Neutrophils and complement are key sentinels of innate immunity and mediators of acute inflammation. Recent studies have suggested that inflammatory processes modulate thrombogenic pathways. To date, the potential cross-talk between innate immunity and thrombosis and the precise molecular pathway by which complement and neutrophils trigger the coagulation process have remained elusive. In this study, we demonstrate that antiphospholipid Ab-induced complement activation and downstream signaling via C5a receptors in neutrophils leads to the induction of tissue factor (TF), a key initiating component of the blood coagulation cascade. TF expression by neutrophils was associated with an enhanced procoagulant activity, as verified by a modified prothrombin time assay inhibited by anti-TF mAb. Inhibition studies using the complement inhibitor compstatin revealed that complement activation is triggered by antiphospholipid syndrome (APS) IgG and leads to the induction of a TF-dependent coagulant activity. Blockade studies using a selective C5a receptor antagonist and stimulation of neutrophils with recombinant human C5a demonstrated that C5a, and its receptor C5aR, mediate the expression of TF in neutrophils and thereby significantly enhance the procoagulant activity of neutrophils exposed to APS serum. These results identify a novel cross-talk between the complement and coagulation cascades that can potentially be exploited therapeutically in the treatment of APS and other complement-associated thrombotic diseases.

The complement system contributes to various immune and inflammatory diseases, including cancer. In this study we investigated the capacity of lung cancer cells to activate complement, and characterized the consequences of complement activation on tumor progression. We focused our study on the production and role of the anaphylatoxin C5a, a potent immune mediator generated after complement activation. We first measured the capacity of lung cancer cell lines to deposit C5 and release C5a. C5 deposition, after incubation with normal human serum, was higher in lung cancer cell lines than in non-malignant bronchial epithelial cells. Interestingly, lung malignant cells produced complement C5a even in the absence of serum. We also found a significant increase of C5a in plasma from patients with non-small cell lung cancer, suggesting that the local production of C5a is followed by its systemic diffusion. The contribution of C5a to lung cancer growth in vivo was evaluated in the Lewis lung cancer model. Syngeneic tumors of 3LL cells grew slower in mice treated with an antagonist of the C5a receptor. C5a did not modify 3LL cell proliferation in vitro but induced endothelial cell chemotaxis and blood-vessels formation. C5a also contributed to the immunosuppressive microenvironment required for tumor growth. In particular, blockade of C5a receptor significantly reduced myeloid-derived suppressor cells and immunomodulators ARG1, CTLA-4, IL6, IL10, LAG3 and PDL1 (B7H1). In conclusion, lung cancer cells have the capacity to generate C5a, a molecule that creates a favorable tumor microenvironment for lung cancer progression.

Purpose PARP inhibition (PARPi) has modest clinical activity in recurrent BRCA mutant (BRCAMUT) high-grade serous ovarian cancers (HGSOC). We hypothesized that PARPi increases dependence on ATR/CHK1 such that combination PARPi with ATR/CHK1 blockade results in increased cell death and tumor regression. Experimental Design Effects of PARPi (olaparib), CHK1 inhibition (CHK1i;MK8776) or ATR inhibition (ATRi;AZD6738) alone or in combination on survival, colony formation, cell-cycle, genome instability and apoptosis were evaluated in BRCA1/2MUT HGSOC cells. Tumor growth in vivo was evaluated using a BRCA2MUT patient-derived-xenograft (PDX) model. Results PARPi monotherapy resulted in a decrease in BRCAMUT cell survival, colony formation and suppressed but did not eliminate tumor growth at the maximum-tolerated dose in a BRCAMUT PDX. PARPi treatment increased pATR and pCHK1 indicating activation of the ATR-CHK1 fork protection pathway is relied upon for genome stability under PARPi. Indeed, combination of ATRi or CHK1i with PARPi synergistically decreased survival and colony formation compared to single agent treatments in BRCAMUT cells. Notably, PARPi led to G2 phase accumulation, and the addition of ATRi or CHK1i released cells from G2 causing premature mitotic entry with increased chromosomal aberrations and apoptosis. Moreover, the combinations of PARPi with ATRi or CHK1i were synergistic in causing tumor suppression in a BRCAMUT PDX with the PARPi-ATRi combination inducing tumor regression and in most cases, complete remission. Conclusions PARPi causes increased reliance on ATR/CHK1 for genome stability and combination PARPi with ATR/CHK1i is more effective than PARPi alone in reducing tumor burden in BRCAMUT models.

Oral vancomycin treatment enhances the direct and abscopal antitumor effects of hypofractionated RT in preclinical melanoma and lung/cervical tumor models. Given the role of the gut microbiota in modulating immune cells that are also known to be involved in the response to RT, we examined whether the microbiota-regulated systemic immune response contributes to the RT-mediated anticancer immune response. The effects of oral vancomycin treatment are localized and impact the gut microbiota directly without any known systemic effects (21-23). Vancomycin (mostly targeting gram-positive bacteria) or a neomycin/metronidazole (Neo/Met) regimen (mostly targeting gram-negative bacteria

Beyond its role in immunity, complement mediates a wide range of functions in the context of morphogenetic or tissue remodeling processes. Angiogenesis is crucial during tissue remodeling in multiple pathologies; however, the knowledge about the regulation of neovascularization by the complement components is scarce. Here we studied the involvement of complement in pathological angiogenesis. Strikingly, we found that mice deficient in the central complement component C3 displayed increased neovascularization in the model of retinopathy of prematurity (ROP) and in the in vivo Matrigel plug assay. In addition, antibody-mediated blockade of C5, treatment with C5aR antagonist, or C5aR deficiency in mice resulted in enhanced pathological retina angiogenesis. While complement did not directly affect angiogenesis-related endothelial cell functions, we found that macrophages mediated the antiangiogenic activity of complement. In particular, C5a-stimulated macrophages were polarized toward an angiogenesis-inhibitory phenotype, including the up-regulated secretion of the antiangiogenic soluble vascular endothelial growth factor receptor-1. Consistently, macrophage depletion in vivo reversed the increased neovascularization associated with C3- or C5aR deficiency. Taken together, complement and in particular the C5a-C5aR axes are potent inhibitors of angiogenesis.