static magnetic field in combination with radiation treatments ^[12]. However this area is understudied and contradictory. Early research in 2D cell culture suggests an alteration of biological response to radiation in combination with SMF ^[11-14]. More recently only a small number of studies have been reported ^[12]. More specifically, there is an identifiable gap in studying SMF and radiotherapy combinations 3D in vitro systems.

The BioProChem Group at the University of Surrey have previously fabricated a 3D porous polymeric scaffolding system to support long term PDAC cell growth and in vivo properties such as dense cellular masses, initiated collagen-1 growth and the development of hypoxic regions ^{[1-2 ]}for the application chemo-radiotherapy screening ^[3]. In addition, the research group produced the first scaffold assisted multicellular model for PDAC ^[15]. Utilising the 3D porous polymeric scaffolding system, here we aim to replicate the PDAC TME to investigate hypoxia-induced radio-resistance and the cellular response of combining a SMF during exposure.

Materials and Methods:

Fabrication of polymeric 3D scaffolds employed the Thermally Induced Phase Separation method ^[1-3]. PANC-1 cells were seeded at 0.5x10⁶ and cultured for 4 weeks before being placed at 5% oxygen in a hypoxic chamber. Radiotherapy exposures were performed using an orthovoltage X-ray unit at the Royal Surrey County Hospital and static magnetic field experiments took place with an MR-Linac at the National Physical Laboratory. Scanning electron microscopy and confocal laser scanning microscopy (CLSM) of multiple scaffold sections, enabled scaffold characterization, allowing analysis of cellular organisation and viability and mapping of (radio-)resistance. More specifically, live/dead analysis as well as Ki67 and acute (HIF-1α) and chronic (PDK1) hypoxic biomarkers quantification/mapping was performed and compared for different SMF-radiotherapy regimes.

Results and Discussion:

This research provides a platform for studying the complex and hypoxic PDAC TME. Growth kinetics and morphological variations in (i) hypoxic and normoxic cultures (ii) different static magnetic field-radiotherapy regimes were identified. These data show for the first time (i) a 3D polymeric scaffold supporting long-term hypoxic PDAC cell culture (ii) a long term post-treatment in vitro tissue characterisation for novel SMF-radiotherapy treatment patterns. This system provides a versatile platform to study hypoxia-associated radio-resistance profiling of PDAC in combination with a static magnetic field.

Significance and Impact:

PDAC is a cancer of unmet needs. As advancing technologies evolve, a realistic replica of the unique and extremely hypoxic TME is required for treatment screening and resistance profiling. This novel hypoxic 3D PU scaffold acts as a platform for biomimicry of the complex and hypoxic PDAC TME. This is the first 3D PU hypoxic scaffold system for PDAC static magnetic field and radiation combination studies.

Acknowledgements:

This research was supported by the Department of Chemical and Process Engineering of the University of Surrey as well as the National Physical Laboratory, the EPSRC and the Royal Society. E.V. is grateful for a Royal Academy of Engineering Industrial Fellowship.

References:

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