Metronomic chemotherapy enhances antitumor effects of cancer vaccine by depleting regulatory T lymphocytes and inhibiting tumor angiogenesis

Abstract

Although cancer vaccines are emerging as innovative methods for cancer treatment, these alone have limited potential for treating measurable tumor burden. Thus, the importance of identifying anticancer strategies with greater potency is necessary. The chimeric DNA vaccine CTGF/E7 (connective tissue growth factor linked to the tumor antigen human papillomavirus 16 E7) generates potent E7-specific immunity and antitumor effects. We tested immune-modulating doses of chemotherapy in combination with the CTGF/E7 DNA vaccine to treat existing tumors in mice. Metronomic low doses of paclitaxel, not the maximal tolerable dose, are synergistic with the antigen-specific DNA vaccine. Paclitaxel, given in metronomic sequence with the CTGF/E7 DNA vaccine enhanced the vaccine's potential to delay tumor growth and decreased metastatic tumors in vivo better than the CTGF/E7 DNA vaccine alone. The two possible mechanisms of metronomic paclitaxel chemotherapy are the depletion of regulatory T cells and the inhibition of tumor angiogenesis rather than direct cancer cell cytolytic effects. Results indicate that combination treatment of metronomic chemotherapy and antigen-specific DNA vaccine can induce more potent antigen-specific immune responses and antitumor effects. This provides an immunologic basis for further testing in cancer patients.

Figure 1

In vivo tumor treatment experiments. (a) Diagrammatic representation of the different treatment regimens of paclitaxel and/or DNA vaccination. (b) Overall survival of mice treated with various doses of paclitaxel only or with CTGF/E7 DNA vaccination. The mice were first inoculated with TC-1 tumor cells and treated with CTGF/E7 DNA vaccine only, CTGF/E7 DNA vaccine combined with various doses of paclitaxel, or paclitaxel only as described in Materials and Methods section. All of the mice vaccinated with CTGF/E7 DNA vaccine in combination with 3 or 6 mg/kg of paclitaxel were alive even after 70 days of tumor challenge and had significantly longer survival durations compared to the other groups. The survival curve of mice vaccinated with CTGF/E7 DNA vaccine plus 25 mg/kg paclitaxel was not longer than those of mice that received 25 mg/kg paclitaxel only (P > 0.05) and was even shorter than those vaccinated with CTGF/E7 DNA only (P < 0.05, Kaplan–Meier test). (c) Tumor volumes of mice treated with various doses of paclitaxel only. The mice were first inoculated with TC-1 tumor cells and treated with respective doses of paclitaxel, and the tumor sizes were calculated as described in Materials and Methods section. Mice treated with 25 mg/kg paclitaxel had smaller tumor volumes than the naive group and the 3 or 6 mg/kg of paclitaxel-treated mice (P < 0.05, one-way ANOVA). (d) Tumor volumes of mice treated with various doses of paclitaxel and CTGF/E7 DNA vaccination. The mice were first inoculated with TC-1 tumor cells and then treated with CTGF/E7 DNA vaccination only or CTGF/E7 DNA vaccination with respective doses of paclitaxel and the tumor sizes were calculated as described in Materials and Methods section. Mice treated with CTGF/E7 DNA vaccine combined with 3 or 6 mg/kg of paclitaxel had significantly smaller tumor volumes than those treated with CTGF/E7 DNA only, or CTGF/E7 DNA vaccine combined with 25 mg/kg of paclitaxel. Pooled data of three experiments (five mice per group) were calculated. Each result represents experiments in triplicate. ANOVA, analysis of variance; CTGF, connective tissue growth factor.

Figure 2

Kinetic changes of various subtypes of lymphocytes. (a) Total numbers of splenocytes in mice treated with various doses of paclitaxel. Mice first received various doses of paclitaxel and their splenocytes were harvested and counted as described in the Materials and Methods section. Total splenocytes of mice treated with 25 mg/kg paclitaxel were significantly lower than those of the other groups treated with PBS or lower doses of paclitaxel after 7 days of drug administration. (b) Bar figures show percentages of CD4⁺ helper T lymphocytes from total splenocytes. Percentages of CD4⁺ helper T lymphocytes in 25 mg/kg paclitaxel group were lower than the other groups after 7 days of drug administration. (c) Bar figures show percentages of cytotoxic CD8⁺ T lymphocytes from total splenocytes. The percentages of CD8⁺ cytotoxic T lymphocytes decreased significantly in the 25 mg/kg paclitaxel-treated group compared to other groups after 21 days of drug administration.

Figure 3

E7-specific immunity in mice that received various doses of paclitaxel and CTGF/E7 chimeric DNA vaccine. The mice were first immunized with CTGF/E7 DNA vaccine only or in combination with various doses of paclitaxel. The mice were then killed and their splenocytes were collected and stained with anti-CD8 and anti-IFN-γ antibodies, and analyzed by flow cytometry as described in the Materials and Methods section. (a) Bar figures show the frequencies of E7-specific CD8⁺ T-cell precursors 7 days after the last immunization. The frequencies of IFN-γ-secreting CD8⁺ T precursors in the CTGF/E7 DNA vaccine and 6 mg/kg paclitaxel-treated group were significantly higher than those in the other groups that received the CTGF/E7 DNA vaccine with 3 or 25 mg/kg paclitaxel (P < 0.001, one-way ANOVA). (b) Bar figures show kinetic changes in frequencies of E7-sepcific CD8⁺ T precursors among splenocytes. The numbers of E7-specific CD8⁺ T precursors in mice vaccinated with CTGF/E7 DNA and 6 mg/kg paclitaxel were higher than in mice that received the CTGF/E7 DNA vaccine only, regardless of the time interval. ANOVA, analysis of variance; CTGF, connective tissue growth factor.

Figure 4

In vivo angiogenesis assay characterizing antiangiogenic effects of various doses of paclitaxel. Matrigel was mixed with heparin and basic fibroblast growth factor, and was injected subcutaneously into the abdominal midline of C57BL/6 or BALB/c nude mice (a total 0.5 mg/mouse) on day 0. The mice were given 25 mg/kg of paclitaxel on days 0, 3, and 7, or 3 mg/kg or 6 mg/kg of paclitaxel every day intraperitoneally from days 0 to 10 and/or CTGF/E7 DNA vaccine on days 0 and 7. They were killed on day 11. The matrigel plugs were retrieved, resected, fixed, embedded in paraffin, sectioned, and stained with hematoxylin and eosin to determine microvessel density (MVD). The remaining half of the matrigel plugs were assayed for hemoglobin content as described in the Materials and Methods section. (a) Representative matrigel plugs: A1, negative; A2, PBS only; A3, 3 mg/kg paclitaxel; A4, 6 mg/kg paclitaxel; and A5, 25 mg/kg paclitaxel. (b) Bar graph shows the MVDs of matrigel plugs in C57BL/6 immunocompetent mice treated with various doses of paclitaxel. The MVDs of matrigel implants in 6-mg/kg paclitaxel-treated mice were significantly lower than those in the other paclitaxel and PBS-treated mice. (c) Bar graph shows matrigel hemoglobin content for C57BL/6 immunocompetent mice treated with various doses of paclitaxel. The hemoglobin concentrations of matrigel implants in 6-mg/kg paclitaxel-treated mice were lowest among the paclitaxel-treated groups. (d) Bar graph shows matrigel hemoglobin content for BALB/c immunocompromised nude mice treated with various doses of paclitaxel: D1, negative; D2, PBS only; D3, 3 mg/kg paclitaxel only; D4, 6 mg/kg paclitaxel only; D5, 25 mg/kg paclitaxel only; and D6, 6 mg/kg paclitaxel and CTGF/E7 DNA vaccine. The hemoglobin contents of matrigel implants in the 6 mg/kg paclitaxel-treated (D4 and D6) groups were significantly lower than those in the other (D2, D3, and D5) groups in BALB/c nude mice. CTGF, connective tissue growth factor.

Figure 5

Lymphocyte subtypes from tumor infiltrating lymphocytes (TILs) and splenocytes. (a) Bar figures show total TILs, CD4⁺ helper, and CD8⁺ cytotoxic T cells from TILs. C57BL/6 mice were inoculated with TC-1 tumor cells intravenously via the tail vein and treated with CTGF/E7 DNA vaccination only, vaccination combined with various doses of paclitaxel, or CD25 antibody, and the TILs from lung tissues were harvested on day 28 after tumor challenge as described in the Materials and Methods section. A1, naive; A2, CTGF/E7 DNA only; A3, CTGF/E7 DNA plus 3 mg/kg paclitaxel; A4, CTGF/E7 DNA plus 6 mg/kg paclitaxel; A5, CTGF/E7 DNA plus 25 mg/kg paclitaxel; A6, CTGF/E7 DNA plus CD25 antibody. The total numbers of TILs in the 3 and 6 mg/kg paclitaxel-treated and CD25 antibody depletion groups were greater than those in the other groups (P < 0.05, one-way ANOVA). However, the 6 mg/kg paclitaxel-treated group had the highest numbers of CD8⁺ cytotoxic T cells from the TILs compared to the other groups (P < 0.05, one-way ANOVA). (b) Bar figures show total Treg cells among TILs. C57BL/6 mice were inoculated with TC-1 tumor cells, treated with CTGF/E7 DNA vaccination only, vaccination combined with various doses of paclitaxel, or CD25 antibody, and their TILs were harvested for further analysis on calculation of CD8⁺ cytotoxic cell/Treg cell ratio from TILs. The ratio of cytotoxic and regulatory T cells was evaluated to determine if it reflected antitumor effects. C57BL/6 mice (five per group) were inoculated with TC-1 tumor cells intravenously and treated with various doses of paclitaxel and/or CTGF/E7 DNA vaccine, and TILs from lung tissues were harvested as described earlier. The TILs were stained with various antibodies and the numbers of cytotoxic T lymphocytes (CD3⁺CD8⁺) and Treg cells (CD4⁺CD25⁺Foxp3⁺) were analyzed and counted by flow cytometry in each group as described earlier. The ratio of cytotoxic T lymphocytes and Treg cells among the TILs from each group was calculated. Day 28 after tumor challenge as described in Materials and Methods section. B1, naive; B2, CTGF/E7 DNA only; B3, CTGF/E7 DNA plus 3 mg/kg paclitaxel; B4, CTGF/E7 DNA plus 6 mg/kg paclitaxel; B5, CTGF/E7 DNA plus 25 mg/kg paclitaxel; B6, CTGF/E7 DNA plus CD25 antibody. Mice treated with CTGF/E7 DNA vaccination and paclitaxel or CD25 antibody had significantly lower percentages of Treg cells/CD4⁺ helper T cells in TILs than those treated with CTGF/E7 DNA only (P < 0.05, one-way ANOVA). The 6 mg/kg paclitaxel-treated group also had the lowest percentage of Treg cells/CD4⁺ helper T cells among the paclitaxel-treated groups. (c) Representative figures show flow cytometry analyses of Treg cells from splenocytes. (d) Bar figures show percentages of Treg cells among CD4⁺ T cells from splenocytes. C57BL/6 mice were inoculated with TC-1 tumor cells, treated with CTGF/E7 DNA vaccination only, vaccination combined with various doses of paclitaxel, or CD25 antibody, and their splenocytes were harvested for further analysis on day 28 after tumor challenge as described in Materials and Methods section. CTGF/E7 DNA vaccinated mice treated with 3 or 6 mg/kg paclitaxel, or CD25 antibody had significantly lower numbers of Treg cells from splenocytes compared to the other groups. Mice treated with 6 mg/kg paclitaxel had the lowest numbers of Treg cells from splenocytes among the three paclitaxel-treated groups. ANOVA, analysis of variance; CTGF, connective tissue growth factor; PBS, phosphate-buffered saline; Treg, T regulatory.

Figure 6

In vivo antibody depletion experiments, in vivo pulmonary metastatic tumor experiments, and ratios of host antitumor and regulatory immunities. (a) In vivo antibody depletion experiments were conducted using a subcutaneous tumor model in C57BL/6 mice. C57BL/6 mice (five per group) were subcutaneously challenged with TC-1 tumor cells and treated with CTGF/E7 DNA vaccination in combination with 6 mg/kg of paclitaxel, and CD4 or CD8 antibody depletion was started on the day of TC-1 tumor challenge as described in the Materials and Methods section. The tumor sizes of mice that received CTGF/E7 DNA vaccination combined with 6 mg/kg paclitaxel and depleted of CD8⁺ T cells grew larger than those even in naive C57BL/6 mice. (b) In vivo experiments using the subcutaneous tumor model in BALB/c nude mice. BALB/c nude mice (five per group) were subcutaneously challenged with TC-1 tumor cells and treated with CTGF/E7 DNA vaccination combined with 6 mg/kg of paclitaxel, and CD4 or CD8 antibody depletion was started on the day of TC-1 tumor challenge as described earlier. BALB/c nude mice that received 6 mg/kg paclitaxel only or in combination with CTGF/E7 DNA vaccination had similar smaller tumors than those of naive or those that received only CTGF/E7 DNA vaccination. (c) Representative figures show pulmonary tumor nodules in C57BL/6 mice: C1, naive; C2, 3 mg/kg paclitaxel; C3, 6 mg/kg paclitaxel; C4, 25 mg/kg paclitaxel; C5, CTGF/E7 DNA; C6, CTGF/E7 DNA and 3 mg/kg paclitaxel; C7, CTGF/E7 DNA and 6 mg/kg paclitaxel; and C8, CTGF/E7 DNA and 25 mg/kg paclitaxel. C57BL/6 mice (five per group) were intravenously challenged with 5 × l0⁴ TC-1 tumor cells/mouse via the tail vein, and paclitaxel and/or CTGF/E7 DNA vaccination were given, and the mice were killed 28 days after tumor challenge in Materials and Methods section to evaluate whether the DNA vaccine combined with chemotherapy also controlled metastatic tumors better than the DNA vaccine only. C57BL/6 mice treated with CTGF/E7 DNA and 6 mg/kg paclitaxel had the fewest pulmonary tumor nodules compared to those treated with CTGF/E7 DNA only, vaccination plus 3 or 25 mg/kg of paclitaxel. (d) Ratios of CD8⁺ T cells/Treg cells from TILs in various groups. C57BL/6 mice were intravenously inoculated with TC-1 tumor cells and treated with various doses of paclitaxel and/or CTGF/E7 DNA vaccine, and TILs from lung tissues were harvested as described in the Materials and Methods section. The TILs were stained with various antibodies and the numbers of cytotoxic T cells (CD3⁺CD8⁺) and Treg cells (CD4⁺CD25⁺Foxp3⁺) in each group were analyzed and counted using flow cytometry as described in Materials and Methods section. The ratios of CD8⁺ T cells/Treg cells were highest in the mice vaccinated with CTGF/E7 DNA and 6 mg/kg paclitaxel compared to those in the other groups. CTGF, connective tissue growth factor.