(460b) Cell Specific Tailoring of the Extracellular Matrix Protein Composition in Advanced Multicellular Pancreatic Cancer Models

Pancreatic Ductal Adenocarcinoma (PDAC) is the 7th leading cause of cancer related deaths worldwide. Furthermore, the survival rate of PDAC is very low, i.e., the 5-year survival rate is only 11% and has barely improved over the last decades^1,2. This is partly attributed to the PDAC’s complex tumour microenvironment (TME). The TME is a cocktail of biomechanical, cellular and biochemical factors which contribute to the progression of the disease and its response (and resistance) to current therapeutic methods. Therefore, to perform advanced and more accurate studies of the disease and its’ treatment resistance mechanisms it is vital to develop robust, in vitro tumour models that can capture various features of the PDAC TME. We have previously developed a poly urethane (PU) based 3D pancreatic cancer model using (i) pancreatic cancer cells (monocellular model) and (ii) pancreatic cancer cells, stellate cells and endothelial cells, i.e., cells abundantly found in PDAC’s in vivo TME (multicellular model).We have shown long term physiological maintenance, feasibility of extracellular matrix (ECM) mimicry, mimicry of PDAC fibrosis/desmoplasia, and we have mapped effect of hypoxia^3-6.

The current work focusses on further development of our scaffold assisted multicellular model via screening and systematically comparing the effect of different ECM proteins on the long-term cell evolution in the two compartments of our model (cancer vs stromal)

METHODS:

PU scaffolds were prepared as per previously published protocols^3-6. Absorption based surface modification of the scaffolds enabled coating with ECM proteins, i.e., laminin, collagen I and/or fibronectin followed by incorporation of different cell types (cancer cells, activated stellate cells and endothelial cells) within the equivalent stromal and cancer compartments. The effect of ECM coatings and their combinations for both model compartments were systematically assessed (>4 weeks). Different cancer cell lines, i.e., PANC-1, BxPC-3 and A-PC-1, were used to validate the robustness of our study and to screen the entire range of the disease (poorly differentiated – well differentiated pancreatic cancer). Imaging of cellular proliferation/spatial organisation and ECM secretion was carried out along with q-PCR assessment of various biomarkers, e.g. EMT or metastatic markers.

RESULTS:

Our study shows that cancer cell and stromal cells show preference to different ECM proteins (including ECM combinations) for optimal growth. All cancer cell lines showed a preference for fibronectin and laminin coated scaffolds in comparison to collagen I, which led to less dense cell aggregates and lower viability 4 weeks after seeding. In contrast, the stromal compartment (containing activated stellate cells and endothelial cells) showed very high viability and dense cell cluster formation in the scaffolds 4 weeks after seeding for all ECM coatings studied. However, the protein coating of the scaffolds affected the stroma functionality in terms of ECM secretion. More specifically, fibronectin coating of the scaffolds resulted in lower collagen-I secretion by the stroma, in contrast to collagen coating which resulted in much higher collagen-I secretion by the stroma. Laminin coating promoted the secretion of both collagen-I and fibronectin by the stroma Furthermore, different ECM combinations affected the cell biomarker upregulation.

CONCLUSION:

Our data show the importance of ECM protein tailoring and combination for the growth, proliferation and functionality of both cancer and stromal cells in terms of ECM protein secretion and EMT and/or ECM biomarker upregulation in vitro. Additionally, our work highlights the importance of developing zonal/spatial cellular and matrix structures in 3D for the accurate mimicry of the tumour tissue architecture, therefore, contributing to the design of more robust PDAC mimicries in vitro.

ACKNOWLEDGMENTS:

E.V. is grateful to the Medical Research Council UK for a New Investigator Research Grant (MR/V028553/1), which also financially supports P.G.

REFERENCES:

[1] Khalaf et al., Clinical Gastroenterology and Hepatology, 2021;19(5)

[2] Aier et al., Cancer Epidemiology, 2019; 58

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

[4] Gupta, et al. RSC Advances.2019; 9 (71).

[5] Wishart et al. Cancers 2021; 13 (23).

[6] Gupta et al. Frontiers in Bioengineering and Biotechnology 2020; 8(290).