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The blood-clotting protein fibrin(ogen) plays a critical role in host defense against invading pathogens, particularly against peritoneal infection by the Gram-positive microbe Staphylococcus aureus. Here, we tested the hypothesis that direct binding between fibrin(ogen) and S. aureus is a component of the primary host antimicrobial response mechanism and prevention of secondary microbe dissemination from the peritoneal cavity. To establish a model system, we showed that fibrinogen isolated from FibγΔ5 mice, which express a mutant form lacking the final 5 amino acids of the fibrinogen γ chain (termed fibrinogenγΔ5), did not support S. aureus adherence when immobilized and clumping when in suspension. In contrast, purified wildtype fibrinogen supported robust adhesion and clumping that was largely dependent on S. aureus expression of the receptor clumping factor A (ClfA). Following peritoneal infection with S. aureus USA300, FibγΔ5 mice displayed worse survival compared to WT mice coupled to reduced bacterial killing within the peritoneal cavity and increased dissemination of the microbes into circulation and distant organs. The failure of acute bacterial killing, but not enhanced dissemination, was partially recapitulated by mice infected with S. aureus USA300 lacking ClfA. Fibrin polymer formation and coagulation transglutaminase Factor XIII each contributed to killing of the microbes within the peritoneal cavity, but only elimination of polymer formation enhanced systemic dissemination. Host macrophage depletion or selective elimination of the fibrin(ogen) β2-integrin binding motif both compromised local bacterial killing and enhanced S. aureus systemic dissemination, suggesting fibrin polymer formation in and of itself was not sufficient to retain S. aureus within the peritoneal cavity. Collectively, these findings suggest that following peritoneal infection, the binding of S. aureus to stabilized fibrin matrices promotes a local, macrophage-mediated antimicrobial response essential for prevention of microbe dissemination and downstream host mortality.

No abstract

Pancreatic ductal adenocarcinoma (PDAC) is a lethal solid tumor malignancy with a 5-year survival rate of 9%. In both patients and animal models of disease, PDAC is associated with robust coagulation system activity. Intriguingly, in addition to being a rich source of procoagulant factors, PDAC tumors highly express fibrinolytic system components. Supporting this concept, urokinase plasminogen activator (uPA) and uPA receptor (uPAR) expression positively correlates with reduced overall patient survival. Here, we tested the hypothesis that the expression and activity of plasminogen activation (PA) system components are functionally linked to PDAC tumor growth and disease progression. We generated C57Bl/6-derived KPC (i.e., KRasG12D, TRP53R172H) PDAC cell lines in which uPA and uPAR were knocked out using CRISPR-Cas9. We then analyzed orthotopic tumor growth and experimental metastasis in mice carrying null or functional mutations in uPA, uPAR, or plasminogen to evaluate the interplay of PA components derived from tumor cells and/or stromal cells in mediating PDAC progression. Although both KPC cell CRISPR variants retained procoagulant function, elimination of tumor cell uPA or uPAR yielded significantly smaller tumors when compared to Cas9 control tumor cells in wildtype mice. Similarly, the growth of WT KPC tumor cells in C57Bl/6 background uPA-KO or uPAR-KO mice also resulted in reduced tumor growth. To our surprise, the metastasis potential of WT KPC tumor cells in uPA-KO or uPAR-KO mice did not change when compared to wildtype mice. Regarding to the uPA/uPAR axis downstream effector plasminogen, the growth of WT KPC tumors in plasminogen-KO mice was also significantly reduced, but not to the same extent as when eliminating uPA or uPAR. In addition, eliminating plasminogen drastically reduced WT KPC tumor cells metastasis potential. In conclusion, our data suggest a mechanism whereby uPA functions through uPAR in both the tumor cell and stromal cell compartments to promote PDAC progression through plasminogen-dependent and -independent mechanisms. Citation Format: Yi Yang, Sara R. Abrahams, Aditi Kothari, Harshi Matada, Keely Davey, Alisa S. Wolberg, Matthew J. Flick. A uPA/uPAR axis in both the tumor cell and stromal compartment drives PDAC disease progression [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-127.