title: The secretome of myofibroblasts and its significance in gastric cancer
Creators: name: Holmberg, Christopher
Publisher: name: University of Liverpool Repository
Year: 2023

description: The microenvironment is important in regulating the behaviour of cancer cells. Myofibroblasts are a key stromal cell type with important roles in defining microenvironments in both health and disease by secreting proteins including growth factors, proteases andextracellular matrix (ECM) components. In the case of gastric cancer, the myofibroblast population is significantly expanded compared with the adjacent non-cancer tissue, and with healthy gastric tissue. It has been shown in other cancers that cancer-associated myofibroblasts (CAMs) can promote tumour development and progression via secreted proteins such as hepatocyte growth factor (HGF) and stromal cell-derived factor 1 (SDF-I). In the stomach it has been shown that changes in the signalling between myofibroblasts and the adjacent epithelial cells are important in mediating the effects of the gastric pathogen Helicobacter pylori. Proteomic studies showed that increased secretion of matrix metalloproteinase 7 (MMP7) by the epithelial cells during H pylori infection causes cleavage of insulinlike growth factor binding protein-5 (IGFBP-5) in the myofibroblast secretome, resulting in increased bioavailability of IGF-II. This thesis is the first comprehensive study of gastric myofibroblast secretomes. The aim of this thesis was to define the differences between gastric CAMs and their adjacent non-cancer tissue myofibroblast (ANM) counterparts. This study made use of the unique availability in the group of cultures of myofibroblasts derived from human gastric carcinoma samples and adjacent noncancer tissue. In vitro assays revealed that CAMs were more migratory than ANMs, both in response to IGF-II and when unstimulated. Comparison of CAM and ANM transcriptomes by gene expression array revealed significant differences, particularly in genes related to transforming growth factor beta (TGF~) signalling. Myofibroblast conditioned media was used to stimulate AGS cells, a gastric cancer cell line. This revealed that CAM conditioned media induced greater AGS migration and invasion than ANM conditioned media. Gene expression array analysis of the stimulated AGS cells showed that these phenotypic differences were accompanied by significant changes in gene expression. Again, many of the differences were in genes involved in TGFp signalling. These experiments suggested that the most important differences between CAMs and ANMs were in their secretomes. Quantitative proteomic analysis of the myofibroblast secretomes and proteomes using the isobaric tagging for relative and absolute quantitation (iTRAQ) system revealed that a large proportion of the secreted proteins were differentially abundant in CAM media compared with ANM media. Most of these proteins, including ECM components and protease inhibitors, were less abundant in the CAM media. Only a small number of proteins, including proteases, were more abundant in CAM media than in ANM media. These results suggested a higher level of proteolytic activity in the CAM secretome. Analysis of the secretome of one CAM and ANM pair by combined fractional diagonal chromatography (COFRADIC) revealed greater proteolytic processing of several proteins in CAM media compared with ANM media. These included proteins with roles in TGFp signalling, ECM components and proteases. One protein which was often identified in proteomic experiments as being less abundant in CAM media than ANM media was TGFp ig-h3. This is a TGFp-induced extracellular protein with a known role in cellular adhesion. It was shown by Western blotting that in several cases Pig-h3 was either less abundant or more degraded in CAM media than ANM media, With proteomic and Western blot data taken together there was evidence for either reduced abundance or increased degradation of ~ig-h3 in 9 out of 11 CAMs compared with their matching ANMs. In vitro assays revealed that ~ig-h3 inhibited AGS cell migration in response to myofibroblast conditioned media. It was also shown that ~ig-h3 could be cleaved by plasmin, and that inhibiting urokinase plasminogen activator (uPA), which can convert plasminogen into active plasmin, also inhibited AGS cell migration in response to myofibroblast conditioned media. Together these data show that there are significant differences between gastric CAMs and ANMs. Many of these differences involve genes and proteins with known roles in TGF~ signalling, and the key differences are in the secretome. These differences, including a higher level of proteolytic activity in CAM media, result in a more migratory response in AGS cells. This suggests that CAMs could be important in defining a microenvironment in cancer which might promote a more aggressive cancer phenotype.