Involvement of dysfunctional lymphatic vessels and CD11b+ macrophages in ascites formation for monoclonal antibody production

Abstract

Ascites formation arises from imbalance between production and drainage of peritoneal fluid, but little is known how peritoneal lymphatic vessels relate to form ascites during the intraperitoneal production of monoclonal antibody by implantation of the myeloma cells in mouse. Here we identify and characterize peritoneal lymphatic vessels in the intraperitoneal myeloma-bearing mice, particularly at the diaphragm, which is the main organ for draining peritoneal fluid. Myeloma cell line FO and pristane (FO+P) were implanted into peritoneal cavity of 8~10 weeks of male BALB/c mice. Profound lymphangiogenesis and lymphatic remodeling occurred in the pleural side of diaphragm, whereas massive and disseminated carcinomatosis was observed in the peritoneal side of diaphragm in the mice implanted with FO+P. The observed lymphangiogenesis was mainly resulted from proliferation of lymphatic endothelial cells (LECs), but not by extension and hypertrophy of LECs. Diaphragmatic lymphatic vessel drainage assay using India ink revealed that lymphatic vessels both on the muscles and central tendon regions were surprisingly non-conductive to peritoneal fluid. Histological assay revealed that massively attached myeloma carcinomatosis and pristane-induced inflammation and fibrosis were main factors to inhibit drainage of peritoneal fluid. Both ELISA assays and the depletion experiment using Clodronate liposome indicated that CD11b+ macrophages play an important role in the formation of the inflammatory-tumor mass and the ascites via inflammatory cytokines. Moreover, the implantation of FO+P markedly induced expression and secretion of VEGF-A from host and implanted myeloma cells. The increased VEGF-A was other prime molecule responsible for the ascites formation through peritoneal vascular leakage. Accordingly, blockade of VEGF-A by treatment of VEGF-Trap significantly suppressed the ascites formation induced by FO+P implantation. Altogether, non-conductive lymphangiogenesis and i...