Macrophage Factor Xa Signaling Promotes Cancer Immune Evasion
Authors: Graf Claudine , Wilgenbus Petra , Kitano Maki , Macher-Göppinger Stephan , Ruf Wolfram 
Published: October 2019

Abstract
Coagulation signaling through protease activated receptors (PARs) participates in inflammation and immunity. In cancer, tissue factor (TF) driven signaling via PAR2 promotes tumor progression, but effective pharmacological strategies to inhibit the PAR2 activating proteases for clinical anti-cancer benefit are currently unknown. To gain a better understanding of signaling by coagulation proteases, we generated PAR2 mouse strains with mutations that abolish canonical proteolysis by all proteases including FVIIa (PAR2 R38E) or create specific resistance to cleavage by the TF-FVIIa-Xa signaling complex (PAR2 G37I) that requires the endothelial cell protein C receptor (EPCR, Procr). As expected from delayed breast cancer development in PAR2-deficient polyoma middle T (PyMT) mice, cancer progression was impaired in completely cleavage-resistant PAR2 R38E relative to wild-type (WT) mice. In contrast, FXa-resistant PAR2 G37I mice displayed normal tumor initiation, but unexpectedly tumor growth was also markedly attenuated compared to WT mice. A similar reduction in transplanted syngeneic tumor growth in mutant PAR2 R38E and PAR2 G37I mice indicated that impaired FXa-PAR2 signaling in the tumor microenvironment (TME), but not by tumor cells, impaired tumor expansion in FXa-resistant PAR2 G37I mice.
Macrophages, but not the studied tumor models, expressed FVII and FX, along with other components of the TF pathway. Tumor-associated macrophage (TAM) phenotypes are determined by complex immune cell alterations in the TME. Exposure of macrophage to tumor cell supernatant resulted in upregulation of immune-suppressive mediators; this effect was significantly attenuated in PAR2 G37I relative to WT macrophages. Accordingly, TAM isolated from spontaneous PyMT breast cancer demonstrated reduced expression of immune modulatory chemokines, as well as immune-suppressive, pro-angiogenic and pro-metastatic mediators in PAR2 mutant relative to WT mice. Thus, PAR2 signaling directly regulates TAM immune-evasive properties. In addition, macrophage-specific deletion of FX in F10flfl-LysMcre mice prevented macrophage polarization in vitro. Consistently, macrophage FX deficiency was sufficient to diminish tumor growth without reducing plasma FX levels in control or tumor-bearing littermate mice. TAM phenotypic changes in macrophage FX-deficient mice recapitulated the anti-tumor effects seen in PAR2 mutant mice and similarly resulted in increased tumor infiltration with cytotoxic anti-tumor T cells, demonstrating that PAR2 activation by cell autonomously synthesized FXa impairs anti-tumor immunity.
We hypothesized that small molecule direct FXa inhibitors with peripheral tissue penetration could interfere with extravascular macrophage FXa signaling in the TME. Akin to FXa-PAR2 signaling-deficient macrophages, therapeutic concentrations of the direct FXa inhibitor rivaroxaban reduced macrophage expression of immune-suppressive mediators. Oral anticoagulation of PyMT mice achieving therapeutic plasma levels of rivaroxaban similarly altered TAM polarization in vivo and improved anti-tumor immunity specifically in macrophage FX-expressing mice. Thus, macrophage synthesized FXa is the primary target for oral FXa anticoagulants in reprogramming TAM phenotypes.
These data show that cell autonomous PAR2 signaling dependent on extrahepatic synthesis of coagulation FX by macrophages controls the immune-evasive phenotype of TAM. While targeting intravascular thrombosis with heparin, the current standard anticoagulant therapy of cancer patients, has limited clinical anti-cancer efficacy, reprogramming of macrophages by direct oral anticoagulants with favorable tissue distribution may enhance cancer immunotherapy and provide anti-inflammatory benefit in other diseases.

Disclosures
No relevant conflicts of interest to declare.