Metronomic Chemotherapy Enhances Antitumor Effects of Cancer ...

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original article

Metronomic Chemotherapy Enhances Antitumor Effects of Cancer Vaccine by Depleting Regulatory T Lymphocytes and Inhibiting Tumor Angiogenesis Chi-An Chen1, Chih-Ming Ho2, Ming-Cheng Chang1, Wei-Zun Sun3, Yu-Li Chen1, Ying-Cheng Chiang1, Ming-Hong Syu1, Chang-Yao Hsieh1 and Wen-Fang Cheng1,4 1 Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei, Taiwan; 2Gynecologic Cancer Center, Department of Obstetrics and Gynecology, Cathay General Hospital, Taipei, Taiwan; 3Department of Anesthesiology, National Taiwan University Hospital, Taipei, Taiwan; 4Graduate Institute of Oncology, College of Medicine, National Taiwan University, Taipei, Taiwan

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. Received 17 August 2009; accepted 5 February 2010; advance online publication 6 April 2010. doi:10.1038/mt.2010.34

Introduction Conventional modalities for cancer treatment are surgery, radiation therapy, and chemotherapy. The metastatic nature of cancer requires that the effect of any treatment be distributed throughout the body. Although chemotherapy is used systemically to destroy any residual or metastatic tumor cells, it cannot discriminate between neoplastic and non-neoplastic cells. Immunotherapy has recently provided an attractive alternative approach, purposely

antigen-specific, with its potential ability to eradicate systemic cancer lesions and differentiate between normal and cancer cells.1 Chemotherapeutic agents suppress host immunity by causing the apoptosis of immunocytes. However, these agents also modulate the immune response to improve antitumor effects.2,3 Kerbel et al.4 recently developed a new strategy for cancer therapy using the concept of metronomic (low-dose) chemotherapy instead of the current maximum tolerated dose (MTD) chemotherapy. Their results reveal that the possible mechanism of metronomic chemotherapy is antiangiogenesis. Paclitaxel is a very effective agent for treating many malignancies, including breast and ovarian carcinomas, lung cancer, head and neck tumors, melanomas, gastric carcinomas, and cervical cancer.5,6 It promotes the assembly of microtubules, inhibits tubulin disassembly7 and DNA synthesis,8 and releases tumor necrosis factor-α9 to cause apoptosis in a variety of cancer cell types.10 Not withstanding MTD chemotherapy, microtubule-interfering agents such as paclitaxel were the first chemotherapeutic agents to have antiangiogenic activity by acting primarily on endothelial cells.11 Paclitaxel, at 3 or 6 mg/kg daily, is effective in inhibiting intratumor angiogenesis in a mouse model.12 Vaccination as a form of specific immunotherapy for cancer has been investigated for years. Tumors that express specific antigens, such as breast and cervical cancer, are considered suitable candidates for immunotherapy.13,14 Encouraging in vitro and animal studies have led to several clinical trials for malignant disorders.15,16 DNA or gene vaccines directed against human papillomavirus E7 tumor antigen protects mice from challenge with E7-expressing tumor cells and pulmonary metastatic tumors.17–19 We investigated the potential benefits of combined chemotherapy and antigen-specific immunotherapy to improve cancer management. Immunotherapy combined with chemotherapy was tested to determine whether it could augment the efficacy of either agent in a rapidly growing, lethal murine cervical tumor model. We also investigated possible mechanisms for the effects of combined immunotherapy and chemotherapy.

Correspondence: Wen-Fang Cheng, Department of Obstetrics and Gynecology, National Taiwan University Hospital, Taipei, Taiwan. E-mail: [email protected] Molecular Therapy

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Immunotherapy Combined With Chemotherapy

Results CTGF/E7 chimeric DNA vaccine with metronomic paclitaxel enhanced survival

CTGF/E7 DNA vaccine only had longer survival than those that received paclitaxel only (P < 0.05, Kaplan–Meier test). However, 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 that received CTGF/E7 DNA only (P < 0.05, Kaplan–Meier test). Mice given CTGF/E7 chimeric DNA vaccine in combination with metronomic paclitaxel survived the longest.

Tumor-bearing mice that received chemotherapy and/or immunotherapy were first tested for possible therapeutic benefits. The survival curves of mice that received connective tissue growth factor (CTGF)/E7 DNA vaccine only, CTGF/E7 DNA vaccine with various doses of paclitaxel, or paclitaxel only are shown in Figure 1b. None of the mice survived after 55 days of tumor challenge, regardless of the amount of paclitaxel received. All of the mice vaccinated with CTGF/E7 DNA vaccine plus 3 or 6 mg/kg of paclitaxel were alive after 70 days of tumor challenge. They also had significantly longer survival durations compared to the other groups (P < 0.05, Kaplan–Meier test). Mice vaccinated with

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Antitumor effects of CTGF/E7 DNA vaccine combined with metronomic chemotherapy Chemotherapeutic effects, measured as tumor volume, in mice with TC-1 subcutaneous tumors, were compared. Mice treated with 25 mg/kg paclitaxel (507.8 ± 31.2 mm3 on day 40) had the

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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.

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In vivo effect of paclitaxel dose and duration The influence of paclitaxel on the quantity of immunocytes was evaluated. Total splenocytes (per 106 cells) of mice in the phosphate-buffered saline (PBS) group (128.0 ± 2.0 × 106 on day 7, 120.0 ± 1.2 × 106 on day 21, 109.0 ± 1.0 × 106 on day 35), 3 mg/kg paclitaxel group (129.0 ± 1.0 × 106 on day 7, 114.0 ± 1.0 × 106 on day 21, 108.0 ± 1.2 × 106 on day 35), and 6 mg/kg paclitaxel group (127.0 ± 0.7 × 106 on day 7, 109.0 ± 0.9 × 106 on day 21, 104.0 ± 1.1 × 106 on day 35) were significantly higher than those of mice injected with 25 mg/kg of paclitaxel (106.0 ± 1.0 × 106 on day 7, 95.0 ± 0.8 × 106 on day 21, 70.0 ± 1.0 × 106 on day 35) after 7 days of drug administration (P < 0.01, one-way ANOVA) (Figure 2a).

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only (255.8 ± 25.1 mm3 on day 47) (P < 0.01, one-way ANOVA). Mice treated with CTGF/E7 DNA vaccine with 6 mg/kg of paclitaxel even had significantly smaller tumor volumes than those treated with CTGF/E7 DNA vaccine with 3 mg/kg of paclitaxel (P < 0.05, one-way ANOVA). Thus, CTGF/E7 DNA vaccination combined with metronomic chemotherapy produced the most potent antitumor effect.

+ Percentage of CD4 helper T Iymphocyte

smallest tumor volume compared to other paclitaxel-treated groups [naive group (751.8 ± 46.5 mm3 on day 40), 3 mg/kg paclitaxel (755.6 ± 49.2 mm3 on day 40), and 6 mg/kg paclitaxel (685.0 ± 49.2 mm3 on day 40)] [P < 0.05, one-way analysis of variance (ANOVA)] (Figure 1c). Combined therapy with antigen-specific DNA vaccine and cytotoxic chemotherapy was further evaluated to determine whether it could generate a more potent in vivo antitumor effect than either therapy alone. Mice that received CTGF/E7 DNA vaccine with 25 mg/kg paclitaxel (753.3 ± 56.6 mm3 on day 47) and those that received 25 mg/kg paclitaxel only (774.9 ± 67.2 mm3 on day 47) had similar tumor volumes (P > 0.05, one-way ANOVA) (Figure 1d). On the other hand, mice treated with CTGF/E7 DNA vaccine only (255.8 ± 25.1 mm3 on day 47) had smaller tumor volumes than those that received the CTGF/E7 DNA vaccine with 25 mg/kg paclitaxel (753.3 ± 56.6 mm3 on day 47) (P 0.05, one-way ANOVA). For CD8+ cytotoxic T cells in the splenocytes, similar to those of CD4+ helper T lymphocytes, 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 (day 21, 10.5 ± 0.2% in the PBS group, 10.1 ± 0.2% in the 3 mg/kg paclitaxel group, 10.4 ± 0.3% in 6 mg/kg paclitaxel group, 8.7 ± 0.2% in the 25 mg/kg paclitaxel group, day 35, 9.7 ± 0.2% in the PBS group, 9.4 ± 0.20% in the 3 mg/kg paclitaxel group, 9.4 ± 0.2% in the 6 mg/kg paclitaxel group, 5.8 ± 0.3% in the 25 mg/kg paclitaxel group) (P < 0.05, one-way ANOVA) (Figure 2c). There was no difference among the PBS, 3 mg/kg paclitaxel, and 6 mg/kg paclitaxel-treated groups (P > 0.05, oneway ANOVA). In vivo, paclitaxel decreased the total quantity of immunocytes in a dose and duration dependent manner.

Effect of metronomic chemotherapy on antigen-specific immunity Antigen-specific immune responses of mice vaccinated with CTGF/ E7 DNA vaccine, after receiving different doses of paclitaxel, were 1,400 1,200

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Effect of metronomic paclitaxel on in vivo angiogenesis We evaluated whether chemotherapy inhibited in vivo tumor growth via an antiangiogenic pathway. The representative figures of matrigel assays in each group of C57BL/6 immunocompetent mice are shown in Figure 4a. The microvessel of matrigel implants in paclitaxel-treated mice (87.0 ± 10.0 in the 3 mg/kg group, 67.0 ± 7.0 in the 6 mg/kg group, and 154.0 ± 12.0 in the 25 mg/kg group) were significantly lower than those in the PBS-treated C57BL/6 mice (265.5 ± 14.5 mg hemoglobin (Hb)/g matrigel) (P < 0.01, ANOVA) (Figure 4b). However, the 6 mg/kg paclitaxel-treated

Number of E7-specific IFN-γ-secreting CD8+ T cell precursors/3.5 x 105 splenocytes

Number of E7-specific IFN-γ-secreting CD8+ T cell precursors/3.5 x 105 splenocytes

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first evaluated 7 days after the last immunization. The frequencies of interferon-γ-secreting CD8+ T precursors were highest in mice treated with CTGF/E7 DNA and 6 mg/kg paclitaxel (1,092.5 ± 61.5) compared to the other groups (for the CTGF/E7 DNA group, 767.0 ± 45.5; for the CTGF/E7 plus 3 mg/kg paclitaxel group, 842.0 ± 33.0; for the CTGF/E7 plus 25 mg/kg paclitaxel group, 508.0 ± 28.0, P < 0.01, one-way ANOVA) (Figure 3a). We also evaluated whether chemotherapy could generate higher and more persistent antigen-specific T-cell immunity when combined with the naked DNA vaccine. The numbers of E7-specific CD8+ T precursors in mice vaccinated with CTGF/E7 DNA and 6 mg/kg paclitaxel were highest among the groups from 7 to 35 days after last immunization (day 21 PBS, 308.0 ± 23.0; 3 mg/kg paclitaxel, 342.0 ± 18.0; 6 mg/kg paclitaxel, 568.5 ± 23.5; 25 mg/kg paclitaxel, 184.5 ± 10.5; day 35 PBS, 150.5 ± 14.8; 3 mg/ kg paclitaxel, 177.5 ± 12.5; 6 mg/kg paclitaxel, 288.0 ± 20.0; 25 mg/ kg paclitaxel, 112.5 ± 9.5, P < 0.01 one-way ANOVA) (Figure 3b). Mice vaccinated with CTGF/E7 DNA with 6 mg/kg paclitaxel had the highest numbers of E7-specific CD8+ T lymphocytes compared to the other groups at 7, 21, and 35 days after last immunization (P < 0.01, one-way ANOVA). Metronomic chemotherapy enhanced antigen-specific immunity.



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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.

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mice had the lowest microvessel density (MVD) among the three paclitaxel-treated groups (P < 0.01, ANOVA) (Figure 4b). Moreover, the Hb contents of matrigel implants in paclitaxeltreated C57BL/6 immunocompetent mice (mg Hb/g matrigel), (25.8 ± 2.6 in the 3 mg/kg group, 16.2 ± 1.9 in the 6 mg/kg group, and 37.0 ± 2.3 in the 25 mg/kg group) were also significantly lower than those in PBS-treated mice (65.0 ± 2.5 mg Hb/g matrigel) (P 0.05, one-way ANOVA). However, the 6 mg/kg paclitaxel-treated group had the highest numbers of CD8+ cytotoxic T cells among the TILs compared to the other groups (Figure 5a). Metronomic paclitaxel increased CD8+ cytotoxic T lymphocytes in the local tumor environment. www.moleculartherapy.org



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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.

For the evaluation of T regulatory (Treg) cells among the TILs, the percentages of Treg cells/CD4+ helper T cells were used to represent the status of Treg cells among the TILs. Mice treated with CTGF/E7 DNA vaccine and various doses of paclitaxel or CD25 Ab had significantly lower percentages of Treg cells/CD4+ helper T cells than those treated with CTGF/E7 DNA only or the naive group (P < 0.05 one-way ANOVA) (Figure 5b). The 6 mg/ kg paclitaxel-treated group also had the lowest percentages of Treg Molecular Therapy

cells/CD4+ helper T cells among the paclitaxel-treated groups (P < 0.05, one-way ANOVA). Treg cells among splenocytes in various vaccinated groups were evaluated. Representative figures of flow cytometric analyses of Treg cells among splenocytes are shown in Figure 5c. CTGF/E7 DNA vaccinated mice treated with 3 or 6 mg/kg paclitaxel, or CD25 antibody had significantly lower numbers of Treg cells among splenocytes compared to the other groups (PBS, 9,350 ± 232; 7



CTGF/E7, 7,310 ± 245; CTGF/E7 plus 3 mg/kg paclitaxel, 4,370 ± 86; CTGF/E7 plus 6 mg/kg paclitaxel, 1,360 ± 88; CTGF/ E7 plus 25 mg/kg paclitaxel, 7,360 ± 351; CTGF/E7 plus CD25 Ab, 80 ± 16, P < 0.01 one-way ANOVA) (Figure 5d). Mice treated with 6 mg/kg paclitaxel had the lowest numbers of Treg cells in the splenocytes among the three paclitaxel-treated groups (P < 0.05, one-way ANOVA).

Essential cells for the antitumor effect of CTGF/E7 DNA combined with 6 mg/kg paclitaxel To determine the subset of lymphocytes important for the antitumor effect of chemotherapy combined with immunotherapy, in vivo Ab depletion experiments were performed. C57BL/6 mice that received the CTGF/E7 DNA vaccine combined with 6 mg/kg paclitaxel depleted of CD4+ T cells had similar smaller tumor sizes compared to those without antibody depletion (P > 0.05, oneway ANOVA). The tumor sizes of mice that received the CTGF/ E7 DNA vaccine combined with 6 mg/kg paclitaxel depleted of CD8+ T cells grew larger than those even in naive C57BL/6 mice (P 800 mm3. For the intravenous therapeutic experiments, C57BL/6 mice (five per group) were challenged with 5 × 104 TC-1 tumor cells/mouse intravenously

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via the tail vein to generate a pulmonary metastatic model as described previously.18 Paclitaxel and/or CTGF/E7 DNA vaccine were administered according to the aforementioned protocols. The mice were killed 28 days after tumor challenge to examine lung weights and pulmonary tumor nodules. In vivo Ab depletion experiment. In vivo Ab depletion was performed as

described previously.40 Briefly, C57BL/6 mice (five per group) were challenged with TC-1 tumor cells subcutaneously, vaccinated with the CTGF/ E7 DNA vaccine, and administered 6 mg/kg of paclitaxel. Ab depletion was started on the day of TC-1 tumor challenge, using mAb GK1.5 for CD4 depletion, mAb 2.43 for CD8 depletion, and mAb PC61 for CD25 depletion.41 Flow cytometry analysis revealed that 95% of the appropriate lymphocyte subsets were depleted, with normal levels of other lymphocyte subsets. Depletion was terminated on the day of euthanasia. Surface marker staining and flow cytometry. Mice (five per group) first received various doses of paclitaxel as described earlier. They were vaccinated with 2 µg of CTGF/E7 DNA on days 7 and 14 after paclitaxel administration. The mice were killed 7, 14, 21, 28, or 35 days after the last DNA immunization and their splenocytes were harvested. The splenocytes were then stained with fluorescein isothiocyanate-conjugated antiCD3, phycoerythrin (PE)-conjugated anti-CD4, PE-conjugated anti-CD8, PE-Cy5-conjugated anti-CD4, or PE-conjugated CD25 Ab (PharMingen, San Diego, CA) for different experiments.40 Cytometric analyses were performed using a Becton Dickinson FACScan (Becton Dickinson, Franklin Lakes, NJ), with CELLQuest software. Intracellular interferon-γ cytokine staining and flow cytometry.

Splenocytes obtained from various vaccinated groups were incubated with 1 µg/ml of major histocompatibility complex I-restricted E7 peptide (aa49–57).40,42,43 Cell surface marker staining of PE-conjugated antiCD8 and fluorescein isothiocyanate-conjugated anti-mouse interferon-γ (PharMingen) were performed44–46 and analyzed by flow cytometry as described earlier. In vivo angiogenesis assay. In vivo angiogenesis was assessed using the

matrigel plug assay with a protocol similar to that described previously.19 Briefly, matrigel (Becton Dickinson) was mixed with heparin (final concentration 50 U/ml) and basic fibroblast growth factor (final concentration, 10 ng/ml). A total of 0.5 ml/mouse of the matrigel mixture was injected subcutaneously into the abdominal midline of C57BL/6 or BALB/c nude mice (five mice/group) 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.

MVD evaluation. The matrigel plugs were resected and half was fixed in 10% formaldehyde, embedded in paraffin, sectioned, and stained with hematoxylin and eosin stains to calculate MVD.19 MVD was determined with ×400 magnification and the mean number of microvessels in the five most vascular fields was calculated and referred to as the MVD. Detection of Hb content in matrigel. The matrigel plugs were resected

as described earlier. Half of these were used to determine the MVD. The remaining half were assayed for Hb content using a Hb detecting kit according to the manufacturer’s instructions (Human GmbH, Wiesbaden, Germany).

Characterization of regulatory T lymphocytes. C57BL/6 mice (five per group) were inoculated intravenously with TC-1 tumor cells (5 × 104/ mouse) via the tail vein and treated with various doses of paclitaxel and/or CTGF/E7 DNA vaccine. TILs from lung tissue and splenocytes were harvested on day 28 after TC-1 tumor challenge. The preparation of single cell suspensions from lung tissues was performed as described previously, with modifications.18 Cell surface marker staining of PE-Cy5-conjugated anti-CD4, PE-conjugated anti-CD25 (PharMingen),

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and fluorescein isothiocyanate-conjugated anti-mouse Foxp3 Abs (eBioscience, San Diego, CA) were performed as described previously.47 The staining was characterized by flow cytometry. Statistical analysis. All of the data were expressed as mean ± SEM, which represented at least two different experiments. Data for intracellular cytokine staining and tumor treatment experiments were evaluated by ANOVA. The event time distributions for different mice in the survival experiments were compared using log rank analysis. Differences were considered significant when P < 0.05.

ACKNOWLEDGMENTS This work was supported in part by the Department of Medical Research of National Taiwan University Hospital and by grants from the National Science Committee of Taiwan (NSC96-2314-B-002-091MY2 and 98-2628-B-002-083-MY3). The E7-specific CD8+ T cell line and TC-1 tumor cell line were kindly provided by Dr Wu of Johns Hopkins Medical Institutes (Baltimore, MD).

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