Treatment of Tumors with Vitamin E Suppresses Myeloid Derived Suppressor Cells and Enhances CD8+ T Cell-Mediated Antitumor Effects

<div><p>Vitamin E has been shown to have strong anticarcinogenic properties, including antioxidant characteristics, making it an ideal candidate for use in combination with immunotherapies that modify the tumor microenvironment. The tumor microenvironment contains immunosuppressive components, which can be diminished, and immunogenic components, which can be augmented by immunotherapies in order to generate a productive immune response. In the current study, we employ the α-tocopherol succinate isomer of vitamin E to reduce immunosuppression by myeloid derived suppressor cells (MDSCs) as well as adoptive transfer of antigen-specific CD8+ T cells to generate potent antitumor effects against the HPV16 E7-expressing TC-1 tumor model. We show that vitamin E alone induces necrosis of TC-1 cells and elicits antitumor effects in TC-1 tumor-bearing mice. We further demonstrate that vitamin E reverses the suppression of T cell activation by MDSCs and that this effect is mediated in part by a nitric oxide-dependent mechanism. Additionally, treatment with vitamin E reduces the percentage of MDSCs in tumor loci, and induces a higher percentage of T cells, following T cell adoptive transfer. Finally, we demonstrate that treatment with vitamin E followed by E7-specific T cell adoptive transfer experience elicits potent antitumor effects in tumor-bearing mice. Our data provide additional evidence that vitamin E has anticancer properties and that it has promise for use as an adjuvant in combination with a variety of cancer therapies.</p></div

Characterization of the T-cell accumulation in tumor-bearing mice treated with Vitamin E.

<p>1×10⁵ TC-1 cells were injected subcutaneously into wild type C57BL/6 mice. 10 days later, mice were treated as in (A) and one day after the last vitamin E treatment, luciferase-expressing E7-specific CD8+ T-cells were adoptively transferred using intravenous injection. (<b>A</b>) Schematic diagram of the treatment regimen. (<b>B</b>) Bar graph depicting the fluorescence intensity in tumor-bearing mice treated with vitamin E or DMSO (mean ± S.D.).</p

Characterization of myeloid-derived suppressor cells in tumor-bearing mice treated with vitamin E.

<p>1×10⁵ TC-1 cells were injected subcutaneously into wild type C57BL/6 mice. 10 days later, mice were treated using the regimen as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103562#pone-0103562-g001" target="_blank">Figure 1C</a>. Tumor tissue was collected from each group of mice 3 days after the last treatment and prepared for flow cytometry analysis to measure CD11b and Gr-1 positive cells. (<b>A</b>) Tumor cells were stained with FITC-Gr-1 antibody and PE-CD11b antibody. (<b>B</b>) Bar graph depicts % of CD11b and Gr-1 positive cells (mean ± SD). Data shown are from one representative experiment of three performed. (<b>C</b>) Tumor cells were stained with Mouse Regulatory T Cell Staining Kit (eBioscience, San Diego, CA) and bar graph depicts % of Foxp3 and CD25 positive cells in CD4 positive cells. (<b>D</b>) Tumor cells were stained with APC-labeled CD11b, FITC labeled F4/80, PE labeled CD206 antibody (eBioscience, San Diego, CA) and bar graph depicts ratio of M1 an M2 cells in CD11b and F4/80 positive cells.</p

Ovarian Cancer Gene Therapy Using HPV-16 Pseudovirion Carrying the HSV-tk Gene

<div><p>Ovarian cancer is the leading cause of death from all gynecological cancers and conventional therapies such as surgery, chemotherapy, and radiotherapy usually fail to control advanced stages of the disease. Thus, there is an urgent need for alternative and innovative therapeutic options. We reason that cancer gene therapy using a vector capable of specifically delivering an enzyme-encoding gene to ovarian cancer cells will allow the cancer cell to metabolize a harmless prodrug into a potent cytotoxin, which will lead to therapeutic effects. In the current study, we explore the use of a human papillomavirus (HPV) pseudovirion to deliver a herpes simplex virus thymidine kinase (HSV-tk) gene to ovarian tumor cells. We found that the HPV-16 pseudovirion was able to preferentially infect murine and human ovarian tumor cells when administered intraperitoneally. Furthermore, intraperitoneal injection of HPV-16 pseudovirions carrying the HSV-tk gene followed by treatment with ganciclovir led to significant therapeutic anti-tumor effects in murine ovarian cancer-bearing mice. Our data suggest that HPV pseudovirion may serve as a potential delivery vehicle for ovarian cancer gene therapy.</p> </div

HPV-16/luc psV preferentially infects ovarian tumors in <i>MISIIR-TAG</i> transgenic mouse.

<p>C57BL/6 mice and <i>MISIIR-TAG</i> transgenic mice were injected with 20 µg of HPV-16/luc psV by intraperitoneal injection 10 weeks after birth. Luminescence images were taken 1 day after HPV-16/luv psV injection. <i>Top,</i> Representative luminescence images of psV-infected C57BL/6 mice or <i>MISIIR-TAG</i> transgenic mice (left) and their harvested organs (right). Note: White arrow indicates only ovarian cancer from MISRII transgenic mice can be infected by HPV-16/luc psV. <i>Bottom,</i> Representative bar graphs of luminescence imaging in MISIIR-TAG transgenic mice or C57BL/6 mice. Data representative of 2 experiments performed.</p

Characterization of myeloid-derived suppressor cells in tumor-bearing mice.

<p>1×10⁵ TC-1 cells were injected subcutaneously into wild type C57BL/6 mice. 20 days later, splenocytes from naïve (control) or tumor-bearing mice were stained with FITC-conjugated Gr-1 antibody and PE-CD11b antibody. (<b>A</b>) Flow cytometry analysis to demonstrate CD11b and Gr-1 positive cells. (<b>B</b>) Bar graph depicts % of CD11b and Gr-1 positive cells (mean ± SD). Data shown are from one representative experiment of three performed. For the characterization of MDSC-mediated CD8+ T cell suppression, MDSCs were purified by magnetic cell sorting using mouse CD11b MicroBeads. The purity of MDSCs was determined by flow cytometry. Purified MDSCs were used in the T cell activation and proliferation experiment as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103562#s2" target="_blank">Materials and Methods</a>. (<b>C</b>) Bar graph depicting the % of IFN-γ positive cells in CD8 positive splenocytes (mean ± S.D.). (<b>D</b>) Representative flow cytometry histograms showing T-cell proliferation after stimulation with CD3 and CD28 Antibody. (<b>E</b>) Bar graph depicting the % of proliferated cells in CD8 and CFSE positive splenocytes (mean ± S.D.).</p

Characterization of the infectivity of HPV pseudovirion in naïve nude mice and human ovarian tumor-bearing nude mice.

<p>5–8 weeks old nude mice were injected intraperitoneally with ES2 human ovarian tumor cells (1×10⁶ cells/mouse). One week later, tumor-bearing mice were intraperitoneally injected with wild-type (wt) HPV-16/Luc psV or mutant L2 (mtL2) HPV-16L1mtL2-Luc pseudovirions (20 µg HPV-16 L1 protein/mouse, equivalent to 120 ng of DNA/mouse). Naïve mice infected with wt or mt HPV-16/luc pseudovirions served as controls. Mice were imaged by non-invasive luminescence imaging 1 day after infection. Data shown are representative of 2 experiments performed.</p

Comparison of HPV pseudovirion infectivity in naïve and murine ovarian tumor-bearing mice.

<p>5–8 weeks old C57BL/6 mice were challenged with MOSEC tumor cells (1×10⁶ cells/mouse). One week later, tumor-bearing mice were intraperitoneally injected with or without HPV-16/luc pseudovirions (20 µg HPV-16 L1 protein/mouse, equivalent to 120 ng of DNA/mouse). Naïve mice infected with or without HPV-16/luc pseudovirions served as a control. Mice were imaged by non-invasive luminescence imaging 1 day after infection. Data shown are representative of 2 experiments performed.</p

<i>In vitro</i> cytotoxicity mediated by HPV16-tk pseudovirions and ganciclovir.

<p>MOSEC-Luc cells were infected HPV16-GFP psV or HPV16/HSV-tk psV at a concentration of 1 µg L1 protein/ml for 48 hours. The infected cells were seeded in 96 well plates and then treated with 0 µg/ml, 0.1 µg/ml, 1 µg/ml, or 10 µg/ml of Ganciclovir for 72 hours. Luciferase expression was examined by the IVIS 200 system (Xenogen Corp., Alameda, CA, USA). Data shown are representative of 2 experiments performed.</p

Characterization and luminescence activity of NKG2D-Fc-GLuc protein.

<p>(A) Gel electrophoresis was used to characterize the size of the purified NKG2D-Fc-GLuc, Con-Fc-GLuc, and NKG2D-Fc proteins. The purity and size of the isolated proteins were characterized by SDS-PAGE, followed by staining with Coomassie brilliant blue. The arrows indicate the proteins of interest. (B) Representative figure of bioluminescent imaging in TC-1 cells incubated with the different purified proteins. TC-1 cells were plated into a 24-well plate. The purified proteins (Con-Fc, NKG2D-Fc, Con-Fc-GLuc, and NKG2D-Fc-GLuc) were added to the TC-1 tumor cells. The binding of NKG2D-Fc-GLuc to TC-1 cells was characterized by bioluminesence imaging using the IVIS Imaging System 200 Series. (C) Histogram depicting the bioluminescence measured in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035141#pone-0035141-g002" target="_blank">Figure 2B</a>. The p-value found was less than 0.001. (D) Representative bioluminescence imaging to demonstrate the specific targeting of NKG2D-Fc-GLuc to TC-1 tumor cells <i>in vivo</i>. TC-1 tumor-bearing C57BL/6 mice were injected intramuscularly with the DNA constructs encoding the Con-Fc-GLuc or NKG2D-Fc-GLuc protein, followed by electroporation. The treated mice were imaged for bioluminescence using the IVIS Imaging System 200 Series. (E) Histogram of the bioluminescence imaging found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035141#pone-0035141-g002" target="_blank">Figure 2D</a>. Note: there is a significant difference in the bioluminescence of the tumors between the mice injected with the DNA constructs for Con-Fc-Glu and NKG2D-Fc-Glu (P-value less than 0.001).</p