(582b) Engineering a Novel Dynamic Multicellular Scaffold Based Model of Pancreatic Ductal Adenocarcinoma

Introduction

With a 5-year survival rate of only 9%, Pancreatic Ductal Adenocarcinoma (PDAC) is 7^thleading cause of cancer related death worldwide ¹. The aggressive nature and high mortality rate of PDAC are attributed to its late diagnosis, heterogeneity in the tumor and the tumor microenvironment and its’ resistance to currently available treatment methods². In depth study of PDAC biology and its resistance to treatment requires the development of biomimetic, niche mimickingin vitro tumor models. Current research focuses on the development of 3D in vitro tumor models to replace 2D culture systems and animal models in order to tide over limitations associated with such systems. Animal models are very informative, however they are complex to generate and use, highly expensive and not reproducible. At the same time, 2D systems are too simplistic, lack structure and cannot provide realistic recapitulation of an actual tissue, consequently being limited in their translational potential. 3D tumor models have been proven to have better in vivo niche mimicking capabilities in comparison to traditional 2D culture systems while mitigating the cost and reproducibility problems associated with animal models. Spheroids/organoid, hydrogels and scaffold-based systems are some of the most well-known 3D systems currently in use within the field of in vitro tumor models including PDAC. Spheroid/organoid systems are currently the most widely used 3D systems for tumor modeling²; however, they have several limitations including lack of long-term culture without requiring resuspension –the latter influences the formed TME, formation of necrotic core and lack of tunability in terms of mechanical properties and architecture. Scaffold systems can overcome such limitations and provide a better niche mimicking model. This is attributed to their tunable mechanical properties, ability to provide structural integrity along with better cell- cell and cell-ECM interactions as well as the feasibility of incorporating multiple cell types in specific spatial orientation³. Additionally, mechanically robust scaffolds can also be easily incorporated within perfusion bioreactors enabling dynamic culture and possible shear stress and vascularization levels mimicry. The latter is particularly important when modelling tumors as they are characterized by different levels of vascularization. Our lab has previously developed a poly urethane (PU) based 3D a pancreatic cancer model using pancreatic cancer cells and appropriate extracellular matrix (ECM) proteins (through scaffold surface modification) which are typically found in the PDAC TME ⁴. Our model has several advantages including long term (> 2 months) culture without requiring resuspension, in situsecretion of extracellular matrix (ECM) proteins, formation of dense cell masses and hypoxic gradients at levels like in vivo. However, the PDAC tumor microenvironment is heterogeneous in cellular nature consisting, additionally to cancer cells, of different cell types like, fibroblasts and endothelial cells, all contributing to the tumor formation, metastasis as well as its response and resistance to treatment.

The aim of our current work is to further advance our mono-culture model via the development of a PU scaffold assisted, multi cellular, robust 3D pancreatic tumor model using pancreatic cancer, stellate and microvascular endothelial cells. Furthermore, incorporation of dynamic flow in the system for vascularization mimicry is achieved with the use of a perfusion bioreactor. It should be stated that most studies on multicellular models for PDAC to date are conducted in spheroid systems and this is the first attempt to generate a mechanically robust multicellular PDAC model using polymeric scaffolds.

Methods

PU scaffolds were prepared using Thermal Induced Phase Separation (TIPS) method. Absorption based surface modification of the scaffolds enabled coating with ECM proteins (collagen and fibronectin) for enhancement of ECM mimicry [2]. Separate protein coatings for the different cells was also considered. Cells were seeded in the scaffolds at a seeding density of 0.5x10⁶ cells/ scaffold for mono cultures and 0.25x10⁶ cells/scaffold, per cell type for the multi-culture systems. Long term culture (4 weeks) was carried out within the scaffolds. A comparative study between static and dynamic 3D culture was performed using a perfusion bioreactor. Various in situ assays including cell viability analysis, Scanning Electron Microscopy for morphology and architectural study, Confocal Laser Scanning Microscopy to map hypoxic gradients, observe spatial orientation of cell distribution and study cell specific markers were carried out at specific time points throughout the culture.

Results & Discussion

We report here for the first time 3D PU scaffold-based co- culture systems involving pancreatic cancer, stellate and endothelial cells to engineer a robust biomimetic in vitro model for PDAC. Coating of various ECM proteins enhanced cell growth rate within the culture system. A composite tri-culture system was also developed on the novel scaffolds and showed characteristics comparable to the current gold standard of in vitro PDAC models. The feasibility of a dynamic culture using perfusion bioreactor was also established, paving the way for in vivo mimicry via vascular mimicry and study of therapeutic agents’ circulation.

Conclusion

Our data show, for the first time, the feasibility of PU scaffolds to support a multicellular tumour growth along with the possibility for a robust ECM mimicry. The feasibility of using our novel multicellular model of PDAC within a perfusion bioreactor was also demonstrated. The lack of development in the survival rate for PDAC brings to the forefront the gaps in our understanding of PDAC biology, PDAC TME, its metastasis and resistance to current treatment methods. The research community needs a robust in vitro model of PDAC TME which can optimise and accelerate such studies. With this work, we contribute in bridging the current gap on PDAC in vitro systems, with our robust, long term niche mimicking model that can be used for high throughput personalized studies, including treatment screening, for pancreatic cancer.

Acknowledgments

Financial supports were received from Chemical and Process Engineering Department, University of Surrey; Impact Acceleration Grant (IAA-KN9149C), University of Surrey; IAA–EPSRC Grant (RN0281J) and the Royal Society. P.G is supported by Commonwealth Rutherford Post-Doctoral Fellowship.

References

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2.H. S. Lee and S. W. Park, Gut and Liver, 2016;10:340-347.

3. Lazzari G. et al. Acta Biomaterialia. 2018;78:296-307

4. Totti, S. et al. Drug discovery today. 2017; 22(4): 690-701.

5. Totti, S. et al. RSC Advances. 2018; 8(37): 20928-20940.