(188an) On the Evaluation of the Efficiency of the Chemotherapeutic Agent Gemcitabine on 3D Polymer Based Pancreatic Cancer Models of Various Extracellular Matrix Compositions

INTRODUCTION:Pancreatic ductal adenocarcinoma is an aggressive disease with an extremely low survival rate¹. This is partly due to the tumour heterogeneity and the high resistance of the disease to chemotherapy, which is currently the most commonly used treatment option for pancreatic cancer². This resistance is partly attributed to the very complex and dense tumour microenvironment (TME) of pancreatic cancer, i.e., dense fibrosis combined with low vascularity, which leads to an increased tolerance to chemotherapeutic agents³. Traditionally used screening systems for chemotherapy studies of pancreatic cancer are (i) animal models and (ii) 2D cultures. Animal models are very informative however, they are complex to generate and use, highly expensive and not reproducible. 2D in vitro systems are very easy to use, cheap and reproducible, however they lack structure and cannot provide realistic recapitulation of an actual tissue, consequently being very limited regarding their translational potential. Recent progress in tissue engineering and the development of three-dimensional (3D) culture systems has enabled a more realistic recapitulation of a 3D tissue/ tumour, including the niches and structure of the TME as well as the extracellular matrix protein (ECM) composition, consequently increasing the accuracy of in vitro models for this malignancy³. Robust optimisation and control of the ECM mimicry in particular, enables more realistic cell-cell and cell-matrix interactions, ECM secretion by the cells and cellular spatial distribution, consequently providing a better mimicry of the tumour evolution and response to treatment^4-7.

The aim of this work was to evaluate the efficiency of the chemotherapeutic drug gemcitabine, which is commonly used as a treatment option for pancreatic cancer⁷, for various ECM compositions, in our previously developed 3D polymer based pancreatic cancer model^4,5. More specifically, surface modification of the scaffolds with various ECM proteins enabled a more realistic TME-ECM mimicry, allowing a more accurate screening of the drug efficiency.

METHODS:Polyurethane based highly porous scaffolds were generated as previously described^4-6. Thereafter,scaffold surface modification took place with different ECM proteins, i.e. fibronectin, laminin, collagen-I, RGD (Arg-Gly-Asp). The concentration of each protein was 25 μg/ mL. PANC-1 pancreatic cancer cells were seeded in the various scaffolds and the culture was monitored for 42 days. After 29 days of culture, when profound differences in cellular spatial distribution and ECM secretion among the ECM coated and uncoated scaffolds were detected⁵, the scaffolds were treated with a range of 5-200 μM of gemcitabine (which was added in the growth medium) for 48 hours. The viability of the cells for all the conditions under study was monitored in situ with the MTS viability assay as well as through live/dead staining and confocal laser scanning microscopy (CLSM) imaging of multiple scaffold sections until the culture end point (42^nd day). Furthermore, in situ sectioning, fluorescent staining and imaging with CLSM enabled the cellular and environmental gradient (oxidative, nutrient stress) spatial determination and the de novo ECM production within the 3D models.

RESULTS:The pancreatic cancer cells retained high viability in the 3D polymeric models until the culture endpoint for all the different protein coatings, with the fibronectin coated model exhibiting the highest cell growth and de novo ECM production by the cells. Additionally, no starvation gradients (LC3 negative), but only hypoxic gradients (HIF-1a positive), the level of which affected the drug efficacy, were detected in both ECM coated and uncoated models. Furthermore, CLSM imaging of multiple scaffold sections revealed that areas of the ECM coated model which presented denser cell aggregates formation and higher amounts of collagen-I secretion than the uncoated, also exhibited differences in cellular viability after the gemcitabine administration.

DISCUSSION & CONCLUSIONS: In this study, we performed in vitro chemotherapy in 3D polymer based pancreatic cancer scaffolds of various ECM compositions. We observed that the ECM presence and composition influenced the cellular growth, spatial arrangement, the ECM de novo production by the cells and the gemcitabine efficiency. Furthermore, the response of the pancreatic cancer cells to gemcitabine was a function of the level of the oxygen gradients present in the 3D model. Similar trends are reported in some animal studies, pointing that this bioinspired 3D model has potential as a low-cost tool for treatment screening of pancreatic cancer.

REFERENCES

1. R. L. Siegel, K. D. Miller and A. Jemal, CA Cancer J Clin, 2018,68, 7-30.
2. H. S. Lee and S. W. Park, Gut and Liver, 2016, 10, 340-347.
3. C. J. Whatcott, R. G. Posner, D. D. Von Hoff and H. Han, in Pancreatic Cancer and Tumor Microenvironment, eds. P. J. Grippo and H. G. Munshi, Trivandrum (India), 2012.
4. S. Totti, S. I. Vernardis, L. Meira, P. A. Perez-Mancera, E. Costello, W. Greenhalf, D. Palmer, J. Neoptolemos, A. Mantalaris and E. G. Velliou, Drug Discov Today, 2017, 22, 690-701.
5. S. Totti, M. Allenby, S. B. D. Santos, A. Mantalaris and E. Velliou, Eur Cells Mater, 2017, 33, 0249.
6. S. Totti, S. B. D. Santos, M. Allenby, A. Mantalaris, M. Ajaz and E. Velliou, European Cells and Materials, 2016, 31 (Supp 1), 441.
7. E. G. Velliou, S. B. D. Santos, M. M. Papathanasiou, M. Fuentes-Gari, R. Misener, N. Panoskaltsis, E. N. Pistikopoulos and A. Mantalaris, Bioprocess Biosyst Eng, 2015, 38, 1589-1600.

ACKNOWLEDGEMENTS:This work was supported by the Chemical and Process Engineering Department of the University of Surrey as well as an Impact Acceleration Grant (IAA-KN9149C) of the University of Surrey, an IAA –EPSRC Grant (RN0281J) and the Royal Society. M.C.A. is grateful for the Imperial College Chemical Engineering Scholarship. M.C.A. S.B.D.S., A.M. acknowledge the support from ERC-BioBlood (no. 340719).