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1.

Figure. From: Combined vaccination with HER-2 peptide followed by therapy with VEGF peptide mimics exerts effective anti-tumor and anti-angiogenic effects in vitro and in vivo.

Figure 8. Combination treatment decreases microvascular density in tumors. Evaluation of vessel density in tumor sections. (A) Vascular staining using anti-CD31 antibody. (B) Effects of combination treatment on the tumor vessel density after quantification with the Image J software. Data represents mean values from four different fields and error bars represents SD of the mean.

Kevin C. Foy, et al. Oncoimmunology. 2012 October 1;1(7):1048-1060.
2.

Figure. From: Combined vaccination with HER-2 peptide followed by therapy with VEGF peptide mimics exerts effective anti-tumor and anti-angiogenic effects in vitro and in vivo.

Figure 4. VEGF induces the expression of VE-cadherin mRNA and treatment with VEGF peptide mimics reverses this induction. (A,B) Total RNA was isolated from matrigel plugs after treatment with peptide inhibitors and used in RT-PCR to measure levels of VE-cadherin expression. GAPDH was measured as an endogenous control and the expression levels were similar in the different treatment groups (A). Addition of VEGF was able to induce VE-cadherin expression but treatment with VEGF peptide mimics inhibited this induction (B).

Kevin C. Foy, et al. Oncoimmunology. 2012 October 1;1(7):1048-1060.
3.

Figure. From: Combined vaccination with HER-2 peptide followed by therapy with VEGF peptide mimics exerts effective anti-tumor and anti-angiogenic effects in vitro and in vivo.

Figure 7. Combination treatment decreases the number of actively dividing cells in a transplantable mouse model of breast cancer. Quantification of the number of actively dividing cells in tumor sections using Ki-67 staining. (A) Tissue sections show the amount of positive cells. The stain is specific for cells that are actively dividing. (B) Quantification of the staining using Image J software. Data represent mean values from four different fields and error bars represent SD of the mean.

Kevin C. Foy, et al. Oncoimmunology. 2012 October 1;1(7):1048-1060.
4.

Figure. From: Combined vaccination with HER-2 peptide followed by therapy with VEGF peptide mimics exerts effective anti-tumor and anti-angiogenic effects in vitro and in vivo.

Figure 6. Effects of combination treatment on tumor size. At the end of treatment, mice were euthanized and tumors extracted and pictures taken using a Nikon camera. After three immunizations BALB/c mice were challenged with TUBO cells and treated with VEGF-P3 and VEGF-P4. Representative photos from different treatment groups at day 39 after the inoculation of cancer cells are reported. (A) untreated; (B) irrelevant; (C) MVF-HER-2; (D) MVF-HER-2+IRRELEVANT; (E) MVF-HER2+VEGF-P3(CYC); (F) MVF-HER2+RI-VEGF-P4(CYC).

Kevin C. Foy, et al. Oncoimmunology. 2012 October 1;1(7):1048-1060.
5.

Figure. From: Combined vaccination with HER-2 peptide followed by therapy with VEGF peptide mimics exerts effective anti-tumor and anti-angiogenic effects in vitro and in vivo.

Figure 3. Inhibition of microvascular-like outgrowth in the aortic ring angiogenesis assay by VEGF peptide mimics. Mouse aortic rings were incubated for 10 d with 100 µg/mL peptides or controls as in Figure 2A. Note the robust outgrowth in controls and the strong inhibition by the peptides. In IV, the insert is a higher magnification showing the limited amount of residual sprouting. Photos were taking using phase contrast microscopy with original magnification at 20X. In (B), outgrowth estimation was done using the brightfield microscope. + indicates presence of growth and table shows the number of rings per treatment group. I-IV are same as in (A).

Kevin C. Foy, et al. Oncoimmunology. 2012 October 1;1(7):1048-1060.
6.

Figure. From: Combined vaccination with HER-2 peptide followed by therapy with VEGF peptide mimics exerts effective anti-tumor and anti-angiogenic effects in vitro and in vivo.

Figure 1. Anti-proliferative effects of combination treatment with anti-HER-2 (266–296) and anti-VEGF-P3 antibodies. (A-C) Inhibition of proliferation with individual anti-HER-2 and anti-VEGF- antibodies in two different cell-lines. BT474 (A) and MDA-468 (B) cells were incubated with HER-2 peptide antibodies, VEGF peptide antibodies, Trastuzumab and unspecific rabbit IgG. Bioconversion of MTT was used to estimate the number of active tumor cells remaining after 3 d. Peptides were added at four different concentrations. The proliferation inhibition rate was calculated using the formula (OD normal Untreated - OD peptides or antibodies)/OD normal untreated x 100. Error bars represent SD. Inhibition of proliferation of combination treatment with anti-HER-2 and anti-VEGF antipeptide antibodies using B-T474 cell lines (C). BT-474 cells were treated in the same manner as above but with HER-2 peptide abs, VEGF peptide abs or combination of both. Trastuzumab and rabbit IgG were used as positive and negative controls. Rate of inhibition was calculated using the same formula above and all results represents the average of three different experiments. Error bars represent SD of the mean.

Kevin C. Foy, et al. Oncoimmunology. 2012 October 1;1(7):1048-1060.
7.

Figure. From: Combined vaccination with HER-2 peptide followed by therapy with VEGF peptide mimics exerts effective anti-tumor and anti-angiogenic effects in vitro and in vivo.

Figure 2. Effects of combination treatment on HER-2 phosphorylation and Antibody dependent cellular cytotoxicity. (A) Inhibition of HER-2 phosphorylation by single and combination treatment with anti-HER-2 and anti-VEGF peptide antibodies. BT-474 cells were incubated with 100 µg/mL of anti-HER-2 266–296 and anti-VEGF-P3 antibodies or combination of both before being exposed to heregulin (HRG, an HER-3 activating ligand) for 10 min and lysed. Phosphorylated HER-2/neu was determined by indirect ELISA and percent inhibition was calculated as in (Fig. 1) above. AG825 (Calbiochem), a potent HER-2 phosphorylation inhibitor, was used as a positive control. (B) Anti-peptide antibodies raised in rabbits are capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC). The target cell line BT474 was coated with 50 μg of purified anti-peptide antibodies from rabbits, unspecific rabbit IgG, unspecific mouse IgG or trastuzumab and then cultured in the presence of human peripheral blood mononuclear effector cells to give an effector:target ratio of 100:1, 20:1, and 4:1 in triplicates. Error bars in panels (A) and (B) represent SD of the mean. Results represent average data from three different experiments with each treatment performed in triplicate.

Kevin C. Foy, et al. Oncoimmunology. 2012 October 1;1(7):1048-1060.
8.

Figure. From: Combined vaccination with HER-2 peptide followed by therapy with VEGF peptide mimics exerts effective anti-tumor and anti-angiogenic effects in vitro and in vivo.

Figure 5. Effects of HER-2 Immunization and VEGF peptide treatment in a transplantable tumor model. (A) Immunization scheme for BALB/c mice. Mice were immunized subcutaneously with 100 μg of MVF-HER-2 three times at three weeks intervals. Two weeks after the third immunization, mice were challenged with TUBO cells and treated weekly with VEGF peptide mimics and irrelevant peptide for 6 weeks. (B) Effects of combination treatment on tumor growth. Wild type BALB/c mice (n = 5), at the age of 5–7 weeks were immunized subcutaneously three times at three weeks intervals with 100 μg of MVF-HER-2 emulsified in ISA720. After immunization, mice were challenged with TUBO cells and treated intravenously with VEGF peptide mimics or an irrelevant peptide. Tumor measurements were performed twice a week using calipers. The data are presented as the average tumor volume per group and are reported as mm3. Results show a statistical significant difference between the group immunized with MVF-HER-2 vs. the group treated with the VEGF peptide mimics (**p < 0.001). There was a significant difference between immunization plus irrelevant peptide vs. immunization plus treatment with VEGF peptide mimics (*p < 0.001). (C) Effects of immunization and treatment on tumor-free survival rates. Results show that immunization with MVF-HER-2 and treatment with VEGF-P4 produced the best results since 40% of the mice (2 out of 5) remained tumor-free at the end of the experiment. There was also a greater delay in onset of tumor development in the case of VEGF-P3 peptide as compared with the MVF-HER-2 immunization alone. (D) Effects of peptide treatment on % tumor weight per body mass. After treatment, tumors were removed and weighed and the results show a significant difference between treated and untreated groups. p values are reported.

Kevin C. Foy, et al. Oncoimmunology. 2012 October 1;1(7):1048-1060.

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