Advantages in use of a cell line are as follows: proliferating cells in culture may share angiogenic antigens with tumor vascular endothelial cells [25], and a cell line is able to supply as many cells as necessary with ease. Here, we demonstrated that vaccination with a syngeneic endothelial cell line Tpit/E inhibited growth and metastasis of B16/F10 melanoma. We also obtained hybridomas secreting specific antibodies to Tpit/E cells from the vaccinated mouse to prove occurrence of the specific immune response to the syngeneic CP673451 mouse endothelial cells. Methods Cell

lines and culture B16/F10 melanoma and SP-2 myeloma cell lines were provided by Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer Tohoku University (Sendai, Japan), and cultured in RPMI-1640 (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum (FBS; Thermo Trace Ltd, Melbourne, Australia), at 37°C in an atmosphere of 95% air and 5% CO2. Tpit/E vascular endothelial cell line OICR-9429 order derived from pituitary gland of temperature sensitive T-antigen transgenic mouse was provided by RIKEN BRC Cell Bank (Tsukuba, Japan), and cultured in HAMF12/DMEM MDV3100 clinical trial (Invitrogen, Carlsbad, CA) with 10% horse serum

(Nichirei, Tokyo, Japan) and 2.5% FBS, at 33°C. Animal models C57BL/6J mice of six to eight weeks were purchased from Tokyo Laboratory Animals Science (Tokyo, Japan). For the subcutaneous tumor model, mice were inoculated with 1 × 105 melanoma cells suspended in 100 μl 50% Matrigel

(BD Biosciences, Bedford, MA)/phosphate buffered saline (PBS) on the back. For the lung metastasis model, 1 × 105 melanoma cells suspended in 100 μl saline were injected in the tail vein. All studies involving mice were approved by the institute’s Animal Study Committee. Vaccination with fixed cells Cultured cells in a sub-confluence condition were harvested and fixed in 0.025% glutaraldehyde/PBS for 20 min at room temperature (RT), followed by washing with PBS for three times as described in a previous paper [23]. Vaccination was performed by inoculating 5 × 106 cells subcutaneously once a week for 8 times before tumor challenge and additional Proteasome inhibitor 4 times afterward (Fig. 1). For control, PBS was injected subcutaneously. Figure 1 Experimental plan for vaccination and tumor challenge. Vaccination was performed once a week for 12 times. Tumor was challenged prior to ninth vaccination on the same day. Computed Tomography Scanning Mice were anesthetized by intrapenetorial injection of 1 mg ketamine hydrochloride and 6.7 μg medetomidine hydrochloride per mouse and subjected to computed tomography scanning using LaTheta LCT-100A in-vivo CT scanner for small animals (Aloka, Tokyo, Japan). For the subcutaneous tumor model, cross-sectional CT scans were taken at 1 mm intervals.