(22e) Treatment Resistance Profiling of a Novel Hypoxic Pancreatic Ductal Adenocarcinoma 3D Organoid System

Introduction and Aim:

Pancreatic ductal adenocarcinoma (PDAC) is a malignant solid tumour with increasing death and incidence rates^[1-2]. Non-specific symptoms, resulting in late diagnosis, high metastatic occurrence^[3]and treatment resistance ^[4], elucidate to a 5 year relative survival rate of just 9% ^[1]. This figure has not significantly improved for over 50 years ^[5]and predictions that this disease will rise to one of the most lethal cancers in coming decades^[6]outline a threat to global health. Thus, the demand for advanced understanding, characterisation, development of screening methods and therapeutics for PDAC is paramount. Treatment options for PDAC are challenged by a notoriously complex tumour microenvironment (TME). More specifically, the PDAC TME is densely hypoxic contributing to high therapeutic resistance ^[10].

Currently only 15% of PDAC patients present as appropriate for curative intervention ^[7]. The chemotherapeutic antimetabolites Gemcitabine and Capecitabine as well as FOLFIRINOX are universal treatment options. Loss of cell proliferation via DNA damage induction elucidates ionising radiation as an attractive therapeutic option for numerous cancer types. However, the use of radiotherapy for PDAC emerged a topic of controversy after the LAP02 2016 ^[8]trial showed radiotherapy resulted in no improvement to overall survival and the ESPAC1 study ^[9]indicated significant damage to organs at risk with deleterious effects on overall survival. Thus research into modern image guided radiotherapeutics is an essential and advancing topic of interest.

The replication of treatment in vitro is imperative to gain understanding in order to optimise therapeutic approaches. The PDAC TME is notoriously complex and challenging to replicate in vitro. 2D cell culture models fail to replicate advanced cellular interactions ^[11]. In comparison to this monolayer, xenografts are difficult to reproduce and show adverse behaviours due to host tissue variances^[11]. 3D cell culture and tissue engineering methods are evolving to bridge this gap, providing a unique approach to develop in vitro platforms ^[12]. The BioProChem Group at the University of Surrey have developed a novel highly porous polymeric scaffolding system for in vitro modelling of PDAC^[12]. This 3D PU scaffold is able to support long term growth of pancreatic cancer cells which form dense cellular masses and secrete significant collagen-I which is a key extracellular matrix protein (ECM) of PDAC. Furthermore, in this model realistic hypoxic gradients are formed with trends similar to the ones reported in in vivo models ^[12]. Overall, this model is readily replicating cell-cell and cell-ECM interactions of the PDAC TME.

The aim of this work is to systematically study the impact of various hypoxic levels on the kinetics and/or treatment response of the developed PDAC organoid model. More specifically, this present work demonstrates exposure dependent oxidative stress gradient effects on the growth kinetics of PDAC in the presence of radiotherapy, indicating radio-resistance. This is the first time oxidative stress dependent radio-resistance has been mapped in a 3D porous PDAC model.

Materials and Methods:

3D Polymeric Scaffolds were fabricated by the Thermally Induced Phase Separation method^[11]. The human PDAC cell line PANC-1 was seeded and cultured in hypoxic levels of 5%, 1%, 0.5% and 0.1%. Radiation exposure was performed by the Gulmay machine at the Royal Surrey County Hospital at delivering 2 Gy, 6 Gy and 8 Gy. Scanning electron microscopy & Confocal Laser Scanning Microscopy (CLSM) were used to evaluate cell viability and cellular distribution within 3D PU scaffolds. Immunofluorescence staining and CLSM enabled the determination of the cellular organisation, the detection of caspase, confirming apoptosis and the distribution HIF-1-alpha, the latter providing indication of the oxidative stress spatial distribution and its relation to treatment resistance.

Results and Discussion:

The 3D PU scaffold sustained prolonged PANC-1 cell growth for a period of 8 weeks. Identification of cellular masses and complex architecture were analysed with scanning electron microscopy revealing in vivo PDAC properties and structural integrity. CLSM revealed the spatial distribution of cell viability. An exposure dependent increase of hypoxic regions was identifiable. Hypoxia levels similar to PDAC tissue were observed via HIF-1-alpha staining. Further analysis revealed that cellular growth kinetics were affected by oxidative stress gradients. A positive correlation was drawn between oxidative stress and the presence of radio-resistance. We identify this as a novel method to map oxidative stress dependent radio-resistance in a 3D porous PDAC model.

Significance and Impact:

The lack of increase in the 5 year relative survival rate for this disease reflects the struggle of early patient diagnosis and treatment developments for this aggressive cancer. As modern image guided technologies evolve, the need for a realistic model of PDAC to replicate treatment options in vitro is essential. This novel hypoxic 3D PU scaffold more readily replicates the complex PDAC TME giving an insight into cell architecture and behaviour. Our findings present in vivo PDAC properties demonstrating a tissue engineering system that is more relevant for clinical application than 2D models. Future work will continue to model the hypoxic PDAC TME to test chemotherapy and chemoradiotherapy, investigating treatment resistance and cross resistance profiling.

Acknowledgments:

This work was supported by the Department of Process and Chemical Engineering of the University of Surrey, the National Physical Laboratory, the EPSRC and the Royal Society. P.G. is grateful for a Commonwealth Rutherford Fellowship.

References:

[1] American Cancer Society. (2019). Cancer Facts and Figures 2019.

[2] Siegel, R. L., et al. (2019). A Cancer Journal for Clinicians.69 (1), 7-34.

[3] Cascinu, S. et al., (2010). Ann. Oncol,21, 55–58.

[4] Li, D., et al. (2004). Lancet,363, 10049–10057.

[5] O’Reilly, D., et al.(2018). Pancreatology, 1–9.

[6] Rahib, L., et al. (2014). Cancer Research, 74(11), 2913–2921.

[7] Blakaj A., et al. (2018). Journal of Gastrointestinal Oncology, 9(6), 1027–1036.

[8] Hammel, P., et al. (2016). JAMA, 315(17), 1844.

[9] Neoptolemos, J. P., et al. (2004). New England Journal of Medicine, 350(12), 1200–1210.

[10] Melstrom, L. G., et al. (2017). Journal of Surgical Oncology,116(1).

[11] Totti, S., et al. (2017). Drug Discovery Today, 22(4).

[12] Totti, S., et al. (2018). RSC Advances, 8(37).