Introduction: Intact intestinal wound healing is the key to successful surgical therapy for patients with colorectal cancer. In particular, anastomotic healing after tumor resection is critical. However, the rate of anastomotic leakage remains high, leading to a significant increase in postoperative and cancer-related morbidity and mortality. A better understanding of the biological processes involved in anastomotic healing is urgently needed. To date, the cellular mechanisms of scar formation and the origin of mesenchymal cells during anastomotic healing have not been elucidated.

Methods: In-depth histological analysis of human and mouse anastomoses at different postoperative time points was performed to reveal the histologic changes of different intestinal layers during the healing process. In vivo staining of the ECM of the serosal layer using fluorophore-coupled N-hydroxysuccinimide (NHS) esters was performed prior to colorectal anastomosis in a murine model to localize the newly formed ECM within the anastomotic scar. Immunofluorescence staining was used to characterize different cell types within the anastomosis.

Results: Histology of human and murine anastomoses showed that the serosal layer was the first to be completely closed during the healing process. Immunofluorescence co-staining of murine anastomoses from mice that had undergone in vivo serosal staining prior to anastomosis formation showed that most of the newly formed ECM within the anastomosis was indeed deposited on top of the serosal layer. A large proportion of cells within the anastomotic scar were positive for the mesothelial cell markers podoplanin (PDPN) and Wilms' tumor protein (WT1) and exhibited a spindle-shaped mesenchymal phenotype in both human and mouse samples.

Conclusions: Our data suggest that serosal scar formation is an important contributor to intestinal anastomotic healing. The large number of PDPN⁺WT1⁺ cells within the anastomotic scar suggests that mesothelial cells contribute to anastomotic healing by transforming into ECM-producing mesenchymal cells through mesothelial-to-mesenchymal transition. Murine lineage-tracing models as well as human in vitro mesothelial cell culture models are currently being established to further the contribution of mesothelial cells to ECM formation during anastomotic healing.

Zoé Clees

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