Comparative antitumor effect of preventive versus therapeutic vaccines employing B16 melanoma cells genetically modified to express GM-CSF and B7.2 in a murine model

Abstract

Cancer vaccines have always been a subject of gene therapy research. One of the most successful approaches has been working with genetically modified tumor cells. In this study, we describe our approach to achieving an immune response against a murine melanoma model, employing B16 tumor cells expressing GM-CSF and B7.2. Wild B16 cells were injected in C57BL6 mice to cause the tumor. Irradiated B16 cells transfected with GM-CSF, B7.2, or both, were processed as a preventive and therapeutic vaccination. Tumor volumes were measured and survival curves were obtained. Blood samples were taken from mice, and IgGs of each treatment group were also measured. The regulatory T cells (Treg) of selected groups were quantified using counts of images taken by confocal microscopy.

Results: one hundred percent survival was achieved by preventive vaccination with the group of cells transfected with p2F_GM-CSF. Therapeutic vaccination achieved initial inhibition of tumor growth but did not secure overall survival of the animals. Classical Treg cells did not vary among the different groups in this therapeutic vaccination model.

Figure 1

Tumor volume in preventive vaccination. Results from inhibition of tumor volume with vaccination groups: (a) Control; (b) B16-p2fØ; (c) B16*; (d) B16-GM-CSF + B7.2/500; (e) B16-B7.2; (f) B16-GM-CSF + B7.2/200; (g) B16-pMok_GM-CSF; (h) B16-GM-CSF. Mice were injected with 10⁵ B16 wild cells in the left leg. We used a vaccination dose of 2 × 10⁵ cells, but also tested other doses in the treatments with B16-GM-CSF + B7.2, expressing the number of cells used with 200 or 500, corresponding to 2 × 10⁵ or 5 × 10⁵ cells, respectively. In the figure, “*” corresponds to the maximum statistical difference, p < 0.001, and “**” to p < 0.01, both with respect to the control group. In turn, “+” corresponds to the maximum statistical difference, p < 0.001, and “++” to p < 0.01, both with respect to the B16-GM-CSF group. Arrows identified as 1, 2 and 3 represent groups B16-GM-CSF + B7.2/200, B16-pMok_GM-CSF and B16-GM-CSF, respectively, with total inhibition of tumor growth during the measurement period.

Figure 2

Survival in preventive vaccination. The plot shows survival of the groups described in Figure 1.

Figure 3

Production of total IgG (a) and IgG1 (b) and IgG2a (c) subtypes against TMP in preventive vaccination. After blood samples were taken from the animals on day −22, day −1 and day 15, with respect to the day of implantation of the tumor (day 0), and the plasma was obtained, the latter was used for enzyme linked immunosorbent assaying (ELISA) specific total IgG (a) and subtypes IgG1 (b) and IgG2a (c) against TMP. The samples were assayed in duplicate to calculate the mean and standard deviation, which is too small to see in the figure. The treatment groups are described in Figure 1. All groups showed significant differences versus the control as a minimum at day −1 and day 15 (p < 0.001).

Figure 4

B16 tumor reimplantation. One year after first vaccination with transfected cells, a reminder vaccine dose was administered to the surviving animals, which were again introduced to the tumor (day 0 = day 365, 10⁵ B16 wild type cells). The plot shows the survival of these animals and a mice control group not previously treated.

Figure 5

Production of total IgG (a) and IgG1 (b) and IgG2a (c) subtypes specific to TMP in reimplantation with preventive vaccination. The mice surviving the first cell vaccine received a vaccine reminder dose and the introduction of tumor 14 days later. Blood was extracted according to the usual pattern (days −22, day −1 and day 15) with respect to implementation of the tumor, day 0. Retrieved plasma was used in the ELISA test to detect IgG anti-TMP, as in Figure 3. The plot shows the production groups vaccinated with B16 cells transfected with p2F_m-GM-CSF, p2F_mGM-CSF + mB7.2 dose 2 × 10⁵ and 5 × 10⁵, p2F_m-B7.2, and the control group.

Figure 6

Inhibition of tumor growth in cell treatment at low doses. C57BL/6 mice (n = 5 per group) were vaccinated after tumor implantation (day 0, 2 × 10⁴ B16 wild type cells) 3, 10 and 17 days. Treatment groups were control (only DMEM) or 5 × 10⁵ or 2 × 10⁶ cells transfected with plasmid p2F (Ø, GM-CSF, GM-CSF + B7.2). The tumor’s size was measured. The symbol * represents a statistically significant difference (p < 0.001) with respect to the control group. In turn, “+” corresponds to the maximum statistical difference, p < 0.001, “++” to p < 0.01, and “+++” to p < 0.05, with respect to the B16-GM-CSF + B7.2/2 group.

Figure 7

Survival in cell treatment at low doses. Mortality among mice treated according to the groups listed in Figure 6 is shown in the figure.

Figure 8

Production of specific total IgG (a) and IgG1 (b) and IgG2a (c) subtypes against TMP in low dose cell treatment. After implanting tumor in animals on day 0 with 2 × 10⁴ B16 cells, three vaccination doses were administered on days 3, 10 and 17, and four blood extractions were performed on days 3, −1, 8, 15 and 22. With the retrieved plasma, ELISA testing was made of samples in duplicate, calculating the mean and standard deviation, which is too small to see in the figure. “*” represents p < 0.001 versus the control group. Groups described in Figure 6.

Figure 9

Inhibition of tumor growth in high dose cell treatment. Mice (n = 5 per group) were vaccinated after tumor implantation (day 0, 2 × 10⁴ B16 cells) day 3, 10 and 17 with 8 × 10⁶ transfected cells, per dose (only DMEM in the control group or irradiated B16 without transfection in B16*), transfecting with plasmids p2FØ, p2F_mGM-CSF + mB7.2, p2F_mGM-CSF, and p2F_mB7.2, respectively. Tumor size was measured and statistical significance was calculated as in the rest of experiments. The symbol “*” represents statistical difference (p < 0.001) with respect to the control group. In turn, “+” corresponds to the maximum statistical difference, p <0.001, and “++” to p < 0.01, both with respect to the B16-B7.2 group.

Figure 10

Representative confocal microscopy images in blood sample 3. Green CD4⁺, red CD25⁺, blue Foxp3⁺. The symbol # shows CD4⁺CD25⁺Foxp3⁺ cells, the symbol * shows CD4⁻CD25⁺Foxp3⁺ cells. Groups described in Figure 9.