Several chemokines are used for immunotherapy against cancers because they can attract immune cells such as dendritic and cytotoxic T cells to augment immune responses. Radiofrequency ablation (RFA) is used to locally eliminate cancers such as hepatocellular carcinoma (HCC), renal cell carcinoma, and lung cancer. Because HCC often recurs even after an eradicative treatment with RFA, additional immunotherapy is necessary. We treated tumor-bearing mice by administering ECI301, an active variant of CC chemokine ligand 3, after RFA. Mice were injected s.c. with BNL 1ME A.7R.1, a murine hepatoma cell line, in the bilateral flank. After the tumor became palpable, RFA was done on the tumor of one flank with or without ECI301. RFA alone eliminated the treated ipsilateral tumors and retarded the growth of contralateral non–RFA-treated tumors accompanied by massive T-cell infiltration. Injection of ECI301 augmented RFA-induced antitumor effect against non–RFA-treated tumors when administered to wild-type or CCR5-deficient but not CCR1-deficient mice. ECI301 also increased CCR1-expressing CD11c+ cells in peripheral blood and RFA-treated tumors after RFA. Deficiency of CCR1 impairs accumulation of CD11c+, CD4+, and CD8+ cells in RFA-treated tumors. Furthermore, in IFN-γ-enzyme-linked immunospot assay, ECI301 augmented tumor-specific responses after RFA whereas deficiency of CCR1 abolished this augmentation. Thus, we proved that ECI301 further augments RFA-induced antitumor immune responses in a CCR1-dependent manner. Cancer Res; 70(16); 6556–65. ©2010 AACR.

Chemokines are a class of candidate molecules for immunotherapy. Chemokines are presumed to play an essential role in the regulation of leukocyte trafficking and dendritic cell-T-cell interactions (14). In animal experiments, intratumoral use of chemokines, such as monocyte chemoattractant protein-1/CC chemokine ligand 2 (CCL2), macrophage inflammatory protein (MIP)-1α/CCL3, or MIP-3α/CCL20, succeeds in decreasing tumorigenesis accompanied by increase in the numbers of tumor-infiltrating dendritic, natural killer, or T cells (57). Thus, application of chemokines in immunotherapy is promising but needs further refinement before they can be used in clinical situations.

Radiofrequency ablation (RFA) is an eradicative treatment against cancers, such as hepatocellular carcinoma (HCC), renal cell carcinoma, and lung cancer. RFA of HCC can generate HCC-specific T cells in peripheral blood (8). Activation of dendritic cells in human peripheral blood is also observed after this treatment (9). Thus, RFA can induce immunogenic tumor cell death and subsequently tumor-specific immune responses (811). However, multicentric development of HCC in the cirrhotic liver frequently results in tumor recurrence even after the apparent curative treatment of HCC by RFA (12). These observations suggest that RFA-induced tumor-specific immune responses are often not sufficient to prevent tumor recurrence. Thus, additional treatment modalities are required to augment HCC-specific immune responses.

CCL3/MIP-1α can augment immune responses but problems arise because of its tendency to form large aggregates at high concentrations when administered systemically. Unlike human naïve CCL3, BB-10010 is generated by a single amino acid substitution of Asp26 to Ala and exhibits similar biological potencies, but rarely forms large aggregates (13). Based on its activity to mobilize bone marrow cells to peripheral blood, randomized clinical trials were performed to examine whether the combined administration of BB-10010 and chemotherapeutic agents can protect against chemotherapy-induced neutropenia. However, the myeloprotective effects of BB-10010 were not sufficient to warrant its use with chemotherapy (14). Concomitantly, several lines of evidence reveal that the administration of human recombinant CCL3 can mobilize activated T-cell and dendritic cell precursors into circulation (15, 16).

ECI301, which has the same amino acid sequence as BB-10010, was generated using the fission yeast (Schizosaccharomyces pombe) expression system. ECI301 can augment irradiation-induced tumor regression when administered systemically to mice bearing multiple subcutaneous tumors (17). Of interest is the fact that the effects were observed in both unirradiated and irradiated tumors. Thus, systemic ECI301 treatment can augment irradiation-induced tumor-specific systemic immunity. These observations prompted us to investigate the effects of ECI301 on RFA-treated mice. Here, we show that ECI301 further augments RFA-induced antitumor immune responses in a CCR1-dependent manner.

Mice

Seven- to 9-week-old specific pathogen–free female BALB/c mice were purchased from Charles River Japan and designated as wild-type (WT) mice. BALB/c-nu/nu mice were purchased from CLEA Japan. CCR1-deficient (CCR1−/−) mice were a gift from Dr. Philip M. Murphy (National Institute of Allergy and Infectious Disease, NIH, Bethesda, MD); CCR5-deficient (CCR5−/−) mice were a gift from Dr. Kouji Matsushima (Department of Molecular Preventive Medicine, Tokyo University, Tokyo, Japan). All mice were backcrossed to BALB/c mice for 8 to 10 generations. All animal experiments were performed under specific pathogen–free conditions in accordance with the Guidelines for the Care and Use of Laboratory Animals of Kanazawa University (Japan).

Tumor cell line

A murine HCC cell line, BNL 1ME A.7R.1 (BNL), was purchased from the American Type Culture Collection in 1998 and kept at low passage throughout the study. The cells were screened for bacteria, fungus, and Mycoplasma contamination by direct culture method in 2006 before start of the study. The cells were cultured in DMEM (Sigma Chemical Co.) containing 10% fetal bovine serum (FBS), 0.1 mmol/L nonessential amino acids, 1 μmol/L sodium pyruvate, 2 mmol/L l-glutamine, 50 μg/mL streptomycin, and 100 units/mL penicillin (Life Technologies, Inc.).

Animal models

ECI301 was generated as previously described and provided by Effector Cell Institute, Inc. (17, 18). The left and right flanks of 7- to 9-week-old female WT, CCR1−/−, CCR5−/−, and nu/nu mice were injected s.c. with 5 × 105 BNL cells in 100 μL of PBS. Fourteen days later, when tumor size reached a diameter of 6 to 8 mm, tumors of one flank were treated using a radiofrequency generator (RITA 500PA, RITA Medical Systems) and needle as described below. On days 0, 2, and 4 after RFA, 20 μg of ECI301 in 100 μL of PBS were injected i.v. via the tail vein, whereas mice treated with RFA alone were injected with 100 μL of PBS. Untreated tumor-bearing mice were used as controls. In another schedule, 2 μg of ECI301 in 100 μL of PBS were injected i.v. from day 0 to day 4 (5 consecutive days). The sizes of non–RFA-treated tumors on the contralateral flank were evaluated twice a week using calipers, and tumor volumes were calculated using the following formula: tumor volume (mm3) = (longest diameter) × (shortest diameter)2 / 2.

RFA-treated or non–RFA-treated tumors were excised at the indicated time intervals for immunohistochemical analysis and quantitative real-time reverse transcription-PCR (RT-PCR). Spleens and peripheral blood were removed from the mice at the indicated time intervals for flow cytometric analysis and enzyme-linked immunospot assay (ELISPOT).

Radiofrequency ablation

Mice were anesthetized by i.p. injection of Somnopentyl (Schering-Plough Animal Health) and carefully shaved in the tumor area. After placing the mice onto an aluminum plate attached with an electricity-conducting pad, an RFA needle of expandable electrode with maximum dimension of 20 mm (70SB 2 cm; RITA Medical Systems) was inserted into the middle of the tumors and expanded at 2 or 3 mm. RFA treatments were done using a radiofrequency generator at a power output of 25 W for 1.5 minutes and the temperature of the needle tips reached 70°C to 80°C.

Immunohistochemical analysis

The removed tumor tissues were embedded in Sakura Tissue-Tek optimum cutting temperature (OCT) compound (Sakura Finetek) as frozen tissues. Cryostat sections of the frozen tissues were fixed with 4% paraformaldehyde in PBS and stained with rat anti-mouse CD4 (BD Biosciences), rat anti-mouse CD8a (BD Biosciences), hamster anti-mouse CD11c (BD Biosciences), and rat anti-mouse F4/80 antibodies (Serotec) overnight at 4°C. The sections were then incubated with biotinylated rabbit anti-rat IgG (DakoCytomation) or biotinylated mouse anti-hamster IgG (BD Biosciences) for 1 hour at room temperature. The immune complexes were visualized using the Catalyzed Signal Amplification System (DakoCytomation) or the Vectastain Elite ABC and DAB substrate kits (Vector Laboratories) according to the manufacturer's instructions. As a negative control, rat IgG (Cosmo Bio) or hamster IgG (BD Biosciences) was used instead of specific primary antibodies. The numbers of positive cells in each animal were counted in 10 randomly selected fields at 400-fold magnification by an examiner without any prior knowledge of the experimental procedures.

Double-color immunofluorescence analysis

Tumor tissues were embedded in OCT compound as frozen tissues. After fixation with 4% paraformaldehyde/PBS, cryostat sections were stained with the combinations of anti-CD4 and goat anti-mouse CCR1 (Santa Cruz Biotechnology), anti-CD8a and anti-CCR1, anti-F4/80 and anti-CCR1, phycoerythrin (PE)-conjugated hamster anti-CD11c (BD Biosciences) and anti-CCR1, anti-F4/80 and goat anti-mouse CCL3 (R&D Systems), and anti-F4/80 and goat anti-mouse CCL4 antibodies (R&D). After extensive washing, AF488 donkey anti-rat IgG (Invitrogen) was used as a secondary antibody to detect CD4+, CD8a+, or F4/80+ cells. Simultaneously, AF546- or AF488-donkey anti-goat IgG (Invitrogen) was used to detect CCR1+, CCL3+, or CCL4+ cells. The sections were observed using a confocal microscope (LSM 510 META, Zeiss).

Quantitative real-time RT-PCR

Total RNA was extracted from the resected tumor using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. After treating the RNA preparations with RNase-free DNase I (Qiagen) to remove residual DNA, cDNA was synthesized as described previously (19). Quantitative real-time PCR was done on a StepOne Real-Time PCR System (Applied Biosystems) using the comparative CT quantification method. TaqMan Gene Expression Assays (Applied Biosystems) containing specific primers and probes [accession numbers: CCL3, Mm00441258_ml; CCL4, Mm00443111_m1; CCL5, Mm01302428_ml; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Mm99999915_g1] and TaqMan Fast Universal PCR Master Mix were used with 10 ng of cDNA to quantify the expression levels of CCL3, CCL4, and CCL5. Reactions were performed for 20 seconds at 95°C followed by 40 cycles of 1 second at 95°C and 20 seconds at 60°C. GAPDH was amplified as an internal control and its CT values were subtracted from the CT values of the target genes (ΔCT). The ΔCT values of tumors after RFA with or without ECI301 were compared with the ΔCT values of tumors of untreated mice.

Enzyme-linked immunospot assay

To prepare tumor lysates, BNL or CT26 cells were suspended in PBS and subjected to four cycles of rapid freezing in liquid nitrogen and thawing at 55°C. The lysate was spun at 15,000 rpm to remove particulate cellular debris. After harvesting murine spleens on day 21 after RFA, mononuclear cells were isolated by centrifugation through a Histopaque-1083 density gradient (Sigma Chemical). ELISPOT was performed using an IFN-γ-ELISPOT kit (Mabtech). Ninety-six-well plates coated with anti-mouse IFN-γ antibody were blocked for 2 hours with RPMI 1640 (Sigma Chemical) containing 10% FBS. Two hundred fifty thousand splenic mononuclear cells were added in triplicate cultures of RPMI 1640 containing 10% FBS together with BNL or CT26 lysates at a tumor cell-to-mononuclear cell ratio of 2:1. After 48 hours of culture, the plates were washed eight times with sterile PBS and further incubated for 2 hours with biotinylated anti-mouse IFN-γ antibody. After another eight washes, alkaline phosphatase–conjugated streptavidin was added to these plates and incubated for 1 hour. Finally, the spots were developed with nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate solution. The number of specific spots was determined by subtracting the number of spots in wells without lysates from the number of spots in wells with tumor lysates. Wells were considered positive if they had more than 10 spots per well and were at least 2-fold greater than control.

Flow cytometric analysis

After harvesting blood samples from mice, mononuclear cells were isolated by centrifugation through a Histopaque-1083 density gradient (Sigma Chemical). The resultant single-cell preparations were stained with various combinations of allophycocyanin (APC)-labeled anti-CD8, APC-labeled anti-CD11c, FITC-labeled anti-CD4 (BD Biosciences), PE-labeled anti-CCR1 (Santa Cruz Biotechnology), and FITC-labeled anti-F4/80 monoclonal antibodies (Serotec). APC-rat IgG, APC-hamster IgG, and FITC-rat IgG were used as isotype controls (BD Biosciences). For each determination, at least 20,000 stained cells were analyzed on a FACSCalibur system (BD Biosciences). The data were expressed as the proportion of positive cells (compared with cells stained with an irrelevant control antibody).

Depletion of macrophages/monocytes

Clodronate liposome was prepared and systemic depletion of monocytes/macrophages was performed as previously described (20, 21). WT mice were i.p. injected with 200 μL of clodronate liposome five times: days −2, 0, 3, 6, and 10 after RFA treatment. Depletion of CD11c-negative monocytes in blood was confirmed by flow cytometry after injection of clodronate liposome.

Statistical analysis

Mean and SD or SE were calculated for the obtained data. Data were analyzed statistically using one-way ANOVA followed by Fisher's protected least significant difference test, except for the data of tumor growth, which were analyzed with two-way ANOVA. P < 0.05 was considered statistically significant.

ECI301 augments RFA-induced antitumor effects

To investigate the effects of RFA against RFA-treated and non–RFA-treated tumors, each bilateral flank of BALB/c mice was injected with 5 × 105 BNL cells. Fourteen days later, when tumor size reached a diameter of 6 to 8 mm, tumors of one flank were treated with RFA. On the day after RFA, ulceration occurred in RFA-treated tumors, and these tumors started to shrink (data not shown). On day 14 after RFA, RFA-treated tumors were covered with scars without any macroscopic tumors (Fig. 1A). Moreover, RFA treatment also retarded the growth of contralateral non–RFA-treated tumors compared with the tumors in untreated mice (Fig. 1B and C). ECI301 (20 μg/mouse) administered on days 0, 2, and 4 after RFA augmented RFA-induced growth retardation of contralateral non–RFA-treated tumors (Fig. 1B and C). Furthermore, non–RFA-treated tumors completely disappeared in 2 of 15 mice treated with RFA and ECI301 but not in the other treatment groups (Fig. 1B and C). Therapeutic effects were observed, even when ECI301 (2 μg/mouse) was injected consecutively for 5 days from day 0 to day 4 after RFA. On the contrary, administration of ECI301 without RFA did not result in a significant decrease in tumor size (Fig. 1C). These observations suggest that ECI301 can augment RFA-induced antitumor effects but fails to induce antitumor effects by itself.

Figure 1.

ECI301-induced augmentation of antitumor effects after RFA. WT mice were injected s.c. with 5 × 105 BNL cells into the left and right flanks. Fourteen days later, when tumors became palpable, tumors of one flank were treated using the RFA generator and needle. On day 0, 2, and 4 after RFA, 20 μg of ECI301 in 100 μL of PBS were injected i.v. into each mouse, whereas mice treated with RFA alone were injected with 100 μL of PBS. Tumor-bearing untreated mice were observed as controls. A, macroscopic appearances of the mice on day 14 after RFA are shown. Arrowheads indicate the scar after RFA. Representative results are from at least 10 mice in each group. B, non–RFA-treated tumor volumes after RFA with or without ECI301 were measured twice a week. Points, mean; bars, SE. *, P < 0.05; ***, P < 0.001. C, volumes of non–RFA-treated tumors on day 14 after RFA. In addition to the groups described in B, tumor volumes were determined in animals receiving 2 μg of ECI301 in 100 μL of PBS i.v. from day 0 to day 4 (5 consecutive days) after RFA and those receiving 20 μg of ECI301 alone without RFA. Columns, mean; bars, SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with untreated mice.

Figure 1.

ECI301-induced augmentation of antitumor effects after RFA. WT mice were injected s.c. with 5 × 105 BNL cells into the left and right flanks. Fourteen days later, when tumors became palpable, tumors of one flank were treated using the RFA generator and needle. On day 0, 2, and 4 after RFA, 20 μg of ECI301 in 100 μL of PBS were injected i.v. into each mouse, whereas mice treated with RFA alone were injected with 100 μL of PBS. Tumor-bearing untreated mice were observed as controls. A, macroscopic appearances of the mice on day 14 after RFA are shown. Arrowheads indicate the scar after RFA. Representative results are from at least 10 mice in each group. B, non–RFA-treated tumor volumes after RFA with or without ECI301 were measured twice a week. Points, mean; bars, SE. *, P < 0.05; ***, P < 0.001. C, volumes of non–RFA-treated tumors on day 14 after RFA. In addition to the groups described in B, tumor volumes were determined in animals receiving 2 μg of ECI301 in 100 μL of PBS i.v. from day 0 to day 4 (5 consecutive days) after RFA and those receiving 20 μg of ECI301 alone without RFA. Columns, mean; bars, SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001, compared with untreated mice.

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Deficiency of CCR1 abrogates increased antitumor effect of ECI301 after RFA

ECI301 uses two distinct chemokine receptors, CCR1 and CCR5. To elucidate the roles of these chemokine receptors, either tumor-bearing CCR1−/− or CCR5−/− mice were similarly treated with RFA plus ECI301. RFA retarded the growth of non–RFA-treated tumors in CCR1−/− mice similar to that in WT mice, but ECI301 failed to further accentuate RFA-induced growth retardation of non–RFA-treated tumors (Fig. 2A). In contrast, ECI301 augmented RFA-mediated inhibition of non–RFA-treated tumors in CCR5−/− mice, resulting in complete tumor eradication in one of six mice (Fig. 2B). These observations indicate that CCR1-expressing, but not CCR5-expressing, cells play an important role in ECI301-induced augmentation of tumor regression after RFA.

Figure 2.

Deficiency of CCR1 abrogates ECI301-augmented antitumor effects after RFA. CCR1−/− or CCR5−/− mice were inoculated with BNL cells and treated as described in the legend to Fig. 1. Non–RFA-treated tumor volumes were measured twice a week in CCR1−/− (A) and CCR5−/− (B) mice. Points, mean; bars, SE. *, P < 0.05; **, P < 0.01.

Figure 2.

Deficiency of CCR1 abrogates ECI301-augmented antitumor effects after RFA. CCR1−/− or CCR5−/− mice were inoculated with BNL cells and treated as described in the legend to Fig. 1. Non–RFA-treated tumor volumes were measured twice a week in CCR1−/− (A) and CCR5−/− (B) mice. Points, mean; bars, SE. *, P < 0.05; **, P < 0.01.

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ECI301 increases CCR1-expressing cells in peripheral blood and RFA-treated tumors after RFA

The reported capacity of CCL3 to mobilize leukocytes into peripheral blood (15, 16) prompted the investigation of the effects of ECI301 on peripheral blood. RFA alone had few effects on the numbers of CCR1-expressing cells, but subsequent ECI301 administration increased the numbers of CCR1-expressing cells in peripheral blood, particularly CD11c+ cells, but not CD4+ or CD8+ cells (Fig. 3). Because immune cells need to accumulate in RFA-treated tumors at an early stage to initiate adaptive immune responses, CCR1 expression by tumor-infiltrating cells in RFA-treated tumors was examined 8 hours after treatment. RFA-induced CD4+, CD8+, CD11c+, and F4/80+ cell infiltrations into RFA-treated tumors were greater than those into tumors of untreated mice. Moreover, ECI301 further increased the numbers of CD4+, CD8+, and CD11c+ cells infiltrating into RFA-treated tumors compared with the numbers of these cells infiltrating into tumors treated with RFA alone (Fig. 4A and B). In RFA-treated tumors, most CD11c+ and F4/80+ cells expressed CCR1, whereas few CD4+ and CD8+ cells expressed CCR1 (Fig. 4C). Furthermore, ECI301-induced CD4+, CD8+, and CD11c+ cell infiltrations into ablated tumors were lesser in CCR1−/− mice than in WT mice, whereas F4/80+ cells infiltrated RFA-treated tumors in CCR1−/− mice and WT mice to a similar extent (Fig. 4D). These observations suggest that ECI301 augments RFA-induced CD4+, CD8+, and CD11c+ cell infiltrations into RFA-treated tumors in a CCR1-dependent manner.

Figure 3.

ECI301 increases CCR1-expressing cells in peripheral blood. Peripheral blood sample was harvested 8 h after RFA. Mononuclear cells were separated and stained with the indicated antibodies as described in Materials and Methods. Columns, mean percentages of CCR1+, CCR1+CD4+, CCR1+CD8+, CCR1+CD11c+, or CCR1+F4/80+ cells (n = 3); bars, SD. *, P < 0.05.

Figure 3.

ECI301 increases CCR1-expressing cells in peripheral blood. Peripheral blood sample was harvested 8 h after RFA. Mononuclear cells were separated and stained with the indicated antibodies as described in Materials and Methods. Columns, mean percentages of CCR1+, CCR1+CD4+, CCR1+CD8+, CCR1+CD11c+, or CCR1+F4/80+ cells (n = 3); bars, SD. *, P < 0.05.

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Figure 4.

ECI301 increases infiltration of CCR1-expressing leukocytes into RFA-treated tumors after RFA. RFA-treated tumors were removed from WT or CCR1−/− mice 8 h after RFA. A, B, and D, immunohistochemical analysis was performed using anti-CD4, anti-CD8a, anti-CD11c, or anti-F4/80 antibodies. A, representative results from three individual WT mice in each group. Original magnification, ×400. Bar, 50 μm. B and D, the numbers of CD4+, or CD8+, CD11c+, or F4/80+ cells were counted. Cell density was determined in 10 randomly chosen tumor areas at 400-fold magnification. Columns, mean (n = 5); bars, SD. **, P < 0.01. C, RFA-treated tumor tissues were processed using double-color immunofluorescence analysis as described in Materials and Methods. Right, digitally merged images. Representative results from three individual animals. Original magnification, ×400. Bar, 50 μm.

Figure 4.

ECI301 increases infiltration of CCR1-expressing leukocytes into RFA-treated tumors after RFA. RFA-treated tumors were removed from WT or CCR1−/− mice 8 h after RFA. A, B, and D, immunohistochemical analysis was performed using anti-CD4, anti-CD8a, anti-CD11c, or anti-F4/80 antibodies. A, representative results from three individual WT mice in each group. Original magnification, ×400. Bar, 50 μm. B and D, the numbers of CD4+, or CD8+, CD11c+, or F4/80+ cells were counted. Cell density was determined in 10 randomly chosen tumor areas at 400-fold magnification. Columns, mean (n = 5); bars, SD. **, P < 0.01. C, RFA-treated tumor tissues were processed using double-color immunofluorescence analysis as described in Materials and Methods. Right, digitally merged images. Representative results from three individual animals. Original magnification, ×400. Bar, 50 μm.

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ECI301 increases intratumoral expression of CCL3 after RFA

We showed that CCR1+ cells were mobilized into blood by i.v. administered ECI301. However, the concentration of ECI301 in blood can go down rapidly as time passes (the peak is 5 minutes, and the half-life is <2 hours),4

4Unpublished data from Effector Cell Institute.

allowing ECI301-mobilized CCR1+ cells to migrate into tissues where chemokines are highly produced. To prove this point, chemokine expression in RFA-treated tumors was examined. RFA plus ECI301 treatment increased CCL3 mRNA expression level 6 hours after RFA. Moreover, 24 hours after treatment, CCL3 and CCL4 mRNA expression levels became almost 10-fold higher in tumors treated with RFA alone than in tumors of untreated mice, and ECI301 further increased the mRNA expression level of these chemokines in RFA-treated tumors (Fig. 5A). CCL3 and CCL4 were detected in tumor-infiltrating F4/80+ cells (Fig. 5B). These observations indicate that RFA treatment causes local production of CCL3 and CCL4 in RFA-treated tumors and ECI301 further increases the expression of these chemokines. As the concentration of ECI301 in blood decreases, chemokines produced locally in RFA-treated tumor can attract CCR1-expressing CD11c+ cells, thereby indirectly inducing CCR1-negative CD4+ and CD8+ cell infiltrations.

Figure 5.

Increased CCL3 expression in RFA-treated tumors. A, real-time RT-PCR was performed on total RNA extracted from RFA-treated tumors of WT mice. The tumors were harvested 6 h (left) or 24 h (right) after RFA. Chemokine mRNA levels were normalized to GAPDH mRNA levels. Columns, mean (n = 3); bars, SD. *, P < 0.05, compared with untreated mice. B, RFA-treated tumors were removed from WT mice on day 1 after RFA plus ECI301 treatment and immunostained with the indicated combinations of antibodies as described in Materials and Methods. Right, digitally merged images. Representative results from three individual animals. Original magnification, ×400. Bar, 50 μm.

Figure 5.

Increased CCL3 expression in RFA-treated tumors. A, real-time RT-PCR was performed on total RNA extracted from RFA-treated tumors of WT mice. The tumors were harvested 6 h (left) or 24 h (right) after RFA. Chemokine mRNA levels were normalized to GAPDH mRNA levels. Columns, mean (n = 3); bars, SD. *, P < 0.05, compared with untreated mice. B, RFA-treated tumors were removed from WT mice on day 1 after RFA plus ECI301 treatment and immunostained with the indicated combinations of antibodies as described in Materials and Methods. Right, digitally merged images. Representative results from three individual animals. Original magnification, ×400. Bar, 50 μm.

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ECI301 augments RFA-induced tumor-specific immune responses accompanied by T-cell infiltrations into non–RFA-treated tumors

Non–RFA-treated tumors were analyzed histologically to clarify the mechanisms underlying the CCR1-dependent inhibitory effect of RFA plus ECI301 treatment against these tumors. Although few CD4+ or CD8+ cells were observed in the tumors of untreated mice, RFA treatment increased the numbers of CD4+ and CD8+ cells in the non–RFA-treated tumors 3 days after RFA. ECI301 further augmented RFA-induced CD4+ and CD8+ cell infiltrations into non–RFA-treated tumors (Fig. 6A and B). However, only a marginal number of CD11c+ or F4/80+ cells infiltrated into non–RFA-treated tumors of mice treated with RFA alone or RFA plus ECI301-treated mice (data not shown). Based on these findings, we hypothesized that ECI301-augmented tumor regression after RFA may be associated with T-cell–mediated antitumor immune responses. To clarify this point, nu/nu mice on a BALB/c background were treated by RFA with or without ECI301. Deficiency of T cells abrogated the tumor-inhibitory effect of ECI301 as well as the RFA-induced antitumor effect (Fig. 6C). Thus, both ECI301- and RFA-induced tumor regressions require T-cell–mediated antitumor immune response.

Figure 6.

ECI301 augments tumor-specific immune responses after RFA. A, non–RFA-treated tumors were removed from WT mice on day 3 after RFA, and immunohistochemical analysis was done using anti-CD4 or anti-CD8a antibodies. Representative results from three individual animals in each group. Original magnification, ×400. Bar, 50 μm. B, the numbers of CD4+ or CD8+ cells were counted. Cell density was determined in 10 randomly chosen tumor areas at 400-fold magnification. Columns, mean (n = 5); bars, SE. **, P < 0.01. C, BALB/c-nu/nu mice (left) and BALB/c-WT mice (right) were inoculated with BNL cells and treated as described in the legend to Fig. 1. In the RFA + ECI301 + clodronate liposome (CL) group, the mice were injected with 200 μL of CL to deplete monocytes/macrophages as described in Materials and Methods. Non–RFA-treated tumor volumes were measured twice a week. Points, mean; bars, SE. D, spleens from WT, CCR1−/−, or CCR5−/− mice were harvested on day 21 after RFA, and mononuclear cells were separated from the spleens for ELISPOT assay as described in Materials and Methods. The number of specific spots was determined by subtracting the number of spots in wells without lysates from the number of spots in wells with tumor lysates. Columns, mean (n = 3); bars, SE. *, P < 0.05; **, P < 0.01, compared with untreated WT mice.

Figure 6.

ECI301 augments tumor-specific immune responses after RFA. A, non–RFA-treated tumors were removed from WT mice on day 3 after RFA, and immunohistochemical analysis was done using anti-CD4 or anti-CD8a antibodies. Representative results from three individual animals in each group. Original magnification, ×400. Bar, 50 μm. B, the numbers of CD4+ or CD8+ cells were counted. Cell density was determined in 10 randomly chosen tumor areas at 400-fold magnification. Columns, mean (n = 5); bars, SE. **, P < 0.01. C, BALB/c-nu/nu mice (left) and BALB/c-WT mice (right) were inoculated with BNL cells and treated as described in the legend to Fig. 1. In the RFA + ECI301 + clodronate liposome (CL) group, the mice were injected with 200 μL of CL to deplete monocytes/macrophages as described in Materials and Methods. Non–RFA-treated tumor volumes were measured twice a week. Points, mean; bars, SE. D, spleens from WT, CCR1−/−, or CCR5−/− mice were harvested on day 21 after RFA, and mononuclear cells were separated from the spleens for ELISPOT assay as described in Materials and Methods. The number of specific spots was determined by subtracting the number of spots in wells without lysates from the number of spots in wells with tumor lysates. Columns, mean (n = 3); bars, SE. *, P < 0.05; **, P < 0.01, compared with untreated WT mice.

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However, CD4+ or CD8+ T cells rarely expressed CCR1 in blood and RFA-treated tumors. CCR1+ cells in RFA-treated tumors were CD11c+ cells and F4/80+ cells, and only the former accumulate in RFA-treated tumors in a CCR1-dependent manner. These findings suggest that CCR1-positive CD11c+ cells may activate antitumor T-cell responses and indirectly induce tumor retardation. Accordingly, we next examined the effect of depletion of monocytes/macrophages on ECI301-augmented tumor regression. I.p. injection of clodronate liposome depleted CD11c-negative monocytes in blood, although it did not change the number of CD11c+ cells (data not shown). Depletion of these CD11c-negative monocytes did not cause any effects on ECI301-enhanced tumor regression, indicating that ECI301-augmented antitumor T-cell immunity was independent of CD11c-negative monocytes (Fig. 6C).

Finally, to prove the presence of systemic adaptive immune responses, IFN-γ ELISPOT assay was performed using mononuclear cells from the spleen. A greater number of spots against BNL cell lysates, but not against CT26 cell lysates, were generated by RFA plus ECI301–treated mice than that by mice treated with RFA alone or untreated mice. Moreover, ablation of CCR1 gene, but not CCR5 gene, reduced the number of spots against BNL cell lysates even when the mice were treated with RFA plus ECI301 (Fig. 6D). These observations suggest that ECI301 can further augment RFA-induced tumor-specific adaptive immune responses and subsequent tumor retardation in a CCR1-dependent manner.

HCC occurs predominantly in individuals with chronic liver disease related to hepatitis B or hepatitis C virus infections (2224). In addition to surgical resection, RFA treatment has been developed to eradicate solitary HCC lesions (25). RFA of HCC induces specific T-cell responses against liver tumors in human and rabbit (8, 11). Moreover, activated dendritic cells were detected in peripheral blood of HCC patients after RFA (9). These previous reports indicate that RFA treatment can induce antitumor immune responses against HCC (811). Likewise, we observed that RFA treatment generated tumor-specific IFN-γ–producing cells and inhibited the growth of non–RFA-treated tumors accompanied by massive T-cell infiltration into these tumors. However, even after successful ablation of HCC lesion by RFA, tumor recurrence often occurs probably because HCC develops in a multicentric manner in the cirrhotic liver (12). These observations indicate that RFA-induced augmentation in immune response may not be sufficient to prevent tumor recurrence. Thus, a novel therapeutic modality is required to further augment RFA-induced tumor-specific immune responses. Here, we showed that combined administration of ECI301 and RFA can augment tumor-specific immune responses against HCC.

Several chemokines are used for immunotherapy against cancers because they can attract immune cells such as dendritic and cytotoxic T cells to augment tumor-specific immune responses (26). However, some chemokines can simultaneously attract myeloid-derived suppressor and regulatory T cells to promote neovascularization and induce immunosuppressive microenvironments (2628). The double-edged activities of chemokines frequently preclude their use for tumor immunotherapy. Moreover, most chemokines exhibit a bell-shaped dose-response curve with a narrow effective dose window. Thus, determination of an optimal dose of chemokines is important to elicit efficient antitumor responses (29). Several lines of evidence show that intratumoral use of CCL3 reduces tumorigenicity (6, 30). Furthermore, there are no reports showing that use of CCL3 can promote tumor progression. We observed that systemic administration of ECI301 without RFA treatment induced neither reduction nor progression of tumors. On the contrary, systemic injection of ECI301 after RFA can inhibit the growth of non–RFA-treated tumors in the contralateral side. ECI301-enhanced tumor regression after RFA was both CCR1 and T-cell dependent, but T cells rarely expressed CCR1 in blood and RFA-treated tumors. Because depletion of monocytes/macrophages did not affect the retardation of ECI301-treated tumors, CCR1-expressing CD11c+ dendritic cells might activate antitumor T-cell responses and indirectly induce tumor retardation via some mechanisms such as antigen presentation and cytokine production. ECI301 mobilized a large number of CCR1+ cells into blood, and these mobilized cells may be attracted into highly CCL3-producing RFA-treated tumors and cause increased number of tumor-infiltrating CCR1+CD11c+ dendritic cells. Thus, CCR1+ precursors in blood and CCR1+CD11c+ tumor-infiltrating dendritic cells might play important roles in ECI301-augmented antitumor effects (3133).

ECI301 could not increase the number of F4/80+ cells in the RFA-treated tumor sites. Accumulation of F4/80+ cells in the tumor treated with ECI301 plus RFA was also independent of CCR1. F4/80+ cells, which might include a large number of macrophages/monocytes, are usually attracted into the tumor by CCL2, CCL4, and CCL5 that are produced in the tumor sites. CCR2, the receptor for CCL2, and CCR5, the receptor for CCL4 and CCL5, might be responsible for migration of monocytes/macrophages (27, 3436). However, it is still unclear whether monocytes/macrophages use CCR2 or CCR5 after massive tumor cell death caused by treatments such as RFA because tumor cell death induces different profiles of chemokine production in the tumors (4). Although ECI301 did not directly induce migration of F4/80+ cells, the mechanism underlying the infiltration of F4/80+ macrophages remains to be elucidated.

Breaking tolerance for tumor cells is necessary for induction of antitumor immunity. Several independent groups have suggested multiple mechanisms underlying immunogenic tumor cell death induced by anticancer chemotherapy or radiation therapy (3740). Anthracyclin causes apoptosis along with translocation of calreticulin to the apoptotic tumor cell surface. Calreticulin exposure augments phagocytosis of apoptotic cancer cells by dendritic cells with an eventual increase in immune response (37, 38). Chemotherapy or irradiation kills tumor cells to release high mobility group box 1 (HMGB1). Released HMGB1 activates dendritic cells after binding to toll-like receptor 4 expressed by these cells (39). Apoptosis induced by local radiation therapy augments MHC class I expression by tumor cells, thereby facilitating their recognition by cytotoxic T cells (40). RFA induces the expression of heat shock proteins 70 and 90 on ablated tumor cells, and these proteins can activate toll-like receptor–expressing antigen-presenting cells (41, 42). In addition, we showed that RFA treatment alone caused local production of CCL3 in RFA-treated tumors accompanied by accumulation of T cells and CD11c+ dendritic cells. These mechanisms may also account for the observed RFA-induced generation of tumor-specific immune responses.

We revealed that combined treatment of ECI301 and RFA augmented antitumor-specific immune responses, thereby inhibiting the growth of non–RFA-treated tumors in a CCR1-dependent manner. Thus, combined treatment of ECI301 and RFA can prevent human HCC from recurring after RFA treatment. The absence of any severe adverse effects in mice (data not shown) further warrants the clinical trial of ECI301 combined with RFA as a treatment regimen for HCC.

No potential conflicts of interest were disclosed.

We thank Dr. Philip M. Murphy (National Institute of Allergy and Infectious Disease, NIH) and Dr. Kouji Matsushima (Tokyo University) for providing us with CCR1−/− and CCR5−/− mice, respectively.

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.

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