About 31 d after tumor cell transplantation, the synergistic indexes of tumor volume, MVD, cell apoptosis and proliferation in the combination therapy group were 1.71 1.15 1.11 and 1.04, respectively (Table ?(Table11). Open in a separate window Figure 4 The combination therapy synergistic inhibition of angiogenesis (A) and proliferation (B), and induction apoptosis (C) at d 31 after tumor cell injection. cFR-1 vaccine alone or with combination therapy, but not in non-immunized mice. In addition, the deposition of auto-antibodies on endothelial cells from mice immunized with cFR-1 was observed by immunofluorescent stain-ing, but not on endothelial cells from control groups. Synergistic indexes of tumor volume, MVD, cell apoptosis and proliferation in the combination therapy group were 1.71 1.15 1.11 and 1.04, respectively, 31 d after tumor cell injection. CONCLUSION: The combination of cFR-1-mediated anti-angiogenesis and low-dose gemcitabine synergistically enhances the anti-tumor activity without overt toxicity in mice. and applied widely in clinical anti-tumor therapy[20-22]. Moreover, studies have also confirmed that the combination of an anti-angiogenic biotherapy with a low-dose gemcitabine strategy can effectively suppress tumor angiogenesis without increased overt toxicity relative to either therapy alone. Thus, in this study, we primarily evaluated the anti-tumor activities of the recombinant cFR-1 protein vaccine in combination with low-dose gemcitabine in a mouse tumor model. MATERIALS AND METHODS Vaccine preparation The lyophilized recombinant proteins of cFR-1 and mouse FGFR-1 (mFR-1) were dissolved in NS and RMC-4550 mixed with an equal volume of aluminum hydroxide adjuvant at 4 mg/mL for 60 min before use in vaccination. Design of animal experiments The CT26 tumor model was established in BALB/c mice to evaluate whether the combination of cFR-1 vaccine and low-dose gemcitabine would improve the anti-tumor efficacy. Six to eight-week-old female mice were transplanted with 1 106 live tumor cells. After tumors had grown for 7 d, RMC-4550 the mice were randomly divided into the following four groups of 10 mice each. Group 1 mice, treated with a combination of cFR-1 vaccine and low-dose gemcitabine (C + G), received cFR-1 vaccine plus low-dose gemcitabine as follows: after d 0 (7 d after tumor cell injection), cFR-1 vaccine was injected subcutaneously (s.c.) once a week for 4 wk with a dose of 10 g per mouse. At d 7 (14 d after tumor cell injection), 20 mg/kg of gemcitabine was injected intraperitoneal (i.p.) at an interval of every 3 d for a total of 4 doses. Group 2 mice, treated with cFR-1 vaccine alone (cFR), received cFR-1 vaccine in a similar scheme as that in group 1, except that it lacked gemcitabine. Group 3 mice, treated with low-dose gemcitabine alone (G), received the same dose of gemcitabine as group 1, but they did not receive the cFR-1 vaccine. Group 4 mice, the untreated group (NS), received sterile NS s.c. as the scheme of vaccination or were treated i.p. as in group RMC-4550 1, respectively. Tumor growth was evaluated every 3 d and tumor volume was estimated using RMC-4550 the formula for an ellipsoid (0.5 length width height). At the end of the experiment, the tumor tissues, major organs RMC-4550 and blood samples of the mice were collected for subsequent histologic and immunologic investigations. All studies involving mice were approved by the Institute’s Animal Care and Use Committee. Western blot analysis Western blot analysis was performed as described pre-viously. Briefly, the recombinant proteins were separated by 12 % SDS-PAGE. Gels were transblotted with the Mini Polyacrylamide Gel System (Bio-Rad, USA) onto a polyvinylidene difluoride membrane. Membrane blots were blocked at 4C in 5% nonfat dry milk, washed and probed with mouse sera at a 1:500 dilution. Blots were then washed and incubated with goat anti-mouse IgG HRP-labeled secondary antibody and then stained with the Vectastain ABC kit (Vector, Burlingame, USA). Enzyme-linked immunospot (ELISPOT) assay The ELISPOT assay for the enumeration of antibody-producing B cells (APBCs) has been described. Briefly, PVDF-bottomed, 96-well filtration plates (Millipore, Bedford, USA) were coated with 30 g/mL of recombinant FGFR-1 protein. Mononuclear cells prepared from spleen were incubated on the plates at 37C for 4 h. IgG bound to the membrane was revealed as spots with alkaline phosphatase-conjugated anti-mouse IgG antibodies. Immunohistochemistry For microvessel density (MVD) and cell proliferation analyses, frozen sections were fixed in acetone, incubated Mouse Monoclonal to Rabbit IgG (kappa L chain) and stained with antibodies reactive to either CD31 or.