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. 2009 Nov 26;59(5):769–777. doi: 10.1007/s00262-009-0797-1

Immunogenic chemotherapy with cyclophosphamide and doxorubicin against established murine carcinoma

Miki Tongu 1,2, Nanae Harashima 1, Takaya Yamada 2, Takayuki Harada 3, Mamoru Harada 1,✉
PMCID: PMC11030296  PMID: 19940990

Abstract

Mitigation of regulatory T cell-mediated immunosuppression and elicitation of immunogenic tumor cell death are crucial events for optimal anti-tumor immune activity in vivo. This study was designed to investigate the potential synergistic activity of the combined use of cyclophosphamide (CP) and doxorubicin (DR), both of which are known to resolve these two issues. BALB/c mice were inoculated subcutaneously with CT-26 carcinoma cells in the bilateral flank and treated with an intraperitoneal injection of a low dose of CP followed by an intratumoral injection of DR into one side of the tumor. We found that, in addition to a significant suppression of growth on the DR-treated side of the tumor, combination therapy suppressed the growth of DR-untreated remote tumors in both tumor-specific and T cell-dependent manners. Mitomycin C showed no such synergistic anti-tumor activity with CP treatment. Combination therapy increased the frequency of interferon (IFN)-γ-producing T lymphocytes specific to a CT-26-associated class I-binding tumor peptide in the tumor-draining lymph nodes. Real-time PCR analysis revealed that combination therapy led to an increase in IFN-γ and tumor necrosis factor-α mRNA expression; however, levels of Foxp3 and transforming growth factor-β within the remote tumor tissues were decreased. In addition, knock down of calreticulin expression in CT-26 cells using small interfering RNA attenuated anti-tumor vaccine effects induced by DR-treated CT-26 cells. These results provide an immunological rationale for the combined use of chemotherapeutic drugs, i.e., CP and DR, and further recommend their use with current cancer vaccines.

Keywords: Cyclophosphamide, Doxorubicin, Regulatory T cells, Calreticulin

Introduction

Because the immune system is capable of recognizing tumor antigens, cancer vaccines are an attractive approach to treating cancer patients [1, 2]. Various types of cancer vaccines have been used clinically [3–6]; however, evaluation of their clinical efficacy thus far has been limited [7]. Accordingly, further improvements in therapy modalities are required.

Recent advances in tumor immunology have discovered crucial mechanisms that must be overcome for optimal anti-tumor immune activity in vivo. First, tumor-bearing states typically involve several types of immunosuppression via immunosuppressive cells such as CD4+ CD25+ regulatory T cells (Treg) and/or myeloid-derived suppressor cells [8, 9]. Specifically, Tregs have received a great deal of attention as suppressive cells in tumor-bearing patients. Further, their presence at local tumor sites correlates with unfavorable prognosis [10, 11]. Although several methods such as treatment with antibodies can relieve Treg-mediated immunosuppression [12–14], a number of reports have revealed that cyclophosphamide (CP) can mitigate Treg-mediated immunosuppression when administered at a low dose [15–20]. Second, presentation of tumor-derived antigens by dendritic cells (DCs) to T cells is a critical step for in vivo elicitation of anti-tumor T cell immunity [21], and some anti-cancer drugs such as anthracyclines are known to exploit this process [22]. In anthracycline-treated dying tumor cells, calreticulin, which is constitutively expressed in the endoplasmic reticulum, migrates to the cell surface, provides phagocytic (i.e., ‘eat me’) signals to DCs, and consequently promotes their uptake [23]. Simultaneously, the dying tumor cells secrete high-mobility group box 1 (HMGB1) protein as a ‘danger’ signal to DCs, resulting in the efficient processing and cross-presentation of tumor antigens by DCs [24]. This immunogenic tumor cell death is crucial for treatment-associated prognoses and for the survival of tumor-bearing hosts [25, 26].

In this study, we investigated the potential synergistic activity of the combined use of CP and doxorubicin (DR) (as an anthracycline drug), against established murine carcinoma cells. Our findings suggest that combination therapy can synergistically elicit anti-tumor immune activity in vivo. These results provide an immunological rationale for the combined use of CP and DR and further recommend their use with current cancer vaccines.

Materials and methods

Mice and tumor cell lines

BALB/c and BALB/c nu/nu female mice (H-2d: 6–7 weeks old) were purchased from CLEA Japan Inc. (Tokyo, Japan) and Japan SLC Inc. (Hamamatsu, Japan), respectively. They were kept under specific pathogen-free conditions. Experiments were performed according to the ethical guidelines for animal experiments of the Shimane University Faculty of Medicine. CT-26 and RENCA are colon carcinoma and renal cell carcinoma cell lines of BALB/c (H-2d) mouse origin, respectively. They were maintained in RPMI 1640 supplemented with 10% fetal bovine serum.

Combination therapy protocol

BALB/c mice were injected subcutaneously (s.c.) with 2 × 105 CT-26 cells into the right flank and with 2 × 105 CT-26 cells or 2 × 105 RENCA cells into the left flank. On day 10, the mice received an intraperitoneal (i.p.) injection of CP (Shionogi Co. Ltd, Osaka, Japan) at a dose of 100 mg/kg. On days 12, 14, and 16, the mice were injected intratumorally (i.t.) into the right-side tumor with either 120 μg of DR (Adriamycin; Kyowa Hakko Co. Ltd, Tokyo, Japan) or 100 μg of mitomycin-C (MMC; Kyowa Hakko Co. Ltd) at a volume of 40 μl. After tumor inoculation, tumor size (mm2) was measured twice weekly.

Real-time PCR

Total RNA was isolated with TRIzol reagent (Invitrogen Corp., Carlsbad, CA) according to the manufacturer’s instructions. First-strand cDNA was generated using the Superscript III First-Strand Synthesis System (Invitrogen) and random primers. The synthesized first-strand cDNA was amplified using Platinum Tag DNA polymerase (Invitrogen) with EXPRESS SYBR GreenER qPCR SuperMixes (Invitrogen). Real-time PCR was carried out in duplicate using the ABI PRISM 7000 Sequence Detection System. Thermal cycling included an initial denaturation step of 2 min at 95°C, followed by 40 cycles of 95°C for 15 s, and 60°C for 1 min. Relative mRNA levels as compared with β-actin were calculated. The following primers (sense and antisense, respectively) were used for interferon (IFN)-γ: 5′-TCAAGTGGCATAGATGTGGAAGAA-3′ and 5′-TGGCTCTGCAGGATTTTCATG-3′; for tumor necrosis factor (TNF)-α: 5′-CATCTTCTCAAAATTCGAGTGACAA-3′ and 5′-TGGGAGTAGACAAGGTACAACCC-3′; for Foxp3: 5′-TGCAGGGCAGCTAGGTACTTGTA-3′ and 5′-TCTCGGAGATCCCCTTTGTCT-3′; for transforming growth factor (TGF)-β1: 5′-AAACGGAAGCGCATCGAA-3′ and 5′-GGGACTGGCGAGCCTTAGTT-3′; and for β-actin: 5′-AGAGGGAAATCGTGCGTGAC-3′ and 5′-CAATAGTGATGACCTGGCCGT-3′.

In vitro culture of tumor-draining lymph node cells, ELISA, and ELISPOT

To test specific T cell responses against a tumor antigen, an H-2Ld-binding peptide (SPSYVYHQF) derived from the envelope protein (gp70) of an endogenous murine leukemia virus was used. This specific peptide is referred to as a CT-26-associated tumor-derived peptide [27] and is designated AH1. As an H-2Ld-binding control peptide, measles virus hemagglutinin (SPGRSFSYF) was used. All peptides showed >80% purity and were purchased from Invitrogen Corp. For enzyme-linked immunosorbent assay (ELISA), tumor-draining lymph node cells were harvested, pooled, and stimulated in vitro with the indicated peptides in the presence of 20 U/ml interleukin (IL)-2 for 3 days at a cell dose of 5 × 105 cells/well in 96-well flat plates. To determine the levels of IFN-γ in the culture supernatants, an ELISA MAX™ Set Deluxe (BioLegend, San Diego, CA) was used. For the enzyme-linked immunosorbent spot assay (ELISPOT), tumor-draining lymph node cells were harvested, pooled, and then cultured for ELISPOT in the presence of the indicated peptide, without IL-2, for 3 days at a cell dose of 5 × 105 cells/well. To determine the frequency of IFN-γ-producing cells in the tumor-draining lymph node cells, HRP ELISpot Kit (KPL Inc., Gaithersburg, MD) was combined with coating and detecting anti-mouse IFN-γ antibodies contained in the ELISA MAX™ Set Deluxe. The numbers of spots were quantified under microscopy. Each data point consisted of three wells.

In vitro analysis of CD4+ Treg-mediated immunosuppression

To examine an effect of CP and/or DR on CD4+ Treg, purified CD4+ T cells (5 × 104) from the tumor-bearing mice that were treated with or without CP and/or DR were cultured with whole naïve lymph node cells (5 × 104) in the presence of Mouse T-Activator CD3/CD28 (Invitrogen Dynal AS, Oslo, Norway) in 96-well flat plates for 3 days. CD4+ T cells were negatively isolated from the tumor-draining lymph node cells using Dynal Mouse CD4 Negative Isolation Kit (Invitrogen Dynal AS). The cell proliferation was assayed with standard thymidine ([methyl-3H]TdR) incorporation and scintillation counting. During the last 6 h of the culture, 12 kBq of [3H]TdR were added to each well. The levels of IFN-γ in the culture supernatants were determined by the ELISA MAX™ Set Deluxe.

Immunoblot

Cell lysates were prepared using a M-PER mammalian protein extraction reagent (Pierce Biotechnology, Rockford, IL), which contains a protease inhibitor cocktail (Nacalai Tesque, Kyoto, Japan) and 5 mM EDTA (pH 8.0), according to the manufacturer’s instructions. After centrifugation, proteins in the supernatant were loaded onto SDS-PAGE NuPAGE 4–12% Bis-Tris gels, transferred to the PVDF membrane using the iBlot dry blotting system (Invitrogen), and incubated with 1× EzBlock buffer (ATTO, Tokyo, Japan) in TBS-Tween for 60 min. After incubation with either anti-calreticulin (StressMarq Bioscience, Victoria, BC, Canada) or anti-β-actin (BioLegend) antibodies, membranes were incubated with the appropriate alkaline phosphatase-conjugated secondary antibodies and were developed by chemiluminescence (Invitrogen).

Protective models

To compare protective anti-tumor activity induced by vaccination with either DR-treated or MMC-treated CT-26 cells, BALB/c mice were injected s.c. with 2 × 105 CT-26 cells into the right flank and simultaneously vaccinated s.c. with DR-treated or MMC-treated 1 × 106 CT-26 cells into the left flank. To inactivate tumor cells, CT-26 cells were cultured in vitro with DR (20 μg/ml) or MMC (10 μg/ml) for 24 h. As a protective model, BALB/c mice were vaccinated s.c. into the left flank with DR-treated 1 × 106 CT-26 cells that were transfected with siRNA. On day 14, the mice were inoculated s.c. with 2 × 105 CT-26 cells into the right flank.

Transfection of siRNA

Transfection of small interfering RNA (siRNA) was performed using X-tremeGENE siRNA Transfection Reagents (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Both calreticulin siRNA and control siRNA were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Six days after transfection, tumor lysates were used for immunoblot. For vaccination with transfected CT-26 cells, DR was added into the culture bottle at a dose of 20 μg/ml 5 days after the transfection and cultured for an additional 24 h.

Statistics

Data were statistically evaluated using unpaired two-tailed Student’s t tests. A P value of less than 0.05 was considered statistically significant.

Results

Anti-tumor effects of combination therapy with CP and DR against s.c. established CT-26

We first determined whether combination therapy with CP and DR would show synergistic anti-tumor activity against s.c. established CT-26 colon carcinoma cells. The tumor growth of each mouse is shown in Fig. 1a, and growth on day 21 is summarized in Fig. 1b. The i.p. injection of CP significantly suppressed bilateral CT-26 growth. I.t. injection of DR led to a slight suppression in growth of the CT-26 cells on the DR-treated side; no effects on remote CT-26 cells were observed. Combination therapy significantly suppressed growth of the CT-26 cells on the DR-treated side. The growth of remote CT-26 cells was also suppressed by approximately 50% compared to CP alone; however, this difference was not statistically significant. In contrast, combination therapy failed to show any synergistic effect on remote RENCA.

Fig. 1.

Fig. 1

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Anti-tumor effects induced by combination therapy with CP and DR. a BALB/c mice were injected s.c. with 2 × 105 CT-26 cells into the right flank and with 2 × 105 CT-26 cells or 2 × 105 RENCA cells into the left flank. On day 10, mice were injected i.p. with CP (100 mg/kg). On days 12, 14, and 16, mice were injected i.t. with DR (120 μg) in a volume of 40 μl into the right tumor. Tumor size (mm2) was measured twice weekly. Each group consisted of five or six mice, and each line represents the tumor growth of one mouse. Arrows and arrowheads represent the injections of CP and DR, respectively. b The mean ± SD of the results on day 21 after tumor inoculation are shown. *P < 0.05 and **P < 0.01 indicate statistical significance. NS Not significant

We next combined CP treatment with another cytotoxic drug, MMC (Fig. 2). Although i.t. injection of MMC significantly suppressed growth of the CT-26 cells on the MMC-treated side, no synergistic effects with CP were observed. More importantly, combination therapy showed no synergistic effects on the growth of remote CT-26 cells.

Fig. 2.

Fig. 2

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No synergistic anti-tumor effects were observed when CP and MMC were combined. a BALB/c mice were injected s.c. with 2 × 105 CT-26 cells into the flank bilaterally. On day 10, mice were injected i.p. with CP (100 mg/kg). On days 12, 14, and 16, mice were injected i.t. with MMC (100 μg) in a volume of 40 μl into the right tumor. Tumor size (mm2) was measured twice weekly. Each group consisted of five or six mice. b The mean ±SD of results on day 21 after tumor inoculation are shown. *P < 0.05 and **P < 0.01 indicate statistical significance

We further performed combination therapy on CT-26-bearing BALB/c nu/nu mice (Fig. 3). The i.p. injection of CP alone suppressed the growth of bilateral CT-26. In sharp contrast to results obtained with BALB/c mice, the i.t. injection of DR significantly suppressed the growth of the CT-26 cells on the DR-treated side. The anti-tumor effects of combination therapy on CT-26 cells on the DR-treated side were similar to levels induced by the DR therapy alone. In contrast, DR treatment had no effect on remote CT-26 cells, and combination therapy also failed to show any synergistic effects against remote CT-26 cells. Taken together, these results indicate that combination therapy with CP and DR can suppress the growth of the DR-treated side tumor and also can elicit systemic tumor-specific T cell immune responses, resulting in growth suppression of the remote tumor.

Fig. 3.

Fig. 3

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T cell-dependent anti-tumor activity induced by combination therapy. a BALB/c nu/nu mice were injected s.c. with 2 × 105 CT-26 cells into the flank bilaterally. On day 10, mice were injected i.p. with CP (100 mg/kg). On days 12, 14, and 16, mice were injected i.t. with DR (120 μg) in a volume of 40 μl into the right tumor. Tumor size (mm2) was measured twice weekly. Each group consisted of five mice. b The mean ± SD of the results on day 21 after tumor inoculation are shown. *P < 0.05 and **P < 0.01 indicate statistical significance

Tumor antigen-specific and class I-restricted T cells in mice treated with combination therapy

We also attempted to detect tumor-specific T cell responses in CT-26-bearing mice that were treated with combination therapy. Because in vitro cultures of spleen and lymph node cells with inactivated CT-26 cells were not able to induce tumor-specific T cell responses (data not shown), we tested the responses to an H-2Ld-binding peptide derived from the envelope protein (gp70) of an endogenous murine leukemia virus, which is known as a CT-26-associated tumor-derived peptide and is designated AH1 [27]. The tumor-draining lymph node cells from mice of each group were pooled and in vitro cultured for 3 days with each of the indicated peptides in the presence of 20 U/ml IL-2 (Fig. 4a). As a result, AH1 peptide-specific IFN-γ production was observed only in lymph node cells from mice treated with combination therapy; however, background IFN-γ production was relatively high. We also cultured spleen cells with the AH1 peptide in the presence of 20 U/ml IL-2 and found no apparent AH1 peptide-specific IFN-γ production (data not shown). Therefore, we next performed an ELISPOT assay to examine the frequency of AH1 peptide-specific T cells (Fig. 4b). The tumor-draining lymph node cells were cultured with the AH1 peptide without IL-2 for 3 days. Although peptide-specific IFN-γ production was observed in the groups that were treated with either CP or DR alone, the tumor-draining lymph node cells from the combination treatment group produced a higher level of IFN-γ in response to the AH1 peptide compared with other groups. Because AH1 is an H-2Ld-binding peptide, these results indicate that combination therapy can efficiently induce tumor antigen-specific and class I-restricted T cells in tumor-draining lymph nodes.

Fig. 4.

Fig. 4

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Induction of AH1 peptide-specific T cells in tumor-draining lymph nodes by combination therapy. BALB/c mice were injected s.c. with 2 × 105 CT-26 cells into the right flank. On day 10, mice were injected i.p. with CP (100 mg/kg). On days 12 and 14, mice were injected i.t. with DR (120 μg) in a volume of 40 μl. a On day 16, tumor-draining lymph nodes were harvested, and pooled cells were stimulated in vitro with each of the indicated peptides (open no peptide, stripe AH1 peptide, closed control peptide) in the presence of 20 U/ml IL-2. After 3 days of culture, levels of IFN-γ in the supernatant were determined by ELISA. *P < 0.05 indicates statistical significance. b On day 16, tumor-draining lymph nodes were harvested, pooled, and then cultured with each of the indicated peptides (open no peptide, stripe AH1 peptide, closed control peptide) without IL-2. After 3 days of culture, the frequency of IFN-γ-producing T cells was determined by ELISOPT. The number of spots was quantified under a microscopy. Each data point consists of three wells. *P < 0.05 indicates statistical significance

Combination therapy can restore mRNA expression of IFN-γ and TNF-α and mitigate immunosuppression in remote tumor sites

Many studies have already shown that the administration of a low dose of CP into tumor-bearing hosts can diminish Treg and consequently mitigate immunosuppression [15–20]. We also attempted to confirm an effect of CP on Treg-mediated immunosuppression (Fig. 5). We compared an immunosuppressive activity of tumor-draining lymph node cells from the mice that were treated with or without CP and/or DR. The results were that CP treatment significantly relieved immunosuppressive capacity of CD4+ T cells in the tumor-draining lymph node cells, resulting in restored proliferation and IFN-γ production by lymphocytes in response to anti-CD3/CD28 stimulation. Although DR treatment alone failed to relieve the immunosuppressive capacity, the CP-induced restoration of immune reactivity was significantly augmented when combined with DR treatment.

Fig. 5.

Fig. 5

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Effect of CP and/orDR on CD4+ Treg-mediated immunosuppression. BALB/c mice were injected s.c. with 2 × 105 CT-26 cells into the right flank. On day 10, mice were injected i.p. with CP (100 mg/kg). On days 12 and 14, mice were injected i.t. with DR (120 μg) in a volume of 40 μl. On day 16, tumor-draining lymph nodes were harvested, and purified CD4+ T cells from each group were cultured with whole naïve lymph node cells in the presence of Mouse T-Activator CD3/CD28 in 96-well flat plates for 3 days. The cell proliferation was assayed with standard [3H]TdR incorporation and scintillation counting. The levels of IFN-γ in the supernatant were determined by ELISA. Each data consists of three wells. *P < 0.05 and **P < 0.01 indicate statistical significance

We next focused on tumor sites because immune responses in the tumor sites are critical for anti-tumor activity in vivo. On day 16, the remote CT-26 cells were removed separately, and mRNA expression of cytokines in each sample was examined by real-time PCR (Fig. 6). We found that CP treatment recovered mRNA expression of IFN-γ and significantly decreased the expression of Foxp3 and TGF-β. The i.t. injection of DR resulted in a decreased mRNA expression of Foxp3, but no definite change was observed in the mRNA expression of other cytokines. In contrast, combination therapy significantly increased the mRNA expression of IFN-γ and TNF-α and suppressed the expression of Foxp3 and TGF-β. These results indicate that combination therapy can restore local production of IFN-γ and TNF-α and also mitigate immunosuppression in remote tumor sites.

Fig. 6.

Fig. 6

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Effects of combination therapy on cytokine and Foxp3 mRNA expression in remote tumor sites. BALB/c mice were injected s.c. with 2 × 105 CT-26 cells into the bilateral flank. On day 10, the mice were injected i.p. with CP (100 mg/kg). On days 12 and 14, the mice were injected i.t. with DR (120 μg) in a volume of 40 μl into the right-side tumor. On day 16, the DR-untreated left-side (remote) tumor tissues were removed separately, and real-time PCR was performed using their RNA. Each group consisted of four mice, and the mean ± SD is shown. *P < 0.05 and **P < 0.01 indicate statistical significance

Partial role of calreticulin in anti-tumor effects induced by vaccination with DR-treated CT-26

As described in “Introduction”, DR can induce immunogenic tumor cell death. Further, calreticulin participates in this process [23]. We compared protective anti-tumor activity after simultaneous vaccination with either DR-treated or MMC-treated CT-26 cells (Fig. 7a). As expected, vaccination with DR-treated CT-26 cells induced more potent protective immunity than did vaccination with MMC-treated CT-26 cells. Next, we tested the participation of calreticulin in the anti-tumor effects induced by combination therapy. Figure 7b shows that protein expression of calreticulin in CT-26 cells is suppressed by transfection with calreticulin siRNA. In a protective model, although all seven mice rejected the CT-26 challenge when they were pre-vaccinated with control siRNA-transfected and DR-treated CT-26 cells, three of seven mice failed to reject challenged CT-26 when they were pre-vaccinated with calreticulin siRNA-transfected and DR-treated CT-26 cells (Fig. 7c). These results suggest that calreticulin plays a partial role in anti-tumor immune activity induced by combination therapy with CP and DR.

Fig. 7.

Fig. 7

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Partial role of calreticulin in anti-tumor effects induced by combination therapy with CP and DR. a BALB/c mice were injected s.c. with 2 × 105 CT-26 cells into the right flank and simultaneously vaccinated s.c. with DR-treated or MMC-treated 1 × 106 CT-26 cells into the left flank. Each group consisted of seven mice. *P < 0.05 indicates statistical significance. b CT-26 cells were transfected with calreticulin or control siRNA. Six days after transfection, the CT-26 cells were harvested, and immunoblot was performed. c As a protective model, BALB/c mice were injected s.c. in the left flank with DR-treated 1 × 106 CT-26 cells that were transfected with calreticulin or control siRNA. On day 14, mice were inoculated s.c. with 2 × 105 CT-26 cells into the right flank. Each group consisted of seven mice. The number in parentheses denotes tumor-rejected mice/total mice

Discussion

Chemotherapy is the most frequent treatment modality for cancer patients. Broadly speaking, its anti-tumor effects in vivo are dose dependent, and the clinically admissible dosage is the maximum dose at which patients can tolerate its adverse effects. When chemotherapeutic drugs are administered to cancer patients at a high dose, remarkable tumor regression can be induced; however, myelosuppressive side effects and immunosuppression are inevitable. In contrast, some chemotherapeutic drugs are known to harness the host’s immune system to fight against cancer. CP is a representative of this drug group and can mitigate Treg-mediated immunosuppression when administered at a low dose [15–20]. In fact, we confirmed that a low dose of CP can relieve CD4+ Treg-mediated immunosuppression and restore lymphocyte proliferation and IFN-γ production (Fig. 5). In addition, Gemicitabin can enhance anti-tumor immunity in tumor-bearing hosts through selective elimination of myeloid-derived suppressor cells [28, 29], and Taxanes, inducing decetaxel, are reported to enhance cell-mediated anti-tumor activity and CD8+ T cell function when combined with cancer vaccines [30, 31]. Finally, anthracycline drugs can induce immunogenic tumor cell death [22, 23] and can kill tumor cells while helping DCs uptake tumor-derived antigens [22]. During this process, calreticulin on the cell surface and secreted HMGB1 protein provide ‘eat me’ and ‘danger’ signals to DCs, respectively [23, 24]. In this study, we attempted to investigate the potential synergistic activities of combined CP and DR use, as an anthracycline drug, against established murine carcinoma cells.

Combination therapy with CP and DR led to anti-tumor activity not only against the DR-treated side tumor, but also against the DR-untreated remote tumor (Fig. 1). Because synergistic activities were not observed either when CT-26 cells and irrelevant RENCA were injected s.c. into each side (Fig. 1a) or when BALB/c nude mice were used (Fig. 3), the induced anti-tumor activity was tumor-specific and T cell dependent. In addition, combination therapy significantly increased the frequency of tumor antigen-reactive and class I-restricted T cells in the tumor-draining lymph nodes (Fig. 4). Furthermore, combination therapy tended to restore local production of IFN-γ and TNF-α and mitigated Treg-mediated immunosuppression within the tumor tissues (Fig. 6). Overall, these results provide an immunological rationale for the combined use of the chemotherapeutic drugs, CP and DR.

Combination therapy restored local production of IFN-γ and TNF-α within the tumor tissues (Fig. 6). We have not determined what types of T cell subsets produced these cytokines. However, considering that combination therapy increased the frequency of IFN-γ-producing and tumor antigen-derived peptide-reactive CD8+ T cells in the tumor-draining lymph nodes (Fig. 4), such CD8+ T cells would accumulate at the tumor sites. In spite the absence of direct data, we hypothesize that combination therapy augmented the response of Th1-type CD4+ T cells, which migrated into tumor sites.

Cell death can be classified into two categories: immunogenic and tolerogenic [32]. In treating cancer, immunogenic tumor cell death is preferable to tolerogenic, and anthracycline drugs have been reported to induce immunogenic cancer cell death [25, 26]. Immunogenic tumor death seems to elicit anti-tumor immune response in vivo. Obeid et al. [23] recently reported that DR-treated dying tumor cells showed an increase in calreticulin transport to the cell surface, and uptake by antigen-presenting cells, such as DC, was promoted. We confirmed the participation of calreticulin in the anti-tumor activity induced by combination therapy with CP and DR. In a protective model, tumor rejection rates decreased when mice were pre-immunized with DR-treated CT-26 cells that were transfected with calreticulin siRNA (Fig. 7c). Although a difference in tumor rejection rate was small, these results suggest a partial role for calreticulin in anti-tumor immune activity induced by combination therapy with CP and DR. In addition, recent report revealed that HMGB1 released form dying tumor cells can provide danger signal to DCs [24]. Furthermore, Ghiringhelli et al. [33] revealed that ATP released from dying tumor cells acts on purinergic receptors on DCs and induces IL-1β-dependent anti-tumor T cell immunity. We did not test the possibility that HMGB1 and ATP might participate in anti-tumor activity induced by the combined therapy, we would like to test this possibility in the future study.

In contrast to a slight decrease in tumor growth as seen with DR, a local injection of MMC significantly suppressed the growth of s.c. established CT-26 cells (Fig. 2). However, when combined with CP treatment, MMC showed no synergistic effects on tumor growth of the remote CT-26 cells on the DR-untreated side. In addition, simultaneous immunization of MMC-treated CT-26 cells showed inferior anti-tumor effects as compared with that of DR-treated CT-26 cells (Fig. 7a). These results suggest that MMC cannot induce immunogenic tumor cell death. The differences observed must be due to the potential to induce cell surface expression of calreticulin; DR-treated, but not MMC-treated, CT-26 cells expressed calreticulin on their cell surface [23]. These results imply that compatibility of chemotherapeutic drugs should be considered from an immunotherapeutical point of view.

The treatment with both CP and DR was so effective in reducing growth of the treated tumors in nude mice compared with CP alone. We suppose that the result just reflects a direct effect of DR on the treated tumors in nude mice. Such direct effect of DR on the treated tumors was not apparent in BALB/c mice. The absence of mature T cells seems to increase the susceptibility of CT-26 cells to local treatment with DR. We have no clear explanation for this observation at present and are planning to elucidate the underlying mechanism(s).

In conclusion, we demonstrated that combined chemotherapy with CP and DR synergistically elicits tumor-specific T cell responses in tumor-bearing hosts. Because both drugs have been used clinically worldwide, this type of immunogenic chemotherapy could be easily combined with the use of current cancer vaccines.

Acknowledgments

We thank Ms. Yoko Ono and Ms. Yoshimi Fujii for their technical assistance. The English in this document has been checked by at least two professional editors, both native speakers of English. This study was supported in part by grants from the Ministry of Education, Science, Sport, Culture, and Technology of Japan (no. 18591449 to M. H. and no. 20790930 to N. H) and from the Shimane University Medical Education and Research Foundation.

References

  • 1.Boon T, Coulie PG, Van der Eynde BJ, Van der Bruggen P. Human T cell responses against melanoma. Annu Rev Immunol. 2006;24:175–208. doi: 10.1146/annurev.immunol.24.021605.090733. [DOI] [PubMed] [Google Scholar]
  • 2.Novellino L, Castelli C, Parmiani G. A listing of human tumor antigens recognized by T cells: March 2004 update. Cancer Immunol Immunother. 2005;54:187–207. doi: 10.1007/s00262-004-0560-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Rosenberg SA, Yang JC, Schwartzentruber DJ, et al. Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma. Nat Med. 1998;4:321–327. doi: 10.1038/nm0398-321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Nestle FO, Alijagic S, Gilliet M, et al. Vaccination of melanoma patients with peptide- or lysate-pulsed dendritic cells. Nat Med. 1998;4:328–332. doi: 10.1038/nm0398-328. [DOI] [PubMed] [Google Scholar]
  • 5.Jager E, Gnjatic S, Nagata Y, et al. Induction of primary NY-ESO-1 immunity: CD8+ T lymphocyte and antibody responses in peptide-vaccinated patients with NY-ESO-1+ cancers. Proc Natl Acad Sci USA. 2000;97:12198–12203. doi: 10.1073/pnas.220413497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Mine T, Sato Y, Noguchi M, et al. Humoral responses to peptides correlate with overall survival in advanced cancer patients vaccinated with peptides based on pre-existing, peptide-specific cellular responses. Clin Cancer Res. 2004;10:929–937. doi: 10.1158/1078-0432.CCR-1117-3. [DOI] [PubMed] [Google Scholar]
  • 7.Rosenberg SA, Yang JC, Restifo NP. Cancer immunotherapy: moving beyond current vaccines. Nat Med. 2004;10:909–915. doi: 10.1038/nm1100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Yamaguchi T, Sakaguchi S. Regulatory T cells in immune surveillance and treatment of cancer. Semin Cancer Biol. 2006;16:115–123. doi: 10.1016/j.semcancer.2005.11.005. [DOI] [PubMed] [Google Scholar]
  • 9.Ostrand-Rosenberg S, Sinha P. Myeloid-derived suppressor cells: linking inflammation and cancer. J Immunol. 2009;182:4499–4506. doi: 10.4049/jimmunol.0802740. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10.Curiel TJ, Coukos G, Zou L, et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med. 2004;10:942–949. doi: 10.1038/nm1093. [DOI] [PubMed] [Google Scholar]
  • 11.Sato E, Olson SH, Ahn J, et al. Intraepithelial CD8+ tumor-infiltrating lymphocytes and a high CD8+/regulatory T cell ratio are associated with favorable prognosis in ovarian cancer. Proc Natl Acad Sci USA. 2005;102:8538–18543. doi: 10.1073/pnas.0509182102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Onizuka S, Tawara I, Shimizu J, Sakaguchi S, Fujita T, Nakayama E. Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor α) monoclonal antibody. Cancer Res. 1999;59:3128–3133. [PubMed] [Google Scholar]
  • 13.Ko K, Yamazaki S, Nakamura K, et al. Treatment of advanced tumors with agonistic anti-GITR mAb and tits effects on tumor-infiltrating Foxp3+CD25+ CD4+ regulatory T cells. J Exp Med. 2005;202:885–891. doi: 10.1084/jem.20050940. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Nishikawa H, Kato T, Hirayama M, et al. Regulatory T cell-resistant CD8+ T cells induced by glucocorticoid-induced tumor necrosis factor receptor signaling. Cancer Res. 2008;68:5948–5954. doi: 10.1158/0008-5472.CAN-07-5839. [DOI] [PubMed] [Google Scholar]
  • 15.Loeffler M, Kruger JA, Reisfeld RA. Immunostimulatory effects of low dose cyclophosphamide are controlled by inducible nitric oxide synthase. Cancer Res. 2005;65:5027–5030. doi: 10.1158/0008-5472.CAN-05-0646. [DOI] [PubMed] [Google Scholar]
  • 16.Liu JY, Wu Y, Zhang XS, et al. Single administration of low dose cyclophosphamide augments the antitumor effects of dendritic cell vaccine. Cancer Immunol Immunother. 2007;56:1597–1604. doi: 10.1007/s00262-007-0305-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Wada S, Yoshimura K, Hipkiss EL, et al. Cyclophosphamide augments antitumor immunity: studies in an autochthonous prostate cancer model. Cancer Res. 2009;69:4309–4318. doi: 10.1158/0008-5472.CAN-08-4102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Ghiringhelli F, Larmonier N, Schmitt E, et al. CD4+ CD25+ regulatory T cells suppress tumor immunity but are sensitive to cyclophosphamide which allows immunotherapy of established tumors to be curative. Eur J Immunol. 2004;34:336–344. doi: 10.1002/eji.200324181. [DOI] [PubMed] [Google Scholar]
  • 19.Ghiringhelli F, Menard C, Puig PE, et al. Metronomic cyclophosphamide regimen selectively depletes CD4+ CD25+ regulatory T cells and restores T and NK effector functions in end stage cancer patients. Cancer Immunol Immunother. 2007;56:641–648. doi: 10.1007/s00262-006-0225-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Roux S, Apetoh L, Chalmin F, et al. CD4+ CD25+ Tregs control the TRAIL-dependent cytotoxicity of tumor-infiltrating DCs in rodent models of colon cancer. J Clin Invest. 2008;11:3751–3761. doi: 10.1172/JCI35890. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Reis E, Sousa C. Activation of dendritic cells: translating innate into adaptive immunity. Curr Opin Immunol. 2004;16:21–25. doi: 10.1016/j.coi.2003.11.007. [DOI] [PubMed] [Google Scholar]
  • 22.Casares N, Pequignot MO, Tesniere A, et al. Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death. J Exp Med. 2005;202:1691–1701. doi: 10.1084/jem.20050915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Obeid M, Tesniere A, Ghiringhelli F, et al. Calreticulin exposure dictates the immunogenicity of cancer cell death. Nat Med. 2007;13:54–61. doi: 10.1038/nm1523. [DOI] [PubMed] [Google Scholar]
  • 24.Apetoh L, Ghiringhelli F, Tesniere A, et al. Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat Med. 2007;13:1050–1059. doi: 10.1038/nm1622. [DOI] [PubMed] [Google Scholar]
  • 25.Apetoh L, Obeid M, Tesniere A, et al. Immunogenic chemotherapy: discovery of a critical protein through proteomic analysis of tumor cells. Cancer Genomics Proteomics. 2007;4:65–70. [PubMed] [Google Scholar]
  • 26.Tesniere A, Apetoh L, Ghiringhelli F, et al. Immunogenic cancer cell death: a key-lock paradigm. Curr Opin Immunol. 2008;20:504–511. doi: 10.1016/j.coi.2008.05.007. [DOI] [PubMed] [Google Scholar]
  • 27.Huang AY, Gulden PH, Woods AS, et al. The immunodominant major histocompatibility complex class I-restricted antigen of a murine colon tumor derives from an endogenous retroviral gene product. Proc Natl Acad Sci USA. 1996;93:9730–9735. doi: 10.1073/pnas.93.18.9730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Suzuki E, Kapoor V, Jassar AS, Kaiser LR, Albelda SM. Gemcitabine selectively eliminate splenic Gr-1+/CD11b+ myeloid suppressor cells in tumor-bearing animals and enhance antitumor immune activity. Clin Cancer Res. 2005;11:6713–6721. doi: 10.1158/1078-0432.CCR-05-0883. [DOI] [PubMed] [Google Scholar]
  • 29.Ko HJ, Kim YJ, Kim YS, et al. A combination of chemoimmunotherapies can effectively break self-tolerance and induce antitumor immunity in a tolerogenic murine tumor model. Caner Res. 2007;49:181–185. doi: 10.1158/0008-5472.CAN-06-4639. [DOI] [PubMed] [Google Scholar]
  • 30.Chan OT, Yang LX. The immunological effects of taxanes. Cancer Immunol Immunother. 2000;49:181–185. doi: 10.1007/s002620000122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Garnett CT, Schlom J, Hodge JW. Combination of docetaxel and recombinant vaccine enhance T-cell responses and antitumor activity: effects of docetaxel on immune enhancement. Clin Cancer Res. 2008;14:3536–3544. doi: 10.1158/1078-0432.CCR-07-4025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Green DR, Ferguson T, Zitvogel L, Kroemer G. Immunogenic and tolerogenic cell death. Nat Rev Immunol. 2009;9:353–363. doi: 10.1038/nri2545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Ghiringhelli F, Apetoh L, Tesniere A, et al. Activation of the NLRP3 inflammasome in dendritic cells induces IL-1β-dependent adaptive immunity against tumors. Nat Med. 2009;15:1170–1178. doi: 10.1038/nm.2028. [DOI] [PubMed] [Google Scholar]

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