Effects of anti-hTM4SF5 monoclonal antibody on mouse HCC cells.

<p><b>A.</b> The expression levels of mTM4SF5 mRNA in the indicated mouse HCC cell lines and normal mouse hepatocytes were analyzed by RT-PCR. <b>B.</b> Detection of mTM4SF5 in BNL-HCC cells and H2.35 cells by anti-hTM4SF5R2-3 peptide monoclonal antibody according to a FACS analysis. Normal mouse IgG was used as a control. <b>C.</b> Reactivity of anti-hTM4SF5R2-3 monoclonal antibody with the mTM4SF5R2-3 peptide. The hTM4SF5R2-3 peptide was immobilized on a plate, and competitive ELISA was performed using increasing amounts of soluble mTM4SF5R2-3 peptide. <b>D.</b> Effect of anti-hTM4SF5R2-3 peptide monoclonal antibody on the growth of BNL-HCC cells and H2.35 cells. Cell growth was measured by an MTT assay. <b>E.</b> Effect of anti-hTM4SF5R2-3 peptide monoclonal antibody on the proliferation of BNL-HCC cells and H2.35 cells. The DNA synthesis activity was monitored by BrdU incorporation assay. Each bar is expressed as the Mean ± SD of three experiments. **<i>P</i><0.01.</p

Involvement of TLR9 on prophylactic efficacy of a vaccine containing hTM4SF5R2-3 peptide and Lipoplex(O) complex.

<p>BALB/c TLR9−/− mice were immunized with a complex of hTM4SF5R2-3 peptide and Lipoplex(O) and the mice were implanted with the BNL-HCC cells (n = 8 per group). <b>A.</b> Effect of TLR9 on serologic response to BNL-HCC cell implantation after vaccination with epitope and Lipoplex(O). The sera were collected, and the amounts of mTM4SF5R2-3 peptide-specific total IgG were assayed using an ELISA kit. Each bar is expressed as the Mean ± SD of 8 mice. <b>B.</b> Tumor volumes were calculated as width²×length/2. The tumor volumes in TLR9−/− mice were compared with the tumor volumes in wild type BALB/c mice in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033121#pone-0033121-g005" target="_blank">Figure 5B</a>. <b>C.</b> Macroscopic appearance of HCC tumor tissues. <b>D.</b> Tumor growth was measured by tumor weight. <b>E.</b> Body weights were measured at the indicated time intervals. <b>F.</b> Histology of normal liver and tumor tissue derived from BNL-HCC cell-implanted mice was observed by staining with hematoxylin and eosin (left panel). An immunohistochemical analysis was performed with anti-hTM4SF5R2-3 monoclonal antibody (right panel). TM4SF5 positive area was expressed as brown color. Scale bars, 500 µm.</p

Prophylactic efficacy of a vaccine containing hTM4SF5R2-3 peptide and Lipoplex(O) complex in HCC implanted mouse.

<p>BALB/c mice were immunized with a complex of hTM4SF5R2-3 peptide and Lipoplex(O). The immunized mice were implanted with the BNL-HCC cells (n = 12 per group; n = 10 per PBS-treated control). <b>A.</b> Induction of a strong serologic response to BNL-HCC cell implantation by vaccination with epitope and Lipoplex(O). The sera were collected, and the amounts of mTM4SF5R2-3 peptide-specific total IgG were assayed using an ELISA kit. Each bar is expressed as the Mean ± SD of 10 or 12 mice. <b>B–D.</b> Tumor formation in mice implanted with BNL-HCC cells was inhibited by vaccination with the hTM4SF5R2-3 peptide and Lipoplex(O) complex 60 days after the implantation of BNL-HCC cells. Tumor volumes were calculated as (length×width²)/2 (<b>B</b>). Macroscopic appearance of HCC tumor tissues (<b>C</b>). Tumor growth was measured by tumor weight (<b>D</b>). <b>E.</b> Body weights were measured at the indicated time intervals. <b>F.</b> Histology of normal liver and tumor tissue derived from BNL-HCC cell-implanted mice was observed by staining with hematoxylin and eosin (H&E, left panel). An immunohistochemical analysis (IHC) was performed with anti-hTM4SF5R2-3 monoclonal antibody (right panel). TM4SF5 positive area was expressed as brown color. Scale bars, 500 µm. **<i>P</i><0.01.</p

Production of IgG by immunization with a complex of hTM4SF5R2-3 peptide and Lipoplex(O).

<p><b>A.</b> Optimal dose of peptide in the complex of hTM4SF5R2-3 peptide and Lipoplex(O). BALB/c mice (n = 3/group) were immunized with increasing amounts of hTM4SF5R2-3 peptide and Lipoplex(O) complex. <b>B.</b> Effect of the injection protocol. We immunized one group of mice with hTM4SF5R2-3 peptide after injection with Lipoplex(O) (Lipoplex(O)/TM4SF5R2-3) and another group with MB-ODN 4531(O) after injection with hTM4SF5R2-3 peptide and the DOPE∶CHEMS complex (DOPE∶CHEMS+TM4SF5/4531(O)) three times with a 10 day interval (n = 3/group). The sera were collected, and titers of the peptide-specific total IgG were assayed with an ELISA kit. These experiments were performed 3 times with similar results. **<i>P</i><0.01 (<i>vs</i> PBS control).</p

Contribution of Th1 differentiation on IgG production.

<p>STAT4 but not STAT6 is required for IgG production by a complex of peptide and Lipoplex(O). <b>A,B.</b> BALB/c mice, BALB/c STAT4−/− mice (<b>A</b>), and BALB/c STAT6−/− mice (<b>B</b>) (n = 3/group) were injected i.p. three times with a 10 day interval with the hTM4SF5R2-3 peptide and Lipoplex(O) complex. The sera were collected, and the amounts of the peptide-specific total IgG, IgG1, and IgG2a were assayed with an ELISA kit. These experiments were performed 3 times with similar results. <b>C.</b> BALB/c mice, BALB/c STAT4−/− mice, and BALB/c STAT6−/− mice (n = 5/group) were injected i.p with hTM4SF5R2-3 peptide and Lipoplex(O) complex, and sera from the mice were harvested at 24 h after injection. The levels of IL-12 in the serum were measured with an ELISA assay. Each bar is expressed as the Mean ± SD of three mice. *<i>P</i><0.05, **<i>P</i><0.01.</p

Therapeutic efficacy of a vaccine containing hTM4SF5R2-3 peptide and Lipoplex(O) complex in HCC implanted mouse.

<p><b>A.</b> Induction of a strong serologic response by vaccination with the hTM4SF5R2-3 peptide and Lipoplex(O) complex or indicated combinations after BNL-HCC cell implantation in mice (n = 12 per hTM4SF5R2-3 peptide+Lipoplex(O) group; n = 8 per PBS-treated control group, Lipoplex(O) group, and DOPE∶CHEMS+hTM4SF5R2-3 peptide group). The sera were collected 10 days after vaccination, and the amounts of mTM4SF5R2-3 peptide-specific total IgG were assayed using an ELISA kit. Each bar is expressed as the Mean ± SD of 8 or 12 mice. <b>B–D.</b> Inhibition of tumor formation in mice vaccinated with the hTM4SF5R2-3 peptide and Lipoplex(O) complex 60 days after the implantation of BNL-HCC cells. Tumor volumes were calculated as width²×length/2 (<b>B</b>). Macroscopic appearance of HCC tumor tissues (<b>C</b>). Tumor growth was measured by tumor weight of the surviving mice (<b>D</b>). <b>E.</b> Body weights were measured at the indicated time intervals. Each bar represents the Mean ± SD of 8 or 12 mice. *<i>P</i><0.05, **<i>P</i><0.01.</p

Survival rate of tumor-bearing mice.

<p>BALB/c mice were immunized with the hTM4SF5R2-3 peptide and Lipoplex(O) complex or indicated combinations after BNL-HCC cell implantation in mice (n = 12 per group; n = 10 per PBS-treated control). After the vaccination, the survival rate was recorded for 100 days.</p