(119c) Chemo-Therapeutic Screening on NOVEL Scaffold Assisted Dynamic in Vitro multicellular Models of Pancreatic Cancer

Introduction: With a 5-year survival rate of only 9%, Pancreatic Ductal Adenocarcinoma (PDAC) is the 7^th leading cause of cancer related death worldwide¹. The aggressive nature and high mortality rate of PDAC are attributed to its late diagnosis, heterogeneity in the tumour and the tumour microenvironment and its resistance to currently available treatment methods². An in-depth study of PDAC’s resistance to current therapeutic methods requires the development of biomimetic, niche mimicking in vitro tumour models. Current research focuses on the development of 3D in vitro tumour models to replace 2D culture systems and animal models in order to tide over limitations associated with such systems. 3D in vitro models are considered to have better in vivo niche mimicking capabilities in comparison to 2D culture systems while mitigating the cost and reproducibility problems associated with animal models. Additionally, as in all tissues, the PDAC tumour microenvironment (TME) is heterogeneous in cellular nature consisting, additionally to cancer cells, different cell types , e.g., stellate cells and endothelial cells, all contributing to the tumour formation, metastasis as well as its response and resistance to treatment. Thus, recent studies have focused on generating multicellular pancreatic cancer models, which are primarily spheroid based^3,4. Spheroids are useful 3D models due to their ease of development, ability to allow for fast analysis and studies. However, it is difficult to maintain spheroid cultures for long time without requiring resuspension, the latter inevitably affecting the formed cell-cell and cell-ECM interactions. As an advancement of our previous works^5,6,7,8, we have recently developed a poly urethane (PU) based 3D hybrid multicellular model of pancreatic cancer model using pancreatic cancer cells, endothelial cells and pancreatic cancer cells, wherein we were able to show long term maintenance of the in vitro model ( 4 weeks), feasibility of extracellular matrix (ECM) mimicry through scaffold coating via passive absorption, formation of dense cellular masses, secretion of ECM proteins⁹. The current work focusses on screening our multicellular scaffold based model for the purposes of therapeutic assessment (chemotherapy). We also look at the effects of a zonal vs a single scaffold cellular and biochemical architecture on chemotherapy assessment in vitro.

METHODS: PU scaffolds were prepared using the 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 mimicry⁴. A zonal cellular and biochemical structure was achieved with (i) endothelial and stellate cells seeded on the outer side of the polymeric scaffold which were coated with collagen I and (ii) pancreatic cancer cells seeded in the inner scaffold coated which were coared with fibronectin. Further to the zonal architecture, a single scaffold based simplistic multicellular model was designed and compared to the zonal model⁸. More specifically, for the simplistic model, a cocktail of the three cell types ( cancer cells, stellate cells and endothelial cells) were added to a singel 5x5x5 mm^3 scaffold and cultured for the relevant time period. For the chemotherapy assessment, 50µM Gemcitabine was applied to both models after 4 weeks of culture, both in static and dynamic (perfusion bioreactor assisted) conditions, followed by 7 days post-treatment monitoring. Various in situ assays for monitoring the cell viability, spatial organisation, ECM production and q-PCR analysis were carried out at specific time points throughout the culture period.

RESULTS & CONCLUSION: We report here the effect of cellular and biochemical factors of our 3D models on the chemotherapy response of pancreatic cancer cells in vitro. Effects of therapeutic agents on cell viability, apoptosis and ECM secretion in our zonal and single scaffold were observed and compared. The importance of a dynamic culture system was also highlighted in this work. Furthermore, the spatial biochemical and cellular configurations of the model affect the response to chemotherapy. Our developed model is a low cost high throughput tool that can be used for personalized studies and treatment screening of pancreatic cancer.

ACKNOWLEDGMENT: The project was supported financially by the 3DBioNet (UKRI). E.V is grateful to the Royal Academy of Engineering for an Industrial Fellowship and to the Medical Research Council UK for a New Investigator Research Grant (MR/V028553/1), which also financially supports P.G.

REFERENCES:

1.Rawla et al. World J Oncol, 2019; 10(1):10-27.

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

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

4. Di Maggio, et al. Pancreatology. 2016; 16, 995–1004.

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

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

7.Gupta, et al. RSC Advances.2019; 9 (71): 41649-41663

8. Wishart , G. et al., Cancers. 2021 13(23):6080

9. Gupta, et al. Frontiers in Bioengineering & Biotechnology. 2020; 8 (290).