PO-255 The role of solid stress in tumor-stromal interactions

Abstract

IntroductionSolid tumours are characterised by an abnormal microenvironment that drives tumour progression. Tumour abnormalities include matrix stiffening and solid stress accumulation in the tumour interior. Several studies have dealt with the effect of solid stress on cancer cells using different experimental setups including the application of a predefined mechanical compression on a cell monolayer that transmits intracellular stress. To date, results indicated that solid stress guides cancer cell migration, however there are no pertinent studies taking into account the effect of solid stress on other cellular components of the tumour microenvironment, such as fibroblasts, and whether is implicated in tumor-stromal interactions.Material and methodsIn order to investigate the effect of solid stress on fibroblasts we employed a transmembrane pressure device to simulate the compressive solid stress encountered in the tumour microenvironment. Briefly, fibroblasts were cultured overnight in the inner chamber of a 24 mm diameter transwell insert with 0.4 µm pores. An agarose cushion was placed on top of the cells and a 24 mm diameter piston of adjustable weight was placed on the top of the agarose gel applying a predefined compressive stress. Next, we set up a novel co-culture system consisting of compressed fibroblasts seeded in the upper chamber of the transwell insert and pancreatic cancer cells (CFPAC-1 or MIA PaCa-2) seeded in the lower chamber of the transwell insert. Cells were allowed to interact for 48 hours. At the end of the co-culture period, a scratch wound assay was performed on cancer cells to study their migratory ability in response to compressed fibroblasts. Total RNA and protein extracts were isolated in order to be subjected in Real Time PCR and Western Blotting.Results and discussionsFibroblasts were exposed to 4.0 mmHg of compressive stress for 48 hours and the expression of a-SMA, one of the most established markers for fibroblast activation was found to be upregulated as revealed by qPCR and Western Blotting. Moreover, we observed that two distinct pancreatic cancer cell lines exhibited higher migratory ability when co-cultured with compressed compared to uncompressed fibroblasts suggesting that fibroblasts-derived factors may act in a paracrine fashion to stimulate the migration of adjacent cancer cells.ConclusionOur results highlight the involvement of biophysical factors, such as solid stress in the activation of normal pancreatic fibroblasts and the migration of pancreatic cancer cells.