Novel stromal cell signalling systems in oesophageal cancer

Myofibroblasts are recognised to play an important role in wound healing and the maintenance of tissue integrity. In addition, they are increasingly recognised to provide a supporting microenvironment for cancer cells. They secrete a variety of chemokines, cytokines, growth factors and proteases that collectively regulate cell proliferation, migration and invasion. Specific chemokines are known to recruit mesenchymal stromal cells (MSCs) to both tumours and normal tissue which may then give rise to myofibroblasts. Proteomic studies by Holmberg and Varro (unpublished observations) identified chemerin as an upregulated chemokine in conditioned media (CM) from oesophageal cancer associated myofibroblasts (CAMs) compared to adjacent tissue myofibroblasts (ATMs). Chemerin is potent chemo-attractant for immune and inflammatory cells. The objectives of this thesis were (a) to functionally characterise the oesophageal myofibroblasts,(b) validate the findings of previous proteomic studies and (c) determine the role of chemerin in MSC migration. Oesophageal CAMs from both squamous and adenocarcinoma tumours were shown to be more proliferative than their paired ATMs, or normal tissue myofibroblasts (NTMs). In addition, CAM conditioned media increased the proliferation and migration of two oesophageal cancer cell lines (OE21 and OE33) and stimulated MSC migration compared to ATM CM. The data suggest oesophageal CAMs promote an aggressive tumour microenvironment. Western blotting and ELISA confirmed increased chemerin secretion by squamous carcinoma CAMs. Chemerin and conditioned media from squamous carcinoma CAMs, stimulated MSC and OE21 cell migration; Chemerin neutralizing antibody reversed these effects and siRNA knockdown of chemerin in CAMs, or of its cognate receptor ChemR23 in MSCs, decreased migratory responses. Studies using pharmacological inhibitors or Western blot of cellular proteins indicated that chemerin stimulated MSCs via PKC, and p42/44, p38 MAP and JNK kinases. Macrophage Inhibitory Factor (MIF) was identified as a putative chemerin target in MSCs and validated by ELISA and Western blot of MSC media and cell extracts. MIF inhibited MSC migration in response to low or moderate concentrations of chemerin, indicating that it might restrain MSC migration in normal tissues but not in cancers where chemerin is elevated. Finally, confirmation of chemerin-chemR23 interactions was obtained using a chemR23 antagonist, CCX832. Chemerin induced MSC and OE21 cell migration was inhibited by CCX832. Moreover, transendothelial migration of MSCs in response to chemerin or CAM conditioned media was reversed by CCX832. Transendothelial migration was also shown to depend on chemerin-stimulated MMP-2 secretion. These findings indicate a molecular mechanism by which MSCs are recruited to tumours. Taken as a whole, this work indicates that myofibroblasts derived from oesophageal cancers differ from those in adjacent or normal tissue. The finding of increased chemerin in these cells is novel and may be relevant to MSC recruitment. Since it is possible to inhibit the effects of chemerin on MSCs using CCX832, there is the potential for a novel therapeutic approach to prevent cancer progression