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P061. Identification of the mesocolic mesothelium as a novel source of fibroblasts - implications for Crohn's disease

S. Sahebally1, M. Kiernan1, C. Dunne1, P. Kiely2, C. Coffey1, 1University of Limerick, Graduate Entry Medical School, 4i Centre for Interventions in Infection, Inflammation and Immunity, Limerick, Ireland, 2University of Limerick, Life Sciences, Materials and Surface Science and Stoke's Institute, Limerick, Ireland

Background

Traditional research in Crohn's disease (CD) has focused on the gastrointestinal lumen as the primary site of pathology. Mesenteric events, even though pathognomonic for CD, have been understudied. Accumulating evidence in non-intestinal fibrotic disorders points to mesothelial-to-mesenchymal transition as a composite source of fibroblasts (FBs). The aims of this study were to harvest mesocolic mesothelium (MM) from CD and oncological resection specimens, and determine if this process could lead to FB-generation ex-vivo.

Methods

Following ethical approval and informed consent, a technique was developed enabling the harvest of MM from both benign (n = 8) and malignant (n = 15) colorectal resections. From these, a single mesothelial cell suspension was prepared and cultured ex-vivo. The cellular composition of the suspension was characterised via immunofluorescence and confocal microscopy with markers targeting collagen-1 (C), vimentin (V), cytokeratin (CK), phalloidin (P), Golgi apparatus (GM-130) and CD45. Following a period in culture this was repeated. Adhesive and proliferatory cellular characteristics were established using a Real Time Cell Analyzer (RTCA, xCELLigence) machine. Data was analysed using SPSSv19 (Chicago, IL).

Results

23 patients [16 males/7 females, mean age 63 (±18.4) years] were recruited. MM was harvested from the ascending (7, 30%) mesocolon, sigmoid (9, 39%) mesocolon or ileal (7, 30%) mesentery. Initial staining demonstrated mesothelial cells (CK+/CD45−). Spindle-shaped cells morphologically resembling FBs appeared in all samples 10.5 (±13.3) days post plating. Dual staining with C and V confirmed these were mainly FBs whilst dual staining with CD45 and C identified a fibrocyte subpopulation. Additional staining with P and GM-130 further corroborated those observations. Mesenteric FBs derived from malignant resections adhered and proliferated faster than those derived from CD (P < 0.0001). FBs were also transfected with a plasmid encoding the SV40 T antigen and hence immortalised. Of note the proliferation rates of malignant FBs, closely resembled that of transfected FBs. Transfected FBs both adhered and proliferated quicker than non-transformed FBs from the same patients (P < 0.0001).

Conclusion

When expanded ex-vivo, the MM loses mesothelioid markers and sequentially acquires fibroblastoid and fibrocytic surface markers. This mesothelial plasticity may represent a novel FB-generating mechanism in CD-associated fibrosis. Mesenteric FBs in CD differ significantly in properties compared to those from colorectal cancer.