A tritherapy combination of a fusion protein vaccine with immune-modulating doses of sequential chemotherapies in an optimized regimen completely eradicates large tumors in mice

Authors

  • Xinxin Song,

    1. Department of Cellular and Molecular Biology, Cancer Institute & Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, People's Republic of China
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  • Wenzhong Guo,

    1. Department of Cellular and Molecular Biology, Cancer Institute & Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, People's Republic of China
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  • Jianfeng Cui,

    1. Department of Cellular and Molecular Biology, Cancer Institute & Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, People's Republic of China
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  • Xinlai Qian,

    1. Department of Cellular and Molecular Biology, Cancer Institute & Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, People's Republic of China
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  • Linan Yi,

    1. Department of Cellular and Molecular Biology, Cancer Institute & Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, People's Republic of China
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  • Mengjiao Chang,

    1. Department of Cellular and Molecular Biology, Cancer Institute & Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, People's Republic of China
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  • Qiliang Cai,

    1. Department of Cellular and Molecular Biology, Cancer Institute & Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, People's Republic of China
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  • Qingzheng Zhao

    Corresponding author
    1. Department of Cellular and Molecular Biology, Cancer Institute & Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, People's Republic of China
    • Department of Cellular and Molecular Biology, Cancer Institute & Hospital, Peking Union Medical College and Chinese Academy of Medical Sciences, PO Box 2258, Beijing 100021, People's Republic of China

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    • Tel.: +86 10 87788433, Fax: +86 10 87777447


  • All authors have declared there are no financial conflicts of interest in regard to this work

Abstract

Tumor-induced immunosuppression plays a critical role in both impeding tumor-specific immune responses and limiting the effects of cancer immunotherapy. Analyses of regulatory cells recruited during the growth of the E7-expressing tumor, TC-1, revealed a high percentage of regulatory T cells (Tregs) as well as myeloid-derived suppressor cells (MDSCs) in spleens and tumors. In this study, we proposed that treatment with immune-modulating doses of cyclophosphamide (CTX) and all-trans retinoic acid (ATRA) would result in a beneficial tumor microenvironment with the suppression of Tregs and MDSCs and, thus, enhance the effect of a human papillomavirus protein vaccine. Our results showed that CTX preconditioning and persistent ATRA treatment along with the vaccine achieved long-term survival and induced long-term memory responses. However, the effect of the antitumor response sharply declined when the tritherapy was initiated after the optimal therapeutic time. The more intensive regimen could rescue the effect of the tritherapy accompanied by the decreased percentage of Tregs and MDSCs in spleens and tumors. Besides, a favorable host environment was created by the reduced secretion of interleukin-10 and 6 and vascular endothelial growth factor (VEGF) in the tumor niche and decreased the expression of phosphorylation-signal transducer and activator of transcription 3 of TC-1 tumors. Our data shed light on the immune-modulating doses of sequential chemoimmunotherapeutic strategy targeting not only the tumor but also its microenvironment, which suggests a potential clinical benefit for the immunotherapy of HPV-associated malignancies.

Over the last decade, despite several promising reports with a vaccine approach for some cancer patients, consistent clinical responses have not been very well obtained so far.1–3 The tumor microenvironment represents a consistently effective barrier to clinical responses.4 The need to design novel strategies based on targeting not only the tumor but also its microenvironment arises urgently. It is widely accepted that immune-modulating doses of chemotherapeutic agents can exert multiple effects on host immune cells in the tumor microenvironment and induce an antitumor immune response.5–7 Thus, the combination of immunotherapy with proper chemotherapeutic regimen may yield the greatest clinical benefit for patients with late-stage disease.

The tumor contrives to benefit from infiltrating cells by modifying their functions to create the microenvironment favorable to tumor progression and, thus, escape from the host immune system. Of all the various escape mechanisms, two have received special attention in recent years, because they appear to be ubiquitous and are clearly associated with disease progression.4, 8 Myeloid-derived suppressor cells (MDSCs) are recruited by means of tumor-derived soluble factors such as vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-β, interleukin (IL)-10, etc., and migrate to the tumor site and block T-cell functions through the production of arginase I and activation of inducible nitric oxide synthase (iNOs).9–11 Another most successful strategy of tumor-induced immune evasion is the recruitment, expansion and activation of CD4+CD25+FoxP3+regulatory T cells (Tregs). They exist naturally at low numbers but have been documented to increase in patients or animals with malignancies and are capable of suppressing both the innate and the adaptive immune responses in the microenvironment through contact-dependent mechanisms or IL-10 and TGF-β secretion.12 Recent reports suggest that oncologic therapies, such as surgery, radiation and chemotherapy, expand Tregs and enhance their suppressor functions.13–15 These cells in the tumor-bearing host creates an immune imbalance that could not be corrected by immunotherapies aimed only at activation of antitumor immune responses.16

Administration of therapeutic concentrations of all-trans retinoic acid (ATRA) results in a substantial decrease in the numbers of MDSCs in patients with cancer and tumor-bearing mice. ATRA has been shown to induce the differentiation of MDSCs into dendritic cells (DCs) and macrophages in vitro and in vivo.17, 18 The main mechanism of ATRA-mediated differentiation involves an upregulation of glutathione synthesis and a reduction in reactive oxygen species levels in MDSCs.19 In our previous study, we have demonstrated that an HPV protein vaccine, mE6Δ/mE7/TBhsp70Δ, restored antitumor immune responses via the correction of aberrant myeloid cell differentiation by ATRA in a mouse TC-1 tumor model, which expresses the E7 oncoprotein from HPV-16, is used as a surrogate for human tumors infected with HPV-16 and is potentially used to test the efficacy of immunotherapy for cancers expressing defined tumor antigens.20

However, several recent studies have shown that ATRA and other agonists of the retinoic acid receptor alpha lead to an increased induction of CD4+ T cells expressing the Treg specification factor, forkhead family transcription factor (FoxP) 3.21, 22 Our own analysis and other studies revealed a high percentage of splenic and tumor-infiltrating MDSCs and Tregs recruited during the growth of TC-1 tumors.23, 24 Cyclophosphamide (CTX) not only decreases the proportion of Tregs but also inactivates the remaining Tregs and has been reported to enhance the efficacy of adoptive transfer of antigen-specific lymphocytes and tumor vaccines. Furthermore, the expression of glucocorticoid-induced TNF receptor (GITR) and the Foxp3 was also downregulated after CTX administration.25–28 In light of these evidences, the most promising and synergistic approaches for cancer immunotherapy will be those designed to augment specific antitumor immunity while simultaneously eliminating relevant suppressive elements in vivo.29 Thus, in this study, we explored a complementary approach with CTX and ATRA to improve the efficacy of the HPV-associated immunotherapy, with possible therapeutic potential of the combination strategy for cancers that express defined tumor antigens in clinical settings.

Abbreviations

ATRA: all-trans retinoic acid; CTX: cyclophosphamide; DC: dendritic cells; ELISPOT: enzyme-linked immunospot; FBS: fetal bovine serum; FITC: fluorescein isothiocyanate; FoxP3: the forkhead family transcription factor 3; G418: Geneticin; GITR: glucocorticoid-induced TNF receptor; HPV: human papillomavirus; i.p.: intraperitoneally; IFN-γ: interferon-γ; IL: interleukin; MDSC: myeloid-derived suppressor cell; PBS: phosphate buffered saline; PE-Cy5: phycoerythrin-Cy5; PE: phycoerythrin; s.c.: subcutaneously; STAT: signal transducer and activator of transcription; TGF: transforming growth factor; Treg: regulatory T cell; VEGF: vascular endothelial growth factor

Material and Methods

Mice and cell lines

Six- to 8-week-old female C57BL/6 mice were purchased from the Experimental Animal Institute of Peking Union Medical College. All animals were maintained under specific pathogen-free conditions, and all procedures were performed according to approved protocols and in accordance with recommendations for the proper care of laboratory animals. TC-1 tumor cells derived from primary epithelial cells of C57BL/6 mice co-transformed with HPV-16 E6, E7 and c-Ha-ras oncogenes were provided by Dr. T.C. Wu from Johns Hopkins University. TC-1 cells were cultured in RPMI 1640 (Gibco-BRL, Carlsbad, CA) containing 10% fetal bovine serum (HyClone, Logan, UT) in the presence of 200 μg/ml of Geneticin (G418) at 37°C with 5% CO2.

Reagents

CTX (Cat. C0768; Sigma-Aldrich, Milwaukee, WI) was dissolved at 10 mg/ml in deionized water. ATRA (Cat. R2625; Sigma-Aldrich) was dissolved at 40 mg/ml in dimethyl sulfoxide (DMSO) and diluted in solvent containing 20 mmol/L NaOH, 0.5% Tween-80 and 3% ethanol (pH 7.4).30

Flow cytometry

For the sample obtained from fresh TC-1 carcinoma mass, separation of the tumor-infiltrating mononuclear cells was carried out by differential gradient centrifugation, and the tumor-infiltrating mononuclear cells were found at the interface of 75% and 100% Ficoll-Hypaque. For samples obtained from spleens, erythrocytes were removed by suspending the cells in lysis buffer, pH 7.2, and then rinsing the cells twice with RPMI 1640. Cells were incubated for 30 min with an optimal concentration of antibodies on ice and then washed twice with cold phosphate buffered saline (PBS). Analysis was performed on a FACScan (EPICS ELITE ESP model; Beckman Coulter, Fullerton, CA), and data were analyzed with the Expo32 software (Beckman Coulter). Isotype-matched FITC- and PE-conjugated IgG were used as a control for nonspecific binding. The following antibodies were used in the experiments: FITC-conjugated anti-mouse Gr-1 (Cat. 11-5931; eBioscience, San Diego, CA), PE/Cy5-conjugated anti-mouse CD11b (Cat. 12-0112; eBioscience), Alexa Fluor® 488 Anti-mouse/rat/human FOXP3 Antibody (Cat. 320011; biolegend, San Diego, CA), PE/Cy5 Anti-mouse CD4 Antibody (Cat. 100409; biolegend), PE-conjugated anti-mouse CD25 (Cat. 102007; biolegend), FITC rat IgG2b isotype control (Cat. 11-4331; eBioscience), PE rat IgG2b isotype control (Cat. 12-4331; eBioscience), Alexa Fluor® 488 Mouse IgG1, κ Isotype Ctrl (Cat. 400133; biolegend), PE/Cy5 Rat IgG2b, κ Isotype Ctrl Antibody (Cat.400609; biolegend) and PE Rat IgG1, κ Isotype Ctrl Antibody (Cat. 400407; biolegend).

Vaccine preparation

HPV-16 mE6Δ/mE7/TBhsp70Δ was prepared as previously described.31 Briefly, the strain BL21 of Eschericia coli (DE3) was used as a host for the production of an N-terminal histidine-tagged mE6Δ/mE7/TBhsp70Δ fusion protein. The amount of the fusion protein in the culture broth reached its maximum 4 h after isopropyl β-D-1-thiogalactopyranoside induction. The purified protein was exchanged into 1× PBS and stored at −70°C.

Therapeutic tumor experiment protocol

On Day 0, mice were injected subcutaneously (s.c.) on the right flank with 1 × 105 TC-1 tumor cells. On Day 11, mice bearing tumor around 60 mm2 were arbitrarily assigned to eight groups as follows: the control group; ATRA group; CTX group; ATRA and CTX group; mE6Δ/mE7/TBhsp70Δ vaccine group; ATRA and vaccine bitherapy group; CTX and vaccine bitherapy group and ATRA; and CTX and vaccine combined tritherapy group. CTX was given intraperitoneally (i.p.) at a dose of 50 mg/kg on Day 8 after tumor challenge. This fusion protein was administered at 100 μg per mouse on Day 11 and then boosted 7 days later at the same dose. ATRA was provided at a dose of 2.5 mg/kg on Day 11 and continued for 14 days. Tumors were monitored every 3 days, and the survival of mice was recorded. Tumor dimensions were determined by measuring with calipers, and the values were inserted into the formula: tumor volume (mm2) = length × width. The number of deaths and moribund animals was assessed at each interval. Moribund animals were sacrificed. All the measurements were obtained in a strictly blinded fashion.

The tumor-free mice from the tritherapeutic group were s.c. rechallenged with 1 × 105 TC-1 cells/mouse on Day 120 after the initial tumor inoculation. As a control, naive mice were treated in the same way. After tumor inoculation, tumor incidence was monitored over time.

Cytotoxicity assay

Erythrocytes were removed by suspending the cells in lysis buffer. The splenocytes from each group were used as effector cells, and TC-1 cells were used as target cells. The Non-Radioactive Cytotoxicity Assay Kit (Promega, Madison, WI) was used to measure the effector cells that acted against TC-1 cells in the ratios of 20:1, 40:1 and 60:1, according to the manufacturer's protocol. Specific lysis was calculated according to the formula: percent specific lysis = [(experimental release value-effector spontaneous release value-target spontaneous release value)/(target maximum release value-target spontaneous release value)] × 100.

Enzyme-linked immunospot (ELISPOT) assay

Briefly, MultiScreen-HA plates (Millipore Corporation, Berford, MA) were precoated with anti-interferon (IFN)-γ-mAbs (BD PharMingen, San Diego, CA) in PBS overnight at 4°C. Splenocytes from immunized mice were plated in wells (3 × 105 cells/well) in complete media and cultured for 16 h at 37°C in the presence of 10 μg/ml HPV-16-derived E7 peptide (H-2Db-restricted, aa 49-57, RAHYNIVTF, purity >90%; Invitrogen, Shanghai, China) or OVA control peptide (H-2Kb-restricted, aa 257-264, SIINFEKL, purity >90%; Invitrogen). Wells were gently rinsed with PBS-containing 0.1% Tween20, then biotinylated anti-IFN-γ-mAbs (BD PharMingen) was added, and the plates were incubated at 37°C for 1 h. Results were visualized using avidin-alkaline phosphatase and 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium substrate (Sigma-Aldrich). The number of spots in each well was scored blindly by two investigators.

Immunohistochemical staining of tumors

Tumor-bearing mice were sacrificed when the control mice became moribund. Tumor tissues recovered from each group were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections were cut 4-μm thick and then were carried out and stained either by standard hematoxylin and eosin staining or immunohistochemical staining with mAbs specific for mouse IL-10 (Cat. BA1201; Boster, Wuhan, China), IL-6 (Cat. BA0562; Boster), VEGF (Cat. BA0407; Boster) and p-signal transducer and activator of transcription (STAT) 3 (Cat. 11045-1; SAB, Pearland, TX) using a standard Non-Biotin HRP detection system (Cat. PV6001; Zsbio, Beijing, China) following the manufacturer's instructions. Pictures were taken using an Olympus BH2 microscope (Olympus, Tokyo, Japan) with a camera (Coolsnap3.3; Photometrics, Tucson, AZ).

Statistical analysis

Microsoft Office Excel 2003 (Microsoft Inc, Redmond, Wash) and SPSS12.0 (SPSS Inc, Chicago, IL) were used for the statistical analyses. Data showing comparisons between two groups were assessed using the Student's t test. Comparisons among more than two groups were done using ANOVA with the appropriate post hoc testing. The χ2 test was used to compare the incidence rate. Prism software (GraphPad Software, San Diego, CA) was used to determine the significance of differences in survival curves with a log-rank test. Differences were considered significant when p < 0.05. Data are presented as average ± SD. Each experiment was performed in triplicate.

Results

MDSCs and Tregs were simultaneously suppressed by immune-modulating doses of ATRA and CTX in C57BL/6 mice bearing TC-1 tumors

To determine the mutual effect of CTX and ATRA on MDSCs and Tregs, we analyzed the proportion of Gr-1+CD11b+MDSCs and CD4+CD25+FoxP3 Tregs when administering immune-modulating doses of CTX on Day 8 and ATRA for 5 days starting on Day 9 after TC-1 tumor inoculation. The sizes of the tumors on Day 8 were 17.2 ± 12.8 mm2 in the control group, 15.2 ± 4.5 mm2 in the group of ATRA, 18.8 ± 11.9 mm2 in the group of CTX and 17.0 ± 10.9 mm2 in the group of ATRA plus CTX. The sizes of the tumors on Day 14 were 79.0 ± 19.5 mm2 in the control group, 78.0 ± 22.1 mm2 in the group of ATRA, 72.2 ± 7.6 mm2 in the group of CTX and 76.6 ± 22.2 mm2 in the group of ATRA plus CTX. There were no significant differences among these groups (Supporting Information Fig. 1).

Splenocytes and tumor-infiltrating mononuclear cells were assessed for the proportion of MDSCs and Tregs on Day 14. We found that mice bearing TC-1 carcinoma had a significantly elevated percentage of Gr-1+CD11b+ MDSCs (Figs. 1ac) and CD4+CD25+FoxP3+ Tregs (Figs. 1d–f) in the spleens and tumors of the control group compared with the normal (naive) mice (p < 0.05). ATRA primarily decreased the proportion of MDSCs in spleens and tumors (p < 0.05), whereas CTX primarily decreased Tregs in spleens (p < 0.01) and tumors (p < 0.05). In particular, the combination of ATRA and CTX suppressed both the MDSCs and Treg populations. The proportion of Gr-1+CD11b+MDSCs in the combined treatment were 5.7 ± 0.9% in spleens (p < 0.01) and 8.1 ± 0.2% in tumors (p < 0.05) compared with 13.5 ± 1.5% and 14.2 ± 2.3%, respectively, in the control group. The proportion of CD4+CD25+FoxP3/CD4+ lymphocytes in the combined treatment were 7.7 ± 1.6% in spleens and 11.9 ± 1.5% in tumors compared with 14.1 ± 1.8% and 31.2 ± 7.7%, respectively, in the control group (p < 0.05).

Figure 1.

The effect of ATRA and CTX on the proportion of MDSCs and Tregs. The normal group indicated the C57BL/6 mice without any treatment. The other mice were inoculated s.c. with 1 × 105 tumor cells/mouse. CTX was administrated i.p. at a dose of 50 mg/kg on Day 8, and ATRA was given i.p. at a dose of 2.5 mg/kg continuously for 5 days starting from Day 9. The control group indicated the mice injected with the solvent. No significant differences in tumor sizes were observed among these groups. Splenocytes and tumor-infiltrating mononuclear cells were harvested on Day 14 and were assessed for the proportion of MDSCs and Tregs. (a) Gating on forward and side scatter, larger cells were analyzed. (b) Representative dotplots of three individual experiments are shown. (c) The proportions of MDSCs in spleen lymphocytes and tumor infiltrating mononuclear cells in the indicated groups were shown. (d) Cells were gated for the CD4 expression. (e) Representative dotplots of three individual experiments are shown. (f) The proportions of CD4+CD25+FoxP3+/CD4+ cells in spleen lymphocytes and tumor-infiltrating mononuclear cells in the indicated groups were shown. *p < 0.05 compared with the normal group; #p < 0.05 and ##p < 0.01 compared with the control group. Each group included five mice. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Sequential immune-modulating doses of ATRA and CTX treatment dramatically improved the effect of the therapeutic HPV vaccine

In the period of 4 weeks observation for naive mice treated with CTX plus ATRA and the vaccine, we did not observe the autoimmune signs such as loss of the hair, weight or lymphopenia and other toxicities in the each group (Supporting Information Table 1). Then, we hypothesized that the strategy of CTX, ATRA and the HPV protein vaccine in sequence would overcome immune evasion and enhance the antitumor immune response. We, therefore, performed the combination tritherapy strategy, as described in “Material and Methods.” The tritherapy was significantly more effective at inhibiting tumor growth than the single treatments or bitherapy strategy and greatly enhanced the antitumor immunity generated by the vaccine. Representative tumor photographs of three individual experiments were shown in Supporting Information Figure 2. On Day 29, tumor growth was inhibited 25% in the vaccination group (254 ± 21 mm2) compared with the control group (339 ± 84 mm2, p < 0.05; Fig. 2a). No significant inhibition was observed in the ATRA-treated group or the CTX-treated group. Tumor growth in mice treated with the vaccine plus ATRA (104 ± 86mm2) or the vaccine plus CTX (93 ± 65 mm2) was also significantly delayed compared with the vaccination group (p < 0.01). Most importantly, six of seven mice (87.5%) in the tritherapy eradicated the tumors (Fig. 2a).

Figure 2.

Administering an HPV therapeutic vaccine in combination with ATRA and CTX resulted in maximal tumor regression and survival. C57BL/6 mice were inoculated s.c. with 1 × 105 tumor cells/mouse, and the tumors were allowed to grow to 60 mm2 before the mice received our vaccination protocol in addition to ATRA and CTX as described in Materials and Methods. (a) Tumor sizes (mm2). (b) Survival curve of mice. (c) Evaluation of the persistent antitumor response of the tritherapy. The tumor-free mice from the tritherapy group were s.c. rechallenged with 1 × 105 TC-1 cells/mouse on Day 120 after the initial tumor inoculation. As a control, naive mice were treated in the same way. Each group included 7–10 mice. Tumor incidence was monitored after tumor inoculation. **p < 0.01 compared with control group; #p < 0.05; and ##p < 0.01 compared with bitherapy group.

Furthermore, all animals in the control group and the vaccine group died within 40 and 60 days, respectively. When the vaccine was combined with either CTX or ATRA, 25% or 50% of mice survived, respectively, >90 days. Importantly, 100% of the mice in the tritherapy group achieved long-term survival, and 87.5% mice had complete tumor regression (Fig. 2b).

On Day 120, the tumor-free mice from the tritherapy group were rechallenged. There were ∼88% mice of the tritherapy still remained tumor-free by the end of the observation period. In contrast, all mice in the control group developed palpable tumors within 9 days (Fig. 2c). This result indicates that the tritherapy induced long-term memory responses.

ATRA and CTX given in sequence with the HPV therapeutic vaccine significantly primed potent E7-specific CD8+ T cell responses

To examine the ability of the tritherapy to generate Cytotoxic T Lymphocytes (CTLs), splenocytes were harvested on Day 28 and were assessed for cytotoxic activity against TC-1 cells (Fig. 3a). Although ATRA or CTX treatment had little effect on CTL activity, the vaccine increased the lysis activity of CTLs against TC-1 cells compared with the control group (31.1 ± 4.6% versus 6.1 ± 1.9% at a ratio of 60:1, p < 0.01; Fig. 3a). CTLs from the combined vaccine and ATRA group (50.6 ± 3.4%) and vaccine and CTX group (40.3 ± 2.4%) had even higher cytolysis ability. More importantly, CTLs from the tritherapy group had the highest CTL activity against the TC-1 cells at 60.3 ± 4.5%, which was significantly higher than the monotherapy (p < 0.01) or bitherapy (p < 0.05) groups. On Day 30, similar results were obtained in an ELISPOT assay that evaluated antigen-specific CTL precursors (Fig. 3b). Collectively, our data suggests that the combination of vaccine, ATRA and CTX generated E7-specific T-cell responses that were significantly higher than any other treatment.

Figure 3.

Specific CTL responses were enhanced by tritherapy in established tumors. C57BL/6 mice were inoculated s.c. with 1 × 105 tumor cells. On Day 28, splenocytes from control, vaccine, ATRA, CTX, ATRA plus CTX, vaccine plus ATRA, vaccine plus CTX and tritherapy groups were tested for (a) cytolytic activity against TC-1 cells and (b) the response of T cells to the specific E7 aa 49-57 or OVA control peptides as evaluated in an ELISPOT assay. **p < 0.01 compared with control group; #p < 0.05 and ##p < 0.01 compared with bitherapy group. Each group included at least three mice.

The efficacy of the tritherapy was rescued beyond the optimal initial time when more intensive regimens were employed with the reduced proportion of Tregs and MDSCs

To investigate the optimal time window, we initiated the tritherapy with CTX on Day 7 (Group 3), Day 10 (Group 2) or Day 13 (Group 1). The vaccination was performed at the corresponding Days 10, 13 or 16, when the tumor size was approximately 60 mm2, 75 mm2 or 100 mm2, respectively, and boosted 7 days later. ATRA was continuously given for 14 days, starting at the day of vaccination. We found that Group 3 represent viable regimens for curing TC-1 tumors when the tumor size is below 100 mm2 and there was no significant difference between Group 1 (375 ± 56 mm2) and the control group (435 ± 53 mm2) on Day 31 (Supporting Information Fig. 4).

The standard tritherapy regimen has a marginal effect, and no cure can be obtained when mice are treated with a more advanced (>100 mm2) lesions. We wondered whether repeated and more intensive regimens could rescue specific immunity and mediate more significant tumor inhibition. Thus, we initiated all the groups of tritherapy with CTX on Day 16 and defined the standard tritherapy group as Tritherapy 1, higher doses of ATRA (5 mg/kg) were used for Tritherapy 2 and both higher doses of ATRA and repeated CTX were given in Tritherapy 3 (Fig. 4a). The vaccination was performed at Day 19 and boosted on Day 26. On Day 34, the tumor growth in Tritherapy 3 (160.9 ± 42.6 mm2) was inhibited 63.2% compared with the control group (457.4 ± 59.6 mm2, p < 0.01). There was a tendency but no significant differences between Tritherapy 2 (346.7 ± 56.7 mm2) and Tritherapy 1 (437 ± 56.9 mm2). Thus, more intensive regimens represent optimal for large TC-1 tumors above 100 mm2 (Fig. 4b).

Figure 4.

More intensive tritherapy restored the effect of tritherapy beyond the optimal initial time with the reduced percents of Tregs and MDSCs. C57BL/6 mice were injected with TC-1 cells on Day 0. Tritherapies 1, 2 and 3 were all initiated on Day 16. At Day 34, mice were sacrificed, and tumors and spleens were harvested. (a) Diagram depicting the various tritherapy strategies initiated on Day 16. (b) The tumor growth curve with the different tritherapy strategies starting on Day 16. (c) Splenocytes and tumor-infiltrating mononuclear cells were analyzed by flow cytometry using anti-Gr-1, anti-CD11b antibodies. Gating on forward and side scatter, larger cells were analyzed. The percentages of Gr-1+CD11b+ cells of splenocytes and tumors from mice in each group are shown. (d) Splenocytes and tumor-infiltrating mononuclear cell were analyzed by flow cytometry using anti-FoxP3, anti-CD4 and anti-CD25 antibodies. We gated on CD4+ cells. **p < 0.01 compared with control group; #p < 0.05 and ##p < 0.01 compared with tritherapy 2.

On Day 34, we found no significant difference in the proportion of MDSCs and Tregs in spleens and tumors between the control group, Tritherapies 1 and 2, which suggested that higher dose of ATRA alone were not enough to enhance the immunity of the standard tritherapy. The percentage of MDSCs and Tregs in spleens and tumors was dramatically reduced in Tritherapy 3 compared with Tritherapy 1 (p < 0.01), which might indicate repeated CTX and higher dose of ATRA together can largely promote the immunity of Tritherapy 1 with the creation of favorable tumor microenvironment.

The enhanced tritherapy regimen reduced the production of the immunosuppressive cytokines in the tumor niche and STAT3 activation in the tumors

To further study whether the different tritherapy regimens affect the immunosuppressive signals in the tumor microenvironment, tumors were harvested from different tritherapy strategies on Day 34 as we described above, and we examined IL-10, IL-6 and VEGF in the tumor niche and (STAT) 3 expression (Fig. 5). We found high levels of IL-10, IL-6, VEGF and p-STAT3 expression in the control group and Tritherapy 1. When intensive regimens were employed in Tritherapy 3, the levels of IL-10, IL-6 and VEGF were reduced whereas STAT3 activation was decreased. The results suggest that the effect of tritherapy was largely enhanced with low levels of IL-10, IL-6 and VEGF in the tumor microenvironment and p-STAT3 in TC-1 tumors with the enhanced strategy.

Figure 5.

The examination of suppressive factors in the tumor and tumor niche from all the tritherapy groups at Day 34. Tumors were harvested from the tritherapy groups, fixed and stained for the expression level of IL-10, VEGF, IL-6 and p-STAT3 in the groups at Day 34 (magnification, ×200). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Discussion

Tumor microenvironment comprises proliferating tumor cells, the tumor stroma, blood vessels, infiltrating inflammatory cells and a variety of associated tissue cells. Accumulations in tumors of Tregs and MDSCs are common features of human tumors, and the frequency as well as suppressor activity of these cells have been linked to poor prognosis in patients with cancer.4 Therefore, diminishing Tregs as well as MDSCs in favor of the host immune response might result in therapeutic benefits. However, most of the research focused on only one single population to increase the effect of the immunotherapy except a few reports,32, 33 and it is hard to affect the immunosuppressive network; to our knowledge, we are the first to target both the proportion of MDSCs and Tregs with a simple method using ancient drugs for new combinations to create favorite tumor microenvironment for advanced TC-1 tumor lesions.

It has been reported that combination of granulocyte colony-stimulating factor, ATRA and CTX for acute myelogenous leukemia achieved complete remission.34 ATRA followed by intensive chemotherapy gives a high complete remission rate in acute promyelocytic leukemia.35 The combination of CTX, ATRA and other drugs appears favorable for chronic myelogenous leukemia.36 Given the combined effect elicited by ATRA and CTX in the field of leukemia, however, it is still unclear whether ATRA plus CTX could be combined with the immunotherapy and applied to solid tumors. Although Mirza et al. and we did not find increased the percentage of Tregs after the addition of ATRA, however, Tregs still were high in the model and ATRA might not able to restore the maximal efficacy of the immunotherapy alone. We, thus, hypothesized that ATRA and CTX in immune-modulating doses could synergistic enhance the immune response.

Mokyr et al.37 demonstrated that the timing between the vaccine injection and CTX administration is crucial to potentiate the antitumor immune response. Kusmartsev et al. and our previous study demonstrated that it was necessary to continuously administer ATRA pellets or injections i.p. to have an efficient therapeutic effect.18, 20 A single administration of CTX controls the regulatory component of the immune system during the priming phase of vaccination. Consistent ATRA treatment favors effector functions by blocking the induction of the regulatory component of the immune system during the effector phase of the immune response. On this basis, we determined the administration strategy for a sequential tritherapy in this study.

Immune-modulating doses of CTX combined with ATRA could act through a mechanism of selective effect against Tregs and MDSCs, respectively, and we did not observe the autoimmune signs such as loss of the hair, weight or lymphopenia and other toxicities in each group. The subsequent tritherapy of CTX and ATRA at immune-modulating doses along with the HPV therapeutic vaccine given sequentially was very promising, and all the mice achieved long-term survival for 3 months and 87.5% mice eradicated the tumors, thus greatly enhancing the immune response generated by the vaccine. The tritherapy generated significant increase in functional E7-specific T cells with elevated IFN-γ secretion and enhanced cytotoxic T-cell activity. We also found CTX and ATRA sequentially, at a modulating dose, enhanced the effect of other profiles of immunotherapy, including peptide vaccination and adoptive therapy. These results suggest that modulating doses of ATRA combined with CTX could enhance the efficacy of other profiles of HPV-associated immunotherapy (Supporting Information Fig. 3).

One interesting question that remains to be studied is the optimal regimen. We performed the tritherapy with different initial time and found Group 3 represented the optimal regimen for curing TC-1 tumors with the dramatically decreased proportion of MDSCs and Tregs of spleens and tumors. The effect of tritherapy declined sharply when the tumor size was above 100 mm2 with high percentages of MDSCs and Tregs even with the application of CTX and ATRA. Studies revealed that the immune system is confronted with persistent exposure to tumor antigens in the late stages of tumors and Tregs and MDSCs may readily expand in vivo after vaccination.13, 38, 39 The immune tolerance seemed very formidable, so the effect of CTX and ATRA in the schedule seemed very limited at this complicated situation. So,we performed intensive regimens with more frequency of CTX and higher doses of ATRA for advanced TC-1 tumors. The strategy of Tritherapy 3 significantly restored the effect of Tritherapy 1, but the tumors could hardly be eradicated. Thus, more intensive regimens represent viable options for advanced TC-1 tumors, and the initial therapeutic time seemed more important for the best therapeutic potential.

The immunosuppressive network in the tumor microenvironment is created by the pathological interactions between cancer cells and host immune cells and promotes tumor growth, protects the tumor from immune attack and attenuates therapeutic efficacy. Modulation of the level cytokines such as IL-6, IL-10 and VEGF is important for controlling immune cell infiltration and ultimately tumor growth.40 STAT3 is a point of convergence for numerous oncogenic signalling pathways and constitutively activated in the tumor microenvironment.29, 41 The constitutive activation of STAT3 can be propagated, in part through STAT3-regulated factors such as VEGF and IL-10, which generates immunosuppression involving both innate and adaptive immunity.42–44

We found TC-1 tumor microenvironment, filled with a large number of MDSCs and Tregs and a large variety of immunosuppressive products such as VEGF, IL-10 and IL-6 secreted, resulting in continuous STAT3 activated, which were consistent with the literatures.45–47 We also found the therapeutic efficacy with different strategies was closely associated with the suppressive factors in TC-1 tumor microenvironment. The levels of these suppressive factors may be predictive markers for animals and patients. Once the suppressive factor increase in the tumor microenvironment, it is implying that more intensive strategies or other proper treatments may be needed. Increasing understanding of the molecular mechanisms that hinder immune attack in the tumor microenvironment will lead to the development of new therapeutic approaches.

The addition of chemotherapy to immunotherapy has shown evidence of efficacy in some preclinical models and in the clinical setting, and exploiting these two modalities safely and effectively remains an ongoing challenge. In this study, we presented a novel potent tritherapy of ATRA, CTX and a fusion protein vaccine and showed that it was able to eradicate TC-1 tumors when administered in an optimized regimen in TC-1 murine model. The addition of ATRA and CTX is feasible and well tolerated, and they are familiar to clinicians.17 Taken together, our data shed light on the sequential combined chemoimmunotherapeutic strategy resulting in a beneficial host microenvironment, and it will be important to test this combination strategy for cancers that express defined tumor antigens in rationally designed clinical trials.

Acknowledgements

We thank Mr. Jietao Song and Mr. Baochun Duan for help with animal care.

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