Innate Immune Cells Induce Hemorrhage in Tumors during Thrombocytopenia

Benoit Ho-Tin-Noé^*, Carla Carbo^*, Mélanie Demers^*, Stephen M. Cifuni^*, Tobias Goerge^* and Denisa D. Wagner^*

From the Immune Disease Institute,^* Boston, Massachusetts; the Program in Cellular and Molecular Medicine, Children’s Hospital Boston, Boston, Massachusetts; the Department of Pathology, Harvard Medical School, Boston, Massachusetts; and the Servei d’Hemoterapia-Hemostasia, Hospital Clinic, Universitat de Barcelona, Barcelona, Spain

Platelets are crucial regulators of tumor vascular homeostasisand continuously prevent tumor hemorrhage through secretionof their granules. However, the reason for tumor bleeding inthe absence of platelets remains unknown. Tumors are associatedwith inflammation, a cause of hemorrhage in thrombocytopenia.Here, we investigated the role of the inflamed tumor microenvironmentin the induction of tumor vessel injury in thrombocytopenicmice. Using s.c. injections of vascular endothelial growth factoror tumor necrosis factor- ${alpha}$ combined with depletion of neutrophils,we demonstrate that enhancing the opening of endothelial celljunctions was not sufficient to cause bleeding in the absenceof platelets; instead, induction of tissue hemorrhage in thrombocytopeniarequired recruitment of leukocytes. Immunohistology revealedthat thrombocytopenia-induced tumor hemorrhage occurs at sitesof macrophage and neutrophil accumulation. Mice deficient inβ₂ or β₃ integrins, which have decreased neutrophiland/or macrophage infiltration in their tumor stroma, were protectedfrom thrombocytopenia-induced tumor hemorrhage, indicating that,in the absence of platelets, stroma-infiltrating leukocytesinduced tumor vessel injury. This injury was independent ofreactive oxygen species generation and of complement activation,as suggested by the persistence of tumor hemorrhage in C3- andnicotinamide adenine dinucleotide phosphate oxidase-deficientthrombocytopenic mice. Our results show that platelets counteracttumor-associated inflammation and that the absence of this plateletfunction elicits vascular injuries by tumor-infiltrating innateimmune cells.