Fibroblast-derived Gremlin1 localises to epithelial cells at the base of the intestinal crypt

// Louise R. Dutton 1 , * , Owen P. Hoare 2 , * , Amy M.B. McCorry 2 , * , Keara L. Redmond 2 , Noor Eisa Adam 1 , 3 , Shannon Canamara 1 , 4 , Victoria Bingham 2 , Paul B. Mullan 2 , Mark Lawler 2 , Philip D. Dunne 2 , ** and Derek P. Brazil 1 , ** 1 Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast, Belfast, Northern Ireland, UK 2 Centre for Cancer Research and Cell Biology, Queen’s University Belfast, Belfast, Northern Ireland, UK 3 Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai Healthcare City, United Arab Emirates 4 Indonesia International Institute for Life-Sciences, University of East Jakarta, Jakarta Timur, Indonesia * These authors contributed equally to this work ** Co-senior authors Correspondence to: Derek P. Brazil, email: d.brazil@qub.ac.uk Philip D. Dunne, email: p.dunne@qub.ac.uk Keywords: Gremlin1; colorectal cancer; fibroblasts; stroma; pathology Received: February 25, 2019     Accepted: June 19, 2019     Published: July 23, 2019 ABSTRACT Gremlin1 (GREM1) is a secreted glycoprotein member of the differential screening-selected gene in aberrant neuroblastoma (DAN) family of bone morphogenetic protein (BMP) antagonists, which binds to BMPs preventing their receptor engagement. Previous studies have identified that stage II colorectal cancer (CRC) patients with high levels of GREM1 gene expression in their tumour tissue have a poorer prognosis. Using a series of in silico and in situ methodologies, we demonstrate that GREM1 gene expression is significantly higher ( p < 0.0001) in CRC consensus molecular subtype 4 (CMS4), compared to the other CMS subtypes and correlates ( p < 0.0001) with levels of cancer-associated fibroblasts (CAFs) within the CRC tumour microenvironment (TME). Our optimised immunohistochemistry protocol identified endogenous GREM1 protein expression in both the muscularis mucosa and adjacent colonic crypt bases in mouse intestine, in contrast to RNA expression which was shown to localise specifically to the muscularis mucosa, as determined by in situ hybridisation. Importantly, we demonstrate that cells with high levels of GREM1 expression display low levels of phospho-Smad1/5, consistent with reduced BMP signalling. Taken together, these data highlight a novel paracrine signalling circuit, which involves uptake of mature GREM1 protein by colonic crypt cells following secretion from neighbouring fibroblasts in the TME.