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Clinical Cancer Research Vol. 12, 7465-7475, December 15, 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Preclinical

Enhancement of Immunologic Tumor Regression by Intratumoral Administration of Dendritic Cells in Combination with Cryoablative Tumor Pretreatment and Bacillus Calmette-Guerin Cell Wall Skeleton Stimulation

Masaru Udagawa1,4, Chie Kudo-Saito1, Go Hasegawa1, Kazuhito Yano2, Aiko Yamamoto1, Masae Yaguchi3, Masahiro Toda3, Ichiro Azuma5, Takehisa Iwai4 and Yutaka Kawakami1

Authors' Affiliations: 1 Division of Cellular Signaling, Institute for Advanced Medical Research; 2 Department of Surgery and 3 Neuro-Immunology Group, Department of Neurosurgery, Keio University School of Medicine; 4 Department of Surgery, Division of Vascular Surgery, Tokyo Medical and Dental University, School of Medicine, Tokyo, Japan; and 5 Department of Biochemistry, Hokkaido College of Pharmacy, Hokkaido, Japan

Requests for reprints: Yutaka Kawakami, Division of Cellular Signaling, Institute for Advanced Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku, Tokyo 160-8582, Japan. Phone: 81-3-5363-3777; Fax: 81-3-5362-9259; E-mail: yutakawa{at}sc.itc.keio.ac.jp.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: We developed an effective immunotherapy, which could induce antitumor immune responses against shared and unique tumor antigens expressed in autologous tumors.

Experimental Design: Intratumoral administration of dendritic cells is one of the individualized immunotherapies; however, the antitumor activity is relatively weak. In this study, we attempted to enhance the antitumor efficacy of the i.t. dendritic cell administration by combining dendritic cells stimulated with Bacillus Calmette-Guerin cell wall skeleton (BCG-CWS) additionally with cryoablative pretreatment of tumors and analyzed the therapeutic mechanisms.

Results: These two modifications (cryoablation of tumors and BCG-CWS stimulation of dendritic cells) significantly increases the antitumor effect on both the treated tumor and the untreated tumor, which was distant at the opposite side, in a bilateral s.c. murine CT26 colon cancer model. Further analysis of the augmented antitumor effects revealed that the cryoablative pretreatment enhances the uptake of tumor antigens by the introduced dendritic cells, resulting in the induction of tumor-specific CD8+ T cells responsible for the in vivo tumor regression of both treated and remote untreated tumors. This novel combination i.t. dendritic cell immunotherapy was effective against well-established large tumors. The antitumor efficacy was further enhanced by depletion of CD4+CD25+FoxP3+ regulatory T cells.

Conclusions: This novel dendritic cell immunotherapy with i.t. administration of BCG-CWS–treated dendritic cells following tumor cryoablation could be used for the therapy of cancer patients with multiple metastases.

Cancer immune responses have recently been evaluated in patients using various immuno-monitoring techniques focusing on identified tumor antigens (1–4). The immunogenicity for each tumor antigen was found to differ among the individual patients, even for the shared antigens. The variation is probably due to quantitative differences in antigen expression by the tumor cells and the patients' immunoreactive ability and HLA type (5). In addition, autologous unique tumor-specific antigens, which stem from genetic alterations, involved in tumor proliferation and survival have frequently been identified in patients who responded well to various immunotherapies (6, 7). Maintenance of the unique antigen-specific CTL detected by HLA tetramer analysis was observed in these patients (8), suggesting that these unique antigens are attractive targets for immunotherapy. These observations emphasize the importance of developing a more effective individualized immunotherapy.

Various individualized immunotherapies have been investigated. Immunization using immunogenic shared antigens that are individually preselected by rapid immunologic assays can induce immune responses even against unique tumor antigens via antigen spreading (9). Other strategies have been attempted to induce immune responses against unique tumor antigens: immunization using modified autologous tumor cells (10, 11) and crude molecules extracted from autologous tumor cells, such as lysates (12), peptides (13), RNA (14), heat shock protein (15), etc. and interventions that augment the immune response into autologous tumors, such as i.t. administration of various immunostimulating reagents (16). Clinical trials of the immunization with identified shared tumor antigens (17) and immunization with dendritic cells pulsed with autologous tumor lysates (18) have been done, and limited antitumor effects were observed. In terms of the immuno-augmenting interventions into individual tumors, we have previously reported that i.t. injection of modified herpes simplex virus–enhanced in vivo generation of tumor antigen-specific T cells through activation of dendritic cells resulted in T cell–mediated tumor regression (19, 20). Subsequently, we have attempted to develop and improve immunotherapy by i.t. administration of dendritic cells in the mouse tumor model.

In this study, we show that cryoablative pretreatment of the dendritic cell–injecting tumor site and use of short-term cultured dendritic cells with toll-like receptor (TLR)–stimulating reagents, such as Bacillus Calmette-Guerin cell wall skeleton (BCG-CWS), enhance the antitumor effects of i.t. dendritic cells through increasing uptake of tumor antigens and subsequent induction of tumor-specific CD8+ CTL. This modified protocol showed that an effect against relatively large tumors at remote sites may, thus, be potentially applied toward future clinical trials concerning cancer patients with multiple metastases.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Mice, tumor cell lines, and reagents. Six- to 8-week-old female BALB/c mice (H-2d), purchased from SLC, Inc. (Tokyo, Japan), were maintained in the specified pathogen-free conditions and used upon approval by the Animal Care and Use Committee at Keio University School of Medicine. A N-nitroso-N-methylurethane–induced murine colon cancer cell line (CT26) was cultured in DMEM supplemented with 10% heat-inactivated FCS and 100 units/mL penicillin/100 µg/mL streptomycin. Fibrosarcoma cell lines Meth A and CMS5a were cultured in RPMI 1640 supplemented with 10% FCS, 55 µmol/L 2-mercaptoethanol, and penicillin/streptomycin. BCG-CWS, which was purified as previously described (21), was either prepared as an oil-in-water emulsion (3 mg/mL) to use in vivo, or prepared as a saline solution (2 mg/mL) for in vitro dendritic cell culture, then homogenized with a Potter homogenizer, and heat-sterilized for 30 min at 60°C as previously reported (22). Dendritic cells were also cultured with 0.1 KE unit/mL OK-432, a low-virulence Su strain of type III, group A Streptococcus pyogenes, kindly provided by Chugai Pharmaceutical (Tokyo, Japan).

Generation of dendritic cells from bone marrow. Bone marrow–derived dendritic cells were generated by modifying the procedure as previously described (23). Cells expressing the surface markers for CD3e, CD11b, Gr-1, B220, and TER119 were depleted from bone marrow cells isolated from the femurs and the tibias of BALB/c mice using Lineage Panel and Streptavidin Plus Magnetic Particles-DM (BD Biosciences, PharMingen, San Diego, CA). The obtained myeloid dendritic cell precursors were cultured in RPMI 1640 supplemented with 10 ng/mL of murine granulocyte macrophage colony-stimulating factor (Peprotech, London, United Kingdom), 10% FCS, 100 units/mL penicillin and 100 µg/mL streptomycin, 55 µmol/L 2-mercaptoethanol, 0.1 mmol/L MEM nonessential amino acids, 10 mmol/L HEPES buffer, and 1 mmol/L MEM sodium pyruvate at 4 x 105 per mL in six-well plates. On days 4 and 6, half of the medium was replaced with fresh complete medium. On day 7, nonadherent cells were harvested and used in subsequent experiments.

Flow cytometric analysis, phagocytosis assay, and interleukin-12/ELISA. Bone marrow–derived dendritic cells generated by 7-day culture of bone marrow cells with granulocyte macrophage colony-stimulating factor were incubated with 15 µg/mL BCG-CWS. For the phenotypic analysis of dendritic cells, phycoerythrin- or FITC-conjugated mouse monoclonal antibodies (mAb) against CD80, CD86. CD11c, class II MHC antigens (I-Ad), and CD54 and CD40 (MBL Co., Ltd., Nagoya, Japan) were used to analyze the stimulated dendritic cells with the isotype-matched control mAb, and stained cells were analyzed with FACSCalibur. Phagocytotic activity of the dendritic cells was evaluated as previously described (24). Twenty microliters of FITC-conjugated dextran (0.05 mmol/L) or 500 µL FITC-conjugated Escherichia coli particles (0.625 mg/mL) purchased from Molecular Probes (Leiden, The Netherlands) were incubated for 1 h with 5 x 105 immature dendritic cells in RPMI 1640 at either 4°C or 37°C. After incubation, cells were harvested, resuspended in PBS buffer, and analyzed by fluorescence-activated cell sorting. The interleukin-12 (IL-12) production of dendritic cells was measured by ELISA (BD Opt EIA Set mouse IL-12 p70) in the culture supernatants.

Cryoablative pretreatment in vivo. Cryoablation was done twice per a mouse at –80°C for 60 s using a Cryomaster (Keeler Ltd., Windsor, Berkshire, United Kingdom), which permitted the CT26 tumors, of which maximum diameters were ~5 mm, to be entirely frozen without damaging the skin.

Evaluation of in vivo antitumor effects. CT26 tumor cells (5 x 105 and 1.0 x 106) were s.c. inoculated into the bilateral flanks of the mice under anesthesia to generate the small size or the large size tumor model, respectively. When the small size tumor models reached ~5 mm or when the large size tumor models reached ~8 mm at the longest diameter, respectively, the right lateral tumor was treated by cryoablation followed by an i.t. injection of dendritic cells. For the small tumor model, the first treatment was conducted 5 days after tumor implantation by initially freezing part of the tumor site –80°C for 60 s twice using the Cryomaster, then giving 2 x 106 bone marrow–derived dendritic cells ex vivo cultured by various reagents, such as BCG-CWS and OK-432, in 50 µL saline directly into the tumor site. The second treatment of the small tumor model on day 8 consisted of only an i.t. administration of bone marrow–derived dendritic cells without a cryoablative pretreatment due to the diminishment of treated right lateral tumor by the previous cryoablative treatment on day 5. For the large tumor model, the tumor site was pretreated, then 2 x 106 bone marrow–derived dendritic cells were given i.t. three times at 7-day intervals. The size of bilateral tumors was measured in three perpendicular dimensions with a vernier caliper every 3rd day. The tumor volume was calculated using the next formula (25):

Formula
where L is the longest diameter, W is the width, and H is the height.

Data are reported as the average tumor area ± SE.

Detection of tumor-specific T-cell responses by IFN-{gamma} release and cytotoxicity assays. Antigen-specific cytokine release was measured using IFN-{gamma} ELISA as previously described (26). Splenocytes of four mice from the population that had received the i.t. BCG-CWS–treated dendritic cells after cryoablation and splenocytes of four mice that had received i.t. saline after cryoablation were isolated on day 26. The splenocytes were restimulated by culturing the cells with 1 µg/mL CT26 immunodominant T-cell epitope AH-1, SPSYVYHQF (27). Five days later, restimulated cells were collected and re-cultured with various stimulators: CT26, Meth A pulsed with AH-1, Meth A, and medium alone as control in 96-well U-bottomed plate. The next day, supernatant was collected, and IFN-{gamma} production was evaluated using Mini kit mouse IFN-{gamma} (ENDOGEN, Rockford, IL).

Next, the 51Cr release assay was done as previously described (26). Briefly, splenocytes were mixed with 2.5 x 104 target cells labeled with 100 µCi Na251CrO4 for 1 h at 37°C at a series of E/T ratio and incubated for 4 h in 96-well U-bottomed plates. Then, 100 µL of the supernatant were collected, and the radioactivities were counted in a gamma counter, and specific lysis was calculated as follows: specific lysis (%) = (experimental release – spontaneous release) / (maximum release – spontaneous release) x 100.

In vivo depletion of lymphocyte subsets. To investigate requirement of effector cells for in vivo antitumor activity induced by the combination therapy, a specific T-cell subset was depleted from mice during the therapy using antibodies. Antibody in ascitic fluid was injected i.p. on days 4, 8, 12, 16, 20, and 24 after CT26 tumor implantation. The following antibodies were used: anti-CD4 (GK1.5, 50 µg per mouse), anti-CD8 (2.43, 50 µg per mouse), anti-CD25 (300 µg per mouse, kindly provided by Dr. Shimon Sakaguchi in Kyoto University and Dr. Eiichi Nakayama in Okayama University in Japan), and the control rat IgG. The cell depletion was validated by flow cytometry (Cellquest Software, BD Biosciences) using Cychrome-conjugated anti-CD4 mAb (BD Biosciences), phycoerythrin-conjugated anti-CD25 mAb (BD Biosciences), and FITC-conjugated anti-FoxP3 mAb (eBioscience, San Diego, CA); >90% of the relevant cell population was depleted.

In vitro CD8+ T-cell proliferation assay with various CD4+ T-cell subsets. Mice were sacrificed 7 days after treatment with i.t. injection with BCG-CWS–treated dendritic cells following cryoablation of tumors on day 5, and the splenic lymphocytes were tested for CD8+ cell proliferation in vitro (day 12). Spleen cells were purified for CD8+ cells, CD4+CD25– cells, or CD4+CD25+ cells using the Isolation kit MicroBeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer's instructions. These cells were validated by flow cytometry with >97% of the relevant cell population. CD8+ cells (1 x 105 per well) were cultured with AH-1 peptide (1 µg/mL), irradiated antigen-presenting cells from naive mice (5 x 105 per well), and/or irradiated either CD25+ or CD25–, CD4+ T cells (1 x 105 per well, CD8/CD4 = 1:1) in 96-well plates for 5 days. The CD8+ cell proliferation was measured using Premix WST-1 reagent (Takara Bio, Inc., Otsu Shiga, Japan) according to the manufacturer's instructions.

Evaluation of in vivo suppressive activity of CD4+ T-cell subsets from tumor-bearing mice. To evaluate the immunosuppressive activity of CD4+ cells, nude mice were implanted with CT26 tumor cells and received the combination therapy following lymphocyte transfer. Nude mice were implanted with CT26 tumor cells on day 0 at the same time. Splenic lymphocytes from the tumor-bearing BALB/c mice were purified into CD8+ cells, CD4+CD25– cells, and CD4+CD25+ cells and transferred the CD8+ cells in combination with CD4+CD25– cells or CD4+CD25+ cells into the tumor-bearing nude mice on day 4 after CT26 tumor implantation. The nude mice were treated with the combination therapy on days 5 and 8, and tumor size was measured twice a week.

Preparation of green fluorescent protein-labeled CT26 and PKH26-labeled dendritic cells and histochemical study. We prepared green fluorescent protein (GFP)–labeled CT26 by transducing the GFP gene with a cell sorter and PKH26 (Sigma-Aldrich, St. Louis, MO)–labeled bone marrow–derived dendritic cells according to the manufacturer's protocol. After applying cryoablation on day 5 to the CT26 tumor site, apoptotic cells were measured by terminal deoxynucleotidyl transferase–mediated nick-end labeling staining, and necrotic cells were examined by microscope and were evaluated by NIH image. Resected lymph nodes were embedded with ornithine carbamyl transferase compound, frozen, and cryosectioned with 6 to 10 µm using a cryostat, then visualized with a fluorescence microscope and a confocal microscope.

Statistical analysis. Statistical analysis was done using the unpaired two-tailed Student's t test. Differences were considered significant when P < 0.05.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Short-term BCG-CWS–treated dendritic cells are more effective than immature dendritic cells or immature dendritic cells and BCG-CWS on the inhibition of untreated remote tumors. In this study, we attempted to establish protocols of immunotherapy by i.t. dendritic cell administration with some modifications. Firts, we investigated the potential antitumor effect of BCG-CWS, which induces dendritic cell maturation through TLR2/TLR4 stimulation. Dendritic cells injected into the tumor should exhibit phagocytotic activity to facilitate tumor antigen uptake and subsequent differentiation, in an immunosuppressive tumor microenvironment, into mature dendritic cells that migrate into draining lymph nodes and activate T cells. Immature dendritic cells cultured with 15 µg/mL BCG-CWS for 6 h maintained phagocytotic activity against dextran and E. coli particles (Fig. 1A ), while showing an increased expression of various antigen-presenting and costimulatory membrane molecules, including MHC class II, CD11c, CD80, CD86, CD54, and CD40, and producing IL-12 (Fig. 1B and C). However, an 18-h culture of immature dendritic cells with BCG-CWS resulted in a slight decrease of phagocytotic activity. Similar results were observed when dendritic cells were stimulated with OK-432 (data not shown). Thus, we used 6-h BCG-CWS–stimulated dendritic cells for further study.


Figure 1
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  		Fig. 1. Generation of dendritic cells with an increased expression of costimulatory molecules and production of IL-12 maintain phagocytotic activity after a 6-h in vitro culture of bone marrow–derived immature dendritic cells. A, pinocytosis of dextran and phagocytosis of E. coli particles by bone marrow–derived dendritic cells after an in vitro culture with (solid line) or without (dotted line) BCG-CWS were maintained for 6 h but slightly decreased after 18 h. B, increased expression of CD86 and CD40 on bone marrow–derived dendritic cells after in vitro culture with BCG-CWS. C, production of IL-12 by bone marrow–derived dendritic cells after in vitro culture with (solid line) or without (dotted line) BCG-CWS. BCG-CWS–stimulated dendritic cell (bullet); media-cultured dendritic cell ({square}). One representative result from two independent experiments (A and B). One representative result from three independent experiments (C).

 
The left lateral tumors were not treated, whereas the right lateral tumors received treatments on days 5 and 8. Treatments consisted of i.t. injections of saline, BCG-CWS alone in an oil-in-water emulsion, immature dendritic cells alone, a combination of BCG-CWS and immature dendritic cells, or BCG-CWS–treated dendritic cells, and results were evaluated in the 5-day small CT26 colon cancer model (~5 mm diameter; Fig. 2A ). Administration of BCG-CWS alone inhibited growth of the treated tumors but did not inhibit growth of the remote untreated tumors. All of the other treatments except for the saline control inhibited both treated and untreated tumors. Simultaneous administration of BCG-CWS and immature dendritic cells did not enhance antitumor effects generated by injecting immature dendritic cells alone. However, BCG-CWS–treated dendritic cells had a significantly stronger antitumor effect than the other treatments on both treated and untreated tumors, indicating that dendritic cells stimulated for 6 h with BCG-CWS are more effective than immature dendritic cells for i.t. dendritic cell immunotherapy.


Figure 2
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  		Fig. 2. Augmentation of antitumor activity of cryoablative (Cryo) tumor pretreatment and i.t. administration of 6-h BCG-CWS–treated dendritic cells (BCG-CWS-DC). A, augmentation of the antitumor effect from i.t. dendritic cells using BCG-CWS. Five groups with six mice in each without cryoablation, saline (x), BCG-CWS in an oil-in-water emulsion ({square}), immature dendritic cells (iDC; {blacksquare}), a combination of BCG-CWS and immature dendritic cells ({triangleup}), and BCG-CWS–treated dendritic cells ({circ}) were evaluated in the 5-d small CT26 colon cancer model. B, cryoablative tumor pretreatment enhanced the antitumor effects of i.t. BCG-CWS–treated dendritic cells. Three groups, cryoablative pretreatment (–80°C, 60 s, two cycles) with i.t. BCG-CWS–treated dendritic cells ({circ}), i.t. BCG-CWS–treated dendritic cells ({triangleup}), and i.t. saline as control ({square}), with six mice in each group were evaluated in the small tumor model. C, BCG-CWS–treated dendritic cells ({circ}) were more effective than immature dendritic cells ({triangleup}) after cryoablative tumor pretreatment. I.t. saline following cryoablation ({square}) did not inhibit the growth of remote tumors. Arrows indicate points of treatments. Similar results were obtained in two independently conducted experiments. Seven mice were used in each group. Points, mean; bars, SE. *, P < 0.05, unpaired t test on day 26 after tumor inoculation. Representative results from two (A and C) or three (B) independent experiments.

 
Cryoablative pretreatment augmented growth inhibition of remote tumors by i.t. administration of BCG-CWS–treated dendritic cells. Seeking further enhancement of the antitumor effects of our modified i.t. dendritic cell protocol, we evaluated a tumor pretreatment that destroys existing tumor cells and thus potentially increasing the uptake of tumor antigens by the injected dendritic cells. We investigated a cryoablative pretreatment in this study. Although cryoablation alone did not inhibit the growth of the remote tumor, the combination of cryoablation with i.t. BCG-CWS–treated dendritic cells significantly suppressed the growth of the untreated tumors compared with i.t. BCG-CWS–treated dendritic cells alone (Fig. 2B). Administration of BCG-CWS–treated dendritic cells after cryoablation inhibited tumor growth more effectively than immature dendritic cells following the cryoablation (Fig. 2C). These results showed that cryoablative tumor pretreatment enhances the antitumor effects of the i.t. BCG-CWS–treated dendritic cells.

Tumor-specific CD8+ CTL are responsible for the antitumor effect on remote tumors upon i.t. administration of BCG-CWS–treated dendritic cells following cryoablation of tumor. To determine the mechanism causing the growth inhibition of the remote tumor when pretreated with cryoablation and i.t. BCW-CWS–treated dendritic cells, we evaluated the in vivo tumor specificity of this treatment using mice that were s.c. implanted with two types of tumors (CT26 and CMS5a) in three distinct sites. In mice implanted with one syngeneic fibrosarcoma CMS5a and two CT26, BCG-CWS dendritic cell treatment of one of the CT26 tumors following cryoablation suppressed the growth of the untreated CT26 without inhibiting the growth of the CMS5a (Fig. 3A ). Similar treatment of a CMS5a tumor in mice implanted with one CT26 and two CMS5a inhibited the growth of the untreated CMS5a without affecting the growth of CT26 (Fig. 3B). These results indicated tumor-specific effects in vivo of this treatment.


Figure 3
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  		Fig. 3. Tumor-specific growth inhibition by i.t. administration of BCG-CWS–treated dendritic cells following cryoablative pretreatment and induction of tumor-specific CTL. A, CT26-specific growth inhibition by i.t. administration of BCG-CWS–treated dendritic cells into CT26 following cryoablation. When right CT26 was treated by i.t. BCG-CWS–treated dendritic cells following cryoablation ({circ}), growth of the left CT26, but CMS5a, was inhibited. Seven mice were used in each group. B, CMS5a-specific growth inhibition by i.t. administration of BCG-CWS–treated dendritic cells into CMS5a following cryoablation. When right CMS5a was treated by i.t. BCG-CWS–treated dendritic cells following cryoablation ({circ}), growth of the left CMS5a, but CT26, was inhibited. I.t. saline following cryoablation as a control ({blacksquare}) was evaluated in both experiments. Seven mice were used in each group. Representative results from two independent experiments (A) or one independent experiment (B). C, CT26-specific IFN-{gamma} release by spleen T cells from mice treated with i.t. BCG-CWS–treated dendritic cells following cryoablation. IFN-{gamma} releases specific for CT26 and its immunodominant T-cell epitope AH-1 were observed in spleen T cells from mice treated with i.t. BCG-CWS–treated dendritic cells following cryoablation (open columns), but not from mice treated with i.t. saline following cryoablation (closed columns). Columns, mean of IFN-{gamma} release by T cells obtained from four mice; bars, SE. Furthermore, induction of CT26-specific CTL from mice treated with i.t. BCG-CWS–treated dendritic cells following cryoablation. Lysis of CT26, but not Meth A, was detected by T cells generated by a single in vitro stimulation with the AH-1 peptide from mice treated with i.t. BCG-CWS–treated dendritic cells ({circ}) or immature dendritic cells ({triangleup}) following cryoablation, but not from mice treated with i.t. saline following cryoablation ({blacksquare}). The cytotoxic activity was significantly higher in the T cells from mice treated with BCG-CWS–treated dendritic cells than immature dendritic cells. One representative result from two independent experiments.

 
Induction of tumor-specific T cells in mice treated with this protocol was then evaluated. IFN-{gamma} production specific for CT26 and its immunodominant endogenous retroviral epitope AH-1 (SPSYVYHQF) were observed in splenic T cells from mice that had received cryoablative pretreatment and i.t. BCG-CWS–treated dendritic cells but not detected in mice treated with cryoablation and saline (Fig. 3C, left). Moreover, lysis of CT26, but not Meth-A fibrosarcoma, was detected by T cells that had been stimulated once in vitro with the AH-1 peptide derived from mice treated with cryoablation and i.t. BCG-CWS–treated dendritic cells or i.t. immature dendritic cells but not by T cells from mice that had received cryoablation and saline. The cytotoxic activity of T cells from mice treated with cryoablation and i.t. BCG-CWS– dendritic cells was significantly higher than mice treated with cryoablation and i.t. immature dendritic cells (Fig. 3C, right). These results infer i.t. BCG-CWS– dendritic cells following cryoablation efficiently induce CT26-specific CTL.

We conducted depletion experiments of T-cell subsets to identify in vivo effector cells. Depletion of CD8+ T cells completely abrogated the antitumor effects of both treated and remote untreated tumors (Fig. 4A ), indicating CD8+ T cells are the main effector cells associated with the in vivo antitumor effect observed in this treatment.


Figure 4
Figure 4
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  		Fig. 4. CD8+ T cells are effector cells responsible for the in vivo tumor regression by i.t. administration of BCG-CWS–treated dendritic cells following cryoablation and the enhancement of the antitumor effect by depletion of CD4+FoxP3+ T cells. A, CD8+ T cells are main effector cells in the in vivo tumor regression by i.t. BCG-CWS–treated dendritic cells following cryoablation. Depletion of CD8+ T cells ({blacktriangleup}) and depletion of CD8+ T cells and CD4+ T cells ({square}) abrogated the antitumor effects of i.t. BCG-CWS–treated dendritic cells following cryoablation of both treated and untreated remote tumors, whereas depletion of CD4+ T cells ({square}) augmented the antitumor effect. I.t. BCG-CWS–treated dendritic cells ({circ}) and i.t. of saline ({blacksquare}) were also included. Five mice were used in each group. Arrows indicate points of treatments. Points, mean; bars, SE. *, P < 0.05, unpaired t test. One representative result from three independent experiments. B, depletion of CD25+ cells enhanced early inhibition of remote untreated left lateral tumor and increased rate of complete regression by i.t. BCG-CWS–treated dendritic cells following cryoablation. I.p. injection of anti-CD25 antibody at day 4 after the CT26 tumor inoculation inhibited early tumor growth of remote left lateral tumor compared with injection of PBS when treated with BCG-CWS–treated dendritic cells after cryoablation, and tumor regressed completely in 3 of 10 mice. Treatment with anti-CD4 antibody regressed tumors in 6 of 7 mice in this experiment. Ps versus PBS are 0.3815 with anti-CD25 antibody treatment and 0.0001 with anti-CD4 antibody treatment. Complete regression was observed in 0 of 7 mice treated with PBS, 3 of 10 mice treated with anti-CD25 antibody, and 6 of 7 mice treated with in anti-CD4 antibody. The enhanced antitumor effect by the anti-CD4 mAb administration is shown in the four independent experiments. Four groups consisted of 7, 7, 10, or 7 mice, respectively. C, presence of CD4+CD25+FoxP3+ T cells capable of inhibiting in vitro CD8+ T-cell proliferation and in vivo antitumor activity of CD8+ T cells from tumor-bearing mice. Splenic CD8+ cells from mice on 7 d after i.t. BCG-CWS–treated dendritic cells following tumor cryoablation were cultured in vitro with irradiated antigen-presenting cells (APC) from naive mice, AH-1 peptide, and irradiated either CD4+ cells with/without CD25+ cells from the treated mice for 5 d, and then the CD8+ cell proliferation was measured by WST assay (top). Splenic CD8+ T cells from BALB/c mice bearing day 4 CT26 tumors are adoptively transferred into nude mice bearing 4-d CT26 tumors with or without co-transfer of either whole CD4+, CD4+CD25– cells, or CD4+CD25+ T cells from the tumor-bearing BALB/c mice. The nude mice were then treated with i.t. BCG-CWS–treated dendritic cells following cryoablation on days 5 and 8, and tumor size was measured twice a week. Bottom, tumor volume on day 17. CD4+CD25+ T cells inhibited both in vitro CD8+ T-cell proliferation and in vivo antitumor activity of CD8+ T cells (P < 0.05). Five mice were used in each group. D, presence of CD4+CD25+FoxP3+ and CD4+CD25–FoxP3+ T cells in tumor-infiltrating cells and decrease of these cells by the anti-CD4 antibody treatment. Phenotype of tumor-infiltrating cells from mice on 7 d after i.t. BCG-CWS–treated dendritic cells with or without the anti-CD4 antibody treatment was analyzed for CD4, CD25, and FoxP3 expression by flow cytometry. FoxP3 expression was observed in CD4+CD25– cells and CD4+CD25+ cells. By the anti-CD4 antibody treatment, CD4+FoxP3+ T cells are almost completely depleted (right). Three mice were used in each group.

 
Enhancement of antitumor effect by depletion of regulatory T cells. Interestingly, CD4+ T-cell depletion with anti-CD4 antibody augmented the antitumor activity of the combination therapy (Fig. 4A). To investigate the possible roles of CD4+CD25+ regulatory T cells (Treg), mice were treated with anti-CD25 antibody on day 4, and tumor regressed completely in 3 of 10 mice, although tumor growth seemed to be a bit enhanced later. The differential effects of anti-CD25 antibody and anti-CD4 antibody may be explained possibly by suppression of CD25+CD8+ effector cells with anti-CD25 antibody treatment or induction of CD4+CD25–FoxP3+ Treg (ref. 28; Fig. 4B).

To further clarify the Treg involvement, the suppressive function of CD4+CD25+ T cells from tumor-bearing mice was evaluated by using in vitro CD8+ proliferation assay and in vivo antitumor assays. In vitro 5-day proliferation of splenic CD8+ T cells from mice treated with the combination therapy upon stimulation with the AH-1 peptide was significantly suppressed by addition of whole CD4+ T cells, CD4+CD25– T cells, or particularly CD4+CD25+ T cells from the treated mice (Fig. 4, top). In vivo growth of 4-day CT26 tumors implanted on nude mice was significantly inhibited when the combination therapy was applied with transfer of splenic CD8+ T cells from BALB/c mice bearing 4-day CT26 tumors. However, this antitumor activity of the CD8+ T cells was significantly suppressed by co-transfer of CD4+CD25+ T cells, but not of CD4+CD25– T cells, from the tumor-bearing mice (Fig. 4B, bottom). These in vitro and in vivo results suggested that at least immunosuppressive CD4+CD25+ Treg cells are involved in this tumor model.

To investigate FoxP3 expression on T cells and effects of the antibody administration on these cells, the phenotype of T cells in peripheral blood, spleen, and tumors of mice treated with the combination therapy was analyzed by flow cytometry with anti-CD4, anti-CD25, or anti-FoxP3 mAb. FoxP3 expression in the tumor-infiltrating T cells was observed in both CD4+CD25+ T cells and CD4+CD25– T cells (Fig. 4D, top right), and these CD4+FoxP3+ T cells were depleted by treatment with anti-CD4 antibody (Fig. 4D, bottom right). Similar expression pattern of FoxP3 was observed in T cells from peripheral blood and spleen (data not shown). These results indicate that CD4+CD25+FoxP3+ Treg, and also CD4+CD25–FoxP3+ Treg, are involved in this tumor model, and inhibition of these Treg further enhances the antitumor effect of the i.t. dendritic cell immunotherapy.

Efficient uptake of tumor-derived molecules by dendritic cells and their migration into draining lymph nodes after cryoablative treatment of tumors. To further understand the mechanism for the augmented antitumor T-cell responses upon the combined cryoablation and i.t. BCG-CWS–treated dendritic cells, tumor histology following cryoablation was examined. Extensive necrosis with some apoptotic cells detected by terminal deoxynucleotidyl transferase–mediated nick-end labeling staining was observed 2 h after two time cryoablations (–80°C, 60 s) of the ~5-mm-diameter s.c. tumors (Fig. 5A ). We evaluated the efficiency of the tumor lysate uptake of dendritic cells when treated with cryoablation. PKH26-labeled dendritic cells (red) did not phagocyte lentivirally GFP-labeled CT26 tumor cells (green; Fig. 5B, top) when cultured together in vitro for 5 h in a 96-well U-bottomed plate (Fig. 5B). However, a closer examination with laser scanning confocal microscopy revealed the tumor-derived GFP proteins (yellow in the merged picture) were located inside most of the dendritic cells when the GFP-labeled CT26 tumor cells were treated with cryoablation (Fig. 5B, bottom).


Figure 5
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  		Fig. 5. Efficient uptake of tumor-derived molecules from necrotic and apoptotic cells induced by cryoablation by dendritic cells and their migration into draining lymph nodes. A, induction of necrosis and apoptosis by cryoablation of tumor. Extensive necrosis with apoptotic cells detected by terminal deoxynucleotidyl transferase–mediated nick-end labeling staining was observed at 2 h after cryoablation (–80°C, 60 s, two cycles; left, x40; right, x200). One representative result from two independent experiments. B, efficient uptake of tumor-derived molecules by dendritic cells after cryoablation. PKH26-labeled dendritic cells (red) did not phagocyte lentiviral GFP-labeled CT26 tumor cells (green), when cultured together in vitro for 5 h in a 96-well U-bottomed plates (top, x400). Most dendritic cells incorporated tumor-derived GFP proteins (yellow at the merged picture) when GFP-labeled CT26 tumor cells were cryoablated and cultured with dendritic cells (bottom, x400). C, migration of given dendritic cells containing tumor-derived molecules in the draining lymph nodes. When a mixture of PKH26-labeled BCG-CWS–treated dendritic cells or immature dendritic cells and cryoablated GFP-CT26 were s.c. injected on flank of mice, BCG-CWS–treated dendritic cells (top) and immature dendritic cells (bottom) containing CT26-derived GFP proteins (yellow) were detected similarly in draining inguinal lymph nodes 6 h after injection. Magnification of all photographs, x100. One representative result from three independent experiments (B and C).

 
When a mixture of 3.0 x 105 PKH26-labeled dendritic cells with or without BCG-CWS treatment and 3.0 x 105 cryoablated GFP-CT26 cells were s.c. injected into the right flank of the mice, dendritic cells containing CT26-derived GFP proteins (yellow) were detected in the draining inguinal lymph nodes after 6 h (Fig. 5C, right). The BCG-CWS–treated dendritic cells (Fig. 5C, top) and immature dendritic cells (Fig. 5C, bottom) seemed to migrate similarly into lymph nodes, indicating that BCG-CWS did not enhance the dendritic cell migration. These results may suggest that cryoablative pretreatment of tumors augment the induction of tumor specific CTL through an enhanced tumor antigen uptake by injected dendritic cells.

Antitumor activity of i.t. given BCG-CWS–treated dendritic cells following cryoablation against relatively large tumors. The strength of the generated antitumor activity upon giving i.t. BCW-CWS–treated dendritic cells following cryoablation was tested in the large tumor models. Seven-day large tumors that were about the size of 8 mm at the longest diameter received three treatments at 7-day intervals, resulting in the complete regression of treated right lateral tumors in all of the mice and untreated left lateral tumors in two of five mice (Fig. 6A ). Because the cryoablative pretreatment was applied only to a part of tumor site, the regression of tumors indicates an immunologic rejection of both treated and untreated tumors. However, this treatment did not induce the complete regression of remote tumors in a larger 14-day tumor model (~11 mm at the longest diameter); nevertheless, significant growth inhibition of the remote tumor was still observed (Fig. 6B). Increasing the frequency of treatments may further decrease the tumor size in this larger tumor model. Furthermore, dendritic cells incubated for 4 h with 10 µg/mL OK-432, a streptococcal crude extract that stimulates various TLRs, also showed a similar antitumor activity as BCG-CWS–treated dendritic cells (Fig. 6B). These results indicate that repeated i.t. BCG-CWS–treated dendritic cells with cryoablative tumor pretreatment is effective for relatively large tumors.


Figure 6
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  		Fig. 6. I.t. administration of BCG-CWS–treated dendritic cells following cryoablation was effective even in the relatively large tumor models. A, tumor regression by i.t. BCW-CWS–treated dendritic cells following cryoablation in the 7-d large tumor models. Three treatments of i.t. BCG-CWS–treated dendritic cells following partial cryoablation to a section of the tumor ({circ}) every week resulted in the complete regression of the treated right lateral tumors in all the mice and the untreated left lateral tumors in 2 of 5 mice in the 7-d large (~8 mm at the longest diameter) tumor model. I.t. saline after cryoablation group ({blacksquare}) was done as a control. Five mice were used in each group. B, growth inhibition of tumors by i.t. BCW-CWS–treated dendritic cells following cryoablation in the 14-d large tumor models. Two treatments of i.t. BCG-CWS–treated dendritic cells ({circ}) or OK-432–treated dendritic cells ({diamondsuit}) following cryoablation to a part of the tumor every week resulted in a similar significant growth inhibition of both treated right and untreated left tumors in the 14-d large tumor models (~11 mm at the longest diameter). I.t. immature dendritic cells following cryoablation ({triangleup}) and i.t. saline following cryoablation ({blacksquare}) were also evaluated. Six mice were used in each group. Arrows indicate the three periods of treatment in both experiments. Points, mean; bars, SE. *, P < 0.05, unpaired t test on days 28 and 35 after 0 tumor implantation cells. One representative result from two independent experiments.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Previous studies by us and other groups indicate the importance of individualized immunotherapy (5). One strategy is the i.t. administration of dendritic cells. Infiltration of dendritic cells in tumor has been correlated with better prognosis in several types of human cancers (29, 30). In mouse models, i.t. immature dendritic cell alone has a weak antitumor effect through antitumor T-cell induction. A variety of modifications has been investigated to increase the weak antitumor effects (31–33), including use of dendritic cells modified with various genes, such as cytokines (34) IL-12 (35) and IFN-{gamma} (36) and chemokine CCL21 (37).

In this study, we have successfully enhanced the antitumor effects of the i.t. dendritic cells by combining a cryoablative tumor pretreatment with short-term cultured dendritic cells with TLR-stimulating BCG-CWS. Although cryoablation alone did not induce reproducible antitumor effect on remote untreated tumors, the combination of cryoablation and i.t. dendritic cell produced stronger antitumor effects on remote tumors. Cryoablation enhanced the uptake of tumor antigens by dendritic cells via necrosis and apoptosis of tumor cells, induction of tumor antigen (AH-1)–specific CTL by the dendritic cells migrated in the draining lymph nodes, and the CD8+ T cell–dependent tumor regression on both treated and untreated tumors. Usage of the BCG-CWS–treated dendritic cells further enhanced the antitumor effects. BCG-CWS, a component of BCG, consisting of mycolic acid, arabinogalactan, and peptidoglycan, was identified as an agonist for TLR2 and TLR4 (38). BCG-CWS has been used clinically for patients with various cancers (39, 40), showing possible antitumor effects without any major adverse effects. Short-term (<6 h) in vitro culture of dendritic cells with BCG-CWS generated dendritic cells that maintained pinocytotic and phagocytotic activities, and these treated dendritic cells subsequently matured completely. After i.t. BCG-CWS–treated dendritic cells, dendritic cells that had incorporated tumor-derived molecules were detected in the draining lymph nodes. The migrated dendritic cells may directly activate CTL, or indirectly activated CTL via lymph node resident dendritic cells. The combination of these three components (i.t. dendritic cell, cryoablative pretreatment, and TLR stimulation with BCG-CWS) significantly increased the antitumor effects sufficiently to regress large tumors at remote sites.

CD4+CD25+ Treg capable of suppressing in vitro CD8+ T-cell proliferation and in vivo antitumor effect of CD8+ T cells are involved in this tumor model. Augmentation of the antitumor effects along with decrease of CD4+FoxP3+ T cells by treatment of mice with anti-CD4 or anti-CD25 mAb indicated that additional Treg inhibitory interventions, including administration of anti-CTLA-4 or GITR mAb, may further improve this i.t. dendritic cell protocols.

Similar approaches have recently been reported concerning enhancing antitumor immune responses. Others investigated tumor pretreatments, including irradiation, ethanol injection, microwave coagulation, radiofrequency ablation, and cryoablation. Combinations of pre-irradiation and i.t. dendritic cell (41), radiofrequency ablation pretreatment and anti-CTLA4 mAb administration (42), and cryoablative pretreatment and i.t. immature dendritic cells (43) resulted in enhanced antitumor immune responses and antitumor effects. The last one is similar to our protocol. They showed the inhibition of 3LL lung cancer metastases and rejection of rechallenged B16 melanoma. In our study, by using BCG-CWS–treated dendritic cells, we further showed strong antitumor effects sufficiently to regress relatively large established tumors at remote sites, and fine analysis on the mechanisms for the antitumor effects revealed the importance of CD8+ tumor antigen-specific CTL and manipulation of immunosuppressive Treg. In terms of dendritic cell–maturating reagents, we observed that other TLR-stimulating reagents, such as OK-432, have similar augmenting effects on antitumor activity. It was reported that combining an active component of OK-432, OK-PSA, and i.t. dendritic cells had better antitumor effect on the LL/2 lung cancer in the C57BL/6 mouse model (44).

In the clinical setting, destruction of human hard tumor is required before dendritic cell administration; thus, irradiation alone may not be a choice of pretreatment. Heat ablations, such as microwave coagulation and radiofrequency ablation, are attractive pretreatments because high temperature denatures tumor proteins with adverse effect, including immunosuppression, while maintaining primary protein structure sufficient enough to generate T-cell epitopes. However, heat ablation is restricted to relatively small tumors because of the high risk of relatively large vessel damage. On the other hand, cryoablation can be applied more safely towards large tumors because its damage mechanism is the occlusion of small vessels (45), although it may induce immunologic tolerance by rapid systemic release of immunosuppressive proteins and tumor antigens. Nevertheless, cryoablation for liver, lung, and renal cell cancers has successfully been applied without major complications in Keio University, and we focused on cryoablation in this study.

Clinical trials of i.t. immature dendritic cells have recently been reported. One protocol for patients with breast cancer and melanoma resulted in partial regression of tumor in which dendritic cells were given, and of some surrounding metastases, but distant metastases did not recede (46). The other trial using immature dendritic cell adenovirally transduced with IL-12 cDNA for patients with pancreatic, colorectal, or primary liver cancer resulted in one partial response in liver metastases of pancreatic cancer by injecting dendritic cell into primary tumor (47). Our current clinical trial investigating i.t. keyhole limpet hemocyanin–pulsed immature dendritic cells for patients with colon and esophageal cancers showed induction of systemic immune response6 even by dendritic cell administrations into large tumors probably with immunosuppressive microenvironments. These clinical observations suggest that there is a relatively weak induction of systemic immune reactions and antitumor effect by i.t. immature dendritic cells alone. Therefore, we are currently conducting clinical trials of the combinatorial immunotherapy consisting of cryoablative tumor pretreatment and i.t. injection of BCG-CWS–treated dendritic cells for patients with metastatic melanoma.


   Acknowledgments
 
We thank Drs. Tsukasa Seya and Takashi Akazawa for instruction on BCG-CWS usage, Drs. Hideaki Tahara and Marimo Sato for advice concerning dendritic cell generation, and Starlyn Okada, Misako Horikawa, and Ryoko Suzuki for preparation of the article.


   Footnotes
 
Grant support: Cancer Translational Research Program and grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Japan Society for Promotion of Science.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

6 Unpublished observations. Back

Received 7/26/06; revised 9/26/06; accepted 10/ 4/06.


   References
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 Materials and Methods
 Results
 Discussion
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