ngiogenesis, the growth of new blood vessels, is essential for tumor growth and metastasis. [1][2][3] In adults, angiogenesis is infrequent due to the balance between inhibitors and stimulators of angiogenesis. But under conditions of hypoxia, tumor cells turn-on the angiogenic switch by secretion of angiogenic stimulators that activate endothelial cells to proliferate and form new blood vessels. 4,5) Proliferation and expression of antigens critical to angiogenesis are two specific characteristics that distinguish angiogenic endothelial cells from quiescent endothelium of normal vasculature. Monoclonal antibodies and synthetic molecules targeting self-angiogenic antigens have been shown to inhibit tumor growth in animal models and were recently used in clinical trials.6-10) However, relatively high doses of these therapeutics have to be administered for long periods of time due to their short biological half-lives. Furthermore, antigens highly specific for angiogenic endothelium still remain to be isolated. These drawbacks should be overcome by active immunization with whole endothelial cells. Recently, active immunization with xenogeneic, but not autologous, endothelial cells was shown to inhibit the growth of experimental tumors in mice.11) However, from the viewpoint of clinical applicability of endothelial vaccines, the use of autologous endothelial cells would be preferable, since xenogeneic immunization may cause a species-specific immune reaction, with undesirable consequences. In the present study, therefore, we aimed to test the usefulness of autologous endothelial vaccine in the treatment of metastatic lesions that are highly dependent on tumor angiogenesis. For this purpose, we immunized BALB/c mice with a vaccine of glutaraldehyde-fixed murine hepatic sinusoidal endothelial cells (HSEs) in a lung metastasis model of colon cancer. We demonstrated that vaccination with autologous HSEs significantly inhibited the development of metastasis through an autoimmune response that was mediated by antibodies and cytotoxic T lymphocytes specifically reactive with endothelial, but not tumor cells. In addition, a vaccine of xenogeneic human umbilical vein endothelial cells (HUVECs) was tested in the same experimental setting, and the effects of both vaccines were compared, both in the animals and in in vitro immunological assays.
Materials and MethodsCell culture. Hepatic sinusoidal endothelial cells (HSEs), a cell line isolated from BALB/c mouse, 12) were kindly provided by Dr. Tatsuro Irimura (Faculty of Pharmacology, the University of Tokyo). HSEs were grown in DMEM supplemented with 10% fetal calf serum (FCS) and 1% antibiotic/antimycotic (i.e., 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate, 250 ng/ml amphotericin B; Life Technologies, Grand Island, NY), in an atmosphere containing 5% CO 2 at 37°C. Human umbilical vein endothelial cells (HUVECs) were isolated from fresh umbilical cords (obtained with patients' informed consent, from the Department of Gynecology and Obstetrics, the University of Tokyo)...
