Organotypic Culture Model of Pancreatic Cancer Demonstrates that Stromal Cells Modulate E-Cadherin, β-Catenin, and Ezrin Expression in Tumor Cells

Abstract

Pancreatic cancer is characterized by an intense stromal reaction. Reproducible three-dimensional in vitro systems for exploring interactions of the stroma with pancreatic cancer cells have not previously been available, prompting us to develop such a model. Cancer cells were grown on collagen/Matrigel and embedded with or without stromal cells (hTERT-immortalized human PS-1 stellate cells or MRC-5 fibroblasts) for 7 days. Proliferation and apoptosis, as well as important cell-cell adhesion and cytoskeleton-regulating proteins, were studied. PS-1 cells were confirmed as stellate based on the expression of key cytoskeletal proteins and lipid vesicles. Capan-1, and to a lesser extent PaCa-3, cells differentiated into luminal structures, exhibiting a central apoptotic core with a proliferating peripheral rim and an apico-basal polarity. Presence of either stromal cell type translocated Ezrin from apical (when stromal cells were absent) to basal aspects of cancer cells, where it was associated with invasive activity. Interestingly, the presence of 'normal' (not tumor-derived) stromal cells induced total tumor cell number reduction (P < 0.005) associated with a significant decrease in Ecadherin expression (P < 0.005). Conversely, ␤-catenin expression was up-regulated (P < 0.01) in the presence of stromal cells with predominant cytoplasmic expression. Moreover , patient samples confirmed that these data recapitulated the clinical situation. In conclusion , pancreatic organotypic culture offers a reproducible, bio-mimetic, three-dimensional in vitro model that allows examination of the interactions between stromal elements and pancreatic cancer cells.

Impact by pancreatic stellate cells on epithelial-mesenchymal transition and pancreatic cancer cell invasion: Adding a third dimension in vitro

“…The organotypic model has previously displayed favourable results for visualisation of cancer cell invasion and interplay with the local microenvironment (Froeling et al, 2009, Linde et al, 2012. The desmoplastic stroma produced by pancreatic stellate cells has been shown to create a treatment barrier around the tumour, limiting bioavailability of drugs (Jaster and Emmrich, 2008, Erkan et al, 2012, Whatcott et al, 2015.…”

The Novel Oncolytic Adenoviral Mutant Ad5-3Δ-A20T Retargeted to αvβ6 Integrins Efficiently Eliminates Pancreatic Cancer Cells

“…We optimized the previously developed 3D coculture models of pancreatic stellate and cancer cell lines (27)(28)(29), for investigating viral infection and spread in this study. These models have more similarities with the tumor microenvironment in patients than traditional 2D monocultures.…”

“…(35,36). To explore efficacy of the Ad5A20 and Ad5A20-477dlTAYT mutants under conditions that are more similar to the tumor microenvironment than 2D monocultures, Panc04.03 or Suit-2 cells were cocultured with transformed nontumorigenic pancreatic stellate cells (PS1) in 3D collagen Matrigels (27)(28)(29). Growth of both cancer cell lines was greatly increased in the presence of PS1 cells, forming ductlike structures when seeded and cultured within the gels (Supplementary Fig.…”

“…The hTERT-immortalized human pancreatic stellate cells (PS1) were a kind gift from Prof. H. Kocher (BCI, QMUL, London, United Kingdom; ref. 27). The human embryonic kidney cells HEK293 and JH293 (Cell Services, Cancer Research UK) and the human lung carcinoma cells A549 (LGC Standards) were used for viral production.…”

“…Organotypic 3D coculture models The development of the 3-dimensional (3D) coculture models (organotypic cultures) for pancreatic adenocarcinoma cells and stromal cells has been previously described in detail elsewhere (27)(28)(29). Briefly, collagen type I was mixed with Matrigel at different ratios, typically 3:1, in Transwells (Corning; SigmaAldrich) and placed in 24-well plates.…”