INTRODUCTION:With a 5-year survival rate of only 11%,Pancreatic Ductal Adenocarcinoma (PDAC) is the 7th leading cause of cancer related death worldwide. The aggressive nature and high mortality rate of PDAC is partly attributed to the tumour microenvironment(TME) and its resistance to currently available treatment methods1.The TME consists of various structural, cellular and protein components. An in-depth study of PDAC biology and its resistance to current therapeutic methods requires the development of biomimetic, niche mimicking in vitro tumour models. 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., all of which are important elements of the pancreatic cancer cellular tissue microenvironment(multicellular model).We were able to show long term maintenance of the in vitro model (> 2 months), feasibility of extracellular matrix (ECM) mimicry, formation of dense cellular masses, secretion of ECM proteins,formation of realistic hypoxic gradients as well as effect of hypoxia2,3,4.
The current work focusses on further advancement of our PU scaffold assisted multicellular model5 via (i) the further optimisation of the ECM composition for the different cell compartments of our model (cancer vs stromal) (ii) the incorporation of immune cells in our model (the role of which is crucial for the disease development).
METHODS:PU scaffolds were prepared as per previously published protocols.Absorption based surface modification of the scaffolds enabled coating with ECM proteins (laminin, collagen I and fibronectin) for enhancement of ECM mimicry. Various cells (cancer, stellate, endothelial and immune) were incorporated in the model as previously described.The effect of different ECM proteins coatings and their combinations for the cancer compartment and the stromal compartment were systematically assessed long term (4 weeks). In situ assays (microscopy) and ex-situ(PCR) assays for monitoring the cell viability (for all cell types), spatial organisation, ECM and biomarker production were carried out at specific time points throughout the culture period.
RESULTS:We report a systematic comparative study to assess the effect of various ECM protein coatings for pancreatic cancer cells and stromal cells on PU scaffolds. Our study shows that cancer cell and stromal cells show preference for different ECM proteins and therefore tailoring the ECM composition (including ECM combinations) is essential for optimal/physiological in vitro cell growth. Within the cancer compartment, different cell lines of pancreatic cancer showed differences in terms of cell growth, secreted ECM proteins and biomarker expression.
CONCLUSION:Our data show the importance of ECM proteins tailoring and combination for the growth, proliferation and cellular protein and biomarker secretion for cancer and stromal cells in vitro. Furthermore, our work highlights the importance of developing zonal/spatial cellular and matrix structures in 3D for the efficient mimicry of the pancreatic cancer tissue in vitro.
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3. Gupta, et al. RSC Advances.2019; 9 (71).
4. Wishart et al. Cancers 2021; 13 (23).
5. Gupta et al. Frontiers in Bioengineering and Biotechnology 2020; 8(290).