IP-10-encoding plasmid DNA therapy exhibits anti-tumor and anti-metastatic efficiency

We report here that the interferon-induced protein of 10 kDa (IP-10 or CXCL10) elicits strong anti-tumor and anti-metastatic responses in mice when administered by plasmid DNA. Intratumoral but not intramuscular IP-10 DNA inoculation resulted in reduced tumor formation of malignant melanoma (B16F10) and Lewis lung carcinoma (LL/2) in C57BL/6 mice. In addition, plasmid DNA-encoding IP-10 substantially reduced the establishment of metastases when injected systemically by the intramuscular route. In contrast to the primary tumor model, the anti-metastatic effect of DNA-encoding IP-10 was primarily mediated by NK cells. Compared to DNA-encoding interleukin-12 (IL-12), therapy with DNA-encoding IP-10 exhibits lower efficacy against primary melanoma tumors but equivalent efficacy against primary Lewis lung tumors and against B16F10 lung metastasis formation. Co-administration of DNA-encoding IP-10 and IL-12 enhanced the anti-tumor activity of IL-12 in the lung metastasis model but had little effect in the local treatment of established subcutaneous tumors. Interestingly, treatment of nude mice lacking T lymphocytes with DNA-encoding IP-10 or IL-12 still resulted in a pronounced reduction of tumor growth or metastasis formation.     

Anti-tumor activity of mesenchymal stem cells producing IL-12 in a mouse melanoma model

Mesenchymal stem cells (MSCs) represent a new tool for delivery of therapeutic agents to tumor cells. In this study, we have evaluated the anti-tumor activity of human MSCs stably transduced with a retroviral vector expressing the cytokine interleukin-12 (IL-12) in a mouse melanoma model. Application of MSC(IL-12) but not control MSCs strongly reduced the formation of lung metastases of B16F10 melanoma cells. The activity of the MSC(IL-12) cells was dependent on the presence of natural killer (NK) cells in this experimental setting. Further, MSC(IL-12) cells elicited a pronounced retardation of tumor growth and led to prolonged survival when injected into established subcutaneous melanoma in a therapeutic regimen. The therapeutic effect of the MSC(IL-12) was in part mediated by CD8(+) T cells, while NK cells and CD4(+) T cells appeared to play a minor role. The anti-tumor effect of MSC(IL-12) cells was of similar efficiency as observed for application of naked plasmid DNA encoding IL-12. The presented data demonstrate that these two different strategies can induce a similar therapeutic anti-tumor efficacy in the mouse melanoma tumor model.     

In vivo elimination of CD25+ regulatory T cells leads to tumor rejection of B16F10 melanoma, when combined with interleukin-12 gene transfer

CD4(+)CD25(+) T cells are an important population that plays a crucial role in the maintenance of peripheral self-tolerance. Recently, it was shown that the elimination of these cells by in vivo administration of anti-CD25 monoclonal antibody (mAb) caused the regression of highly immunogenic tumors in syngeneic mice. In this study, we examined whether B16F10 melanoma cells regressed with the elimination of CD25(+) regulatory T cells. We found the melanoma cells were not affected at all by in vivo anti-CD25 mAb administration alone but tumor rejection resulted in all mice when the administration was combined with IL-12 gene transfer to tumor cells. In vivo, depletion of natural killer (NK) cells or CD8(+) T cells cancelled the tumor rejection. NK-cell depletion allowed IL-12-transfected B16F10 melanoma (B16/IL-12) to grow from an early stage and resulted in a more rapid tumor growth of B16/IL-12 than that in mice without administration of anti-CD25 mAb. On the other hand, CD8(+) T-cell depletion did not affect the tumor growth in the early phase but allowed B16/IL-12 to grow in rather a late phase and resulted in almost the same degree of tumor growth as in mice without administration of anti-CD25 mAb. In a previous study, we showed that the elimination of CD4(+) T cells enhanced the antitumor effect of B16/IL-12 and induced vitiligo-like coat color alteration. Therefore, we also examined the frequency of the change to a vitiligo-like coat color in mice showing tumor rejection caused by CD25(+) T-cell elimination to compare with the mechanism enhancing the antitumor effects by cell elimination. The elimination of CD25(+) T cells did not induce vitiligo-like coat color changes, though that of CD4(+) T cells induced the change in 60% of mice. Furthermore, we confirmed that elimination of CD25(+) T cells did not affect the T-helper (Th) 1/Th2 cytokine profile, while that of CD4(+)T cells abrogated the Th2 cytokines (IL-4 and IL-10) and resulted in a Th1-dominant cytokine profile in the tumor-draining lymph nodes (TDLNs) of B16/IL-12-bearing mice. These results indicate that in vivo depletion of CD25(+) regulatory T cells is a potent useful adjuvant in immunotherapy of B16F10 melanoma, when combined with IL-12 gene transfer and that the enhancement of the antitumor effect by CD25(+) T-cell depletion is mediated through CD8(+) T cells and may differ from the enhancing mechanism caused by CD4(+) T-cell depletion.     

Topical CpG enhances the response of murine malignant melanoma to dacarbazine

Malignant melanoma is a potentially fatal skin cancer that is increasing in incidence. Standard chemoimmunotherapy consisting of dacarbazine (DTIC) given with IFN-alpha has had disappointing results. We describe a chemoimmunotherapy protocol for cutaneous melanoma that combines the administration of DTIC with the topical application of CpG oligodinucleotide (ODN). Subcutaneous B16 melanoma tumors in C57BL/6 mice were treated with intraperitoneal injections of DTIC followed by the topical application of CpG-ODN over the tumors. This therapeutic approach abrogated the growth of established tumors and significantly enhanced survival. Topical CpG application was more effective than intratumoral CpG. Cell depletion studies indicated that the antitumor effect was dependent on both CD4(+) and CD8(+) cells but not on natural killer (NK) cells. Tumor-specific cytotoxic T-lymphocyte activity was generated in treated animals and was highest in topically treated animals. Immunohistochemical analysis revealed that DTIC, but not CpG, enhanced tumor cell apoptosis. Further, topical CpG induced an expansion of a B220(+)CD8(+) subset of dendritic cells and a subset of NK1.1(+) CD11c(+) cells within the tumors. By enhancing both tumor cell death and local immune activation, DTIC/topical CpG chemoimmunotherapy induced an effective T-cell-dependent host-immune response against melanoma.     

Intracutaneous genetic immunization with autologous melanoma-associated antigen Pmel17/gp100 induces T cell-mediated tumor protection in vivo

Using the differentiation antigen Pmel17/gp100 to genetically immunize C57BL/6 mice (H-2(b)), we and colleagues noticed that only mice that had received the human homolog but not animals injected with the murine counterpart were protected against the growth of syngeneic B16 melanoma cells. The goal of this study was to determine whether the state of nonresponsiveness to the autoantigen Pmel17/gp100 can be broken by immunization with a plasmid DNA construct encoding the autologous form of the molecule. A construct containing the murine form of Pmel17 was administered intradermally to DBA/2 mice (H-2(d)), which were then investigated for the presence of Pmel17/gp100-specific immunity. We show that administration of plasmid DNA coding for the autologous melanoma-associated antigen Pmel17/gp100 protects DBA/2 mice against the growth of Pmel17-positive M3 melanoma cells but not against Pmel17-negative M3 melanoma cells or unrelated P815 mastocytoma cells. Cell depletion experiments demonstrated that this protective effect is mediated by T lymphocytes. The notion that Pmel17/gp100 represents the biologically relevant target in this system was supported by the observations (i) that recipients of Pmel17/gp100 DNA mount an antigen-specific cytotoxic T lymphocyte response and (ii) that M3 tumors growing in mice immunized with autologous Pmel17/gp100 had lost expression of this melanoma-associated antigen whereas M3 melanomas appearing in control-vector-treated animals were still Pmel17/gp100-positive. These results indicate that intracutaneous genetic immunization with autologous melanoma-associated antigen Pmel17/gp100 encoding plasmid DNA can lead to protection against melanoma cells as a result of the induction of a melanoma-associated antigen-specific and protective T-cell-mediated immune response. J Invest Dermatol 115:1082-1087 2000     

Bioluminescent imaging of melanoma in live mice

Melanoma is highly resistant to conventional chemotherapeutic agents and novel therapeutic approaches are needed. Current animal models of melanoma in animals are sub-optimal. The most commonly used homograft model is the B16 mouse melanoma. Evaluation of potential melanoma therapies with this model is limited by the inaccuracy of caliper measurement of subcutaneous tumors, of counting lung nodules in metastasis models, and the indirect nature of "survival" curves when studying brain metastases. We have developed and characterized an accurate, sensitive, and reproducible bioluminescent B16 melanoma model that allows for serial, real-time analyses of tumor burden in live mice. We demonstrate that this model is applicable to subcutaneous tumors, lung metastases, and intracranial tumors and offers a solution to many of the limitations of previous models. As proof of principle, we use this model to show the efficacy of a live, Listeria monocytogenes vaccine expressing the melanoma antigen tyrosinase-related protein-2 to protect mice against intravenous B16 melanoma challenge. Additionally, we extend our approach to include the human A375 melanoma model and are able to show in vivo differences between sub-lines with varying metastatic potential. These models represent an accurate and reproducible means for in vivo melanoma monitoring in preclinical studies.     

腺病毒载体介导的内皮抑素基因治疗小鼠黑素瘤的实验研究

目的　探讨腺病毒载体介导的内皮抑素基因（Ad-mES）在体外和体内的生物学活性。方法　不同感染复度（MOI）的腺病毒体外感染靶细胞;RT-PCR法检测目的基因的表达;MTT法检测Ad-mES对靶细胞生物活性的影响。观察各组小鼠黑素瘤的生长、转移和生存率;免疫组化法鉴定肿瘤组织内内皮抑素蛋白的表达。电子透射电镜观察肿瘤组织内皮细胞、肿瘤细胞的凋亡情况。结果　腺病毒体外能够有效感染靶细胞,MOI为10,20,50,100,200,500时,B16F10细胞和ECV304细胞的腺病毒感染率分别为15.6%、35%、73%、88%、95.2%、97%和19%、35%、80%、90%、97%、98.5%。靶细胞明确表达内皮抑素基因;Ad-mES对B16F10细胞的增殖没有影响;而Ad-mES能抑制ECV304细胞的增殖,且随MOI增大,抑制内皮细胞增殖效果越强。瘤细胞接种后第8天,各组成瘤率100%。开始出现小鼠死亡的最早日：PBS组第16天、Ad-GFP组第18天、Ad-mES单剂、重复治疗组均在第20天。结论 Ad-mES体外和体内均影响靶细胞的生物学活性;Ad-mES治疗组小鼠平均生存时间延长（P < 0.05）,肿瘤体积增长减慢（P < 0.05）。     

腺病毒载体介导的内皮抑素基因治疗小鼠黑素瘤的实验研究

目的 探讨腺病毒载体介导的内皮抑素基因(Ad-mES)在体外和体内的生物学活性.方法 不同感染复度(MOI)的腺病毒体外感染靶细胞;RT-PCR法检测目的基因的表达;MTT法检测Ad-mES对靶细胞生物活性的影响.观察各组小鼠黑素瘤的生长、转移和生存率;免疫组化法鉴定肿瘤组织内内皮抑素蛋白的表达.电子透射电镜观察肿瘤组织内皮细胞、肿瘤细胞的凋亡情况.结果 腺病毒体外能够有效感染靶细胞,MOI为10,20,50,100,200,500时,B16F10细胞和ECV304细胞的腺病毒感染率分别为15.6%、35%、73%、88%、95.2%、97%和19%、35%、80%、90%、97%、98.5%.靶细胞明确表达内皮抑素基因;Ad-mES对B16F10细胞的增殖没有影响;而Ad-mES能抑制ECV304细胞的增殖,且随MOI增大,抑制内皮细胞增殖效果越强.瘤细胞接种后第8天,各组成瘤率100%.开始出现小鼠死亡的最早日:PBS组第16天、Ad-GFP组第18天、Ad-mES单剂、重复治疗组均在第20天.结论 Ad-mES体外和体内均影响靶细胞的生物学活性;Ad-mES治疗组小鼠平均生存时间延长(P<0.05),肿瘤体积增长减慢(P<0.05).     

Endogenous μ-opioid peptides modulate immune response towards malignant melanoma

Abstract: Opioids exert major effects not only in the central nervous system but also in immune responses. We investigated the effects of μ‐opioid peptides, secreted by tumor cells, on anti‐tumor immune responses. For this purpose, tumor growth was studied in wild‐type and μ‐opioid receptor–deficient (MOR?/?) mice injected with B16 melanoma cells. The ability of these cells to produce opioids was studied by Western blots in vitro. Finally, biopsy material from human melanomas was investigated by immunohistochemistry for ß endorphin expression. Injection of B16 melanoma cells, producing endogenous ß endorphin, in the flank of MOR?/? mice revealed a profound reduction in tumor growth, paralleled by a significantly higher infiltration of immune cells into the tumors, when compared to tumor growth after injection of B16 melanoma cells into wild‐type mice. Opioids present in B16 cell supernatant significantly reduced the proliferation of normal but not MOR?/? leucocytes. Immunohistochemical analyses of biopsies from human melanoma tissues showed a positive correlation between expression of ß endorphin and tumor progression. Our data provide evidence that μ‐opioid peptides may play a major role in cancer progression by modulating immune response. This finding may have implications for the future optimization of immunointerventions for cancer.     

Tumor-derived CCL20 affects B16 melanoma growth in mice

BackgroundChemokine ligand-20 (CCL20) expressed in the epidermis is a potent impetus for the recruitment of CC-chemokine receptor 6 (CCR6)-expressing subsets of DCs, B-cells and memory T-cells into the skin. CCL20 and CCR6+ immune cells have been detected in chronic inflammatory skin diseases and several malignancies, including melanoma. Yet, the functional contribution of the CCR6/CCL20 axis for melanoma progression remains controversial.ObjectiveThe functional contribution of CCR6-expressing immune cell subsets and local CCL20 in the tumor microenvironment for the immune control of melanoma was studied.MethodsHomeostatic and inducible CCL20 secretion of murine (B16, Ret) and human (A375, C32) melanoma cells was analyzed by ELISA. To assess the functional relevance of CCR6/CCL20 interactions on local tumor progression, prestimulated or retrovirally transduced B16/F1 melanoma cells overexpressing CCL20 (B16-CCL20) were injected subcutaneously into C57BL/6 Wt mice and congenic CCR6-deficient (CCR6^−/−) mice. Infiltrating leucocytes were examined by flow cytometry in tumors and draining lymph nodes (DLNs).ResultsMelanoma cell lines up-regulate CCL20 secretion upon stimulation with pro-inflammatory cytokines in vitro. While only moderate changes in phenotype and composition of leucocytes were detected in advanced tumors and DLNs, mice injected with CCR6+ B16-CCL20 cells developed smaller tumors compared to B16-Control injected littermates, with CCR6-/- mice displaying the most pronounced reduction in tumor growth and incidence.ConclusionOur results suggest that CCR6/CCL20 interactions and individual independent effects of CCL20 and CCR6 in the microenvironment may be essential for melanoma progression and suggest a decisive role of this chemokine axis for melanoma pathogenesis beyond chemoattraction.     

Ultraviolet A exposure might increase metastasis of mouse melanoma: a pilot study

BACKGROUND: The major sources of long-wave ultraviolet A radiation (UVA; 320-400 nm) exposure are extensive sunbathing and tanning in solaria. While the carcinogenic effects of mid-wave ultraviolet B radiation (UVB; 280-320 nm) are well recognized, the potentially hazardous effects of UVA are less understood. Several studies have shown that a variety of physiological processes in the cell are modified by UVA exposure, some of which might be involved in the regulation of tumor metastasis. In this study we suggest that UVA radiation could lead to the increase of metastatic capability of melanoma cells in mice. METHOD/RESULT: A pilot in vivo study was executed using C57BL/6 mice and syngeneic B16 melanoma cell lines. Mice were intravenously (i.v.) injected with either B16-F1 or B16-F10 melanoma cells into the tail vein and then immediately exposed to UVA. Fourteen days after melanoma injection, lungs were collected and the quantity and quality of metastases were determined under a dissecting microscope. As an outcome of the pilot study we observed that i.v. injected melanoma cells formed more lung metastases in the UVA-exposed mice in comparison with the control mice. CONCLUSION: This result suggests that the UVA exposure of mice, with melanoma cells present in blood circulation, increases the formation of melanoma metastases in lungs. Further studies should determine whether a similar pro-metastatic effect, as observed in mice, could occur in humans and whether other than melanoma tumors might be susceptible.     

Histologic progression of B16 F10 metastatic melanoma in C57BL/6 mice over a six week time period: distant metastases before local growth

B16F10 murine metastatic melanoma in the tails of C57BL/6 mice after subcutaneous injection is a well-established model. However, the histologic progression from injected cells to established local growth of melanoma has not been studied systematically. We therefore have investigated the histologic changes and growth of B16F10 melanoma at the injection site over a six-week time period. One million B16F10 melanoma cells were injected subcutaneously into the dorsal aspect of tails of C57/BL6 mice. Mice were sacrificed at zero, 12, 24, 48, 72 and 96 hours, and at one, two, three, four, five and six weeks. Sections were stained with Hematoxylin and Eosin and immunostained with antibodies to S100. Beginning at time zero, melanoma cells were detected between the dermis and the myofascial bundle of the tail. At week four, distant metastases were clinically evident in the inguinal region, though injection site tumors did not become evident until week six. Histological analysis showed melanoma cells at the injection site at all time periods and no injection site tumor until week six. Indeed, the injection site tumors arose two weeks after distant metastases were clinically apparent. A progression of S100 positivity was also observed. S100 immunostaining was negative in all injection site of B16F10 cells until the cells underwent a morphologic change from small and monomorphic at the injection site, to large, pleomorphic cells at week six in the clinically evident injection site tumors. Inguinal metastases were also S100 positive at week four, though injection site cells were still S100 negative. We conclude that in this particular established model for melanoma, local growth at the injection site may occur after the development of regional metastases. This may prove to be a good model for investigation of local growth of tumor cells and their interaction with metastatic lesions.     

Modulation of Growth of Melanoma

Combined heparin-cortisone treatment induces regression of growth in a variety of murine tumors including melanoma. We injected 92 inbred C 57 b1/6 male mice each with 5 X 10(5) melanoma cells (B16, B16 F1, and B16 A6 lines) with different metastatic potential. Heparin (400 U/ml) and cortisone acetate (250 mg/kg SC injections) were given daily. Control experiments were performed both with the administration of no drugs and with administration of cortisone alone. Plasminogen activator activity, which is notoriously related to tumor growth, was evaluated using fibrin plate technique in 10 fragments taken before and 20 days after the combined heparin-cortisone treatment of B16 F1 and B16 A6 melanomas. The combined heparin-cortisone treatment slowed tumor growth, but no tumour regression was observed. Cutaneous fibrinolytic activity appeared increased in all specimens after the treatment.     

Combined treatment with intratumoral injection of dendritic cells and topical application of imiquimod for murine melanoma

BACKGROUND: The maturation state of dendritic cells is one of the factors that affect their capacity to induce antigen-specific cytotoxic T lymphocytes. Topical cutaneous application of imiquimod can induce the maturation and migration of cutaneous dendritic cells. OBJECTIVES: To evaluate the synergistic effect of topical application of imiquimod plus intratumoral injection of syngeneic bone marrow-derived dendritic cells in the treatment of melanoma. METHODS: For the B16F10 melanoma model, naive C57BL/6 mice were inoculated intradermally with 2x10(3) B16F10 melanoma cells in the right upper flank. Four groups (untreated control, dendritic cells alone, imiquimod alone and imiquimod plus dendritic cells) were included in the animal study, with five mice in each group. Tumour size was measured every 2 weeks, and histochemical and immunohistochemical staining carried out. ELISpot and PKH assays were performed to assess immune activity. RESULTS: Combined treatment of topical application of imiquimod and intratumoral injection of dendritic cells led to significant tumour regression, in contrast to partial eradication of the tumours with imiquimod or dendritic cells alone. CONCLUSION: These findings suggest that combination therapy with topical application of imiquimod and intratumoral administration of dendritic cells is a potent strategy for the treatment of melanoma.     

The efficacy of retroviral herpes simplex virus thymidine kinase gene transfer and ganciclovir treatment on the inhibition of melanoma growth in vitro and in vivo

Abstract  One approach to gene therapy of cancer is based on the insertion of a suicide gene into tumor cells and subsequent activation of the suicide mechanism. We used the herpes simplex virus thymidine kinase (HSVtk) gene followed by ganciclovir (GCV) treatment. The goal of our experiments was to determine the effectiveness of HSVtk gene therapy in malignant melanoma. B16BL6 murine melanoma cells retrovirally transduced with the HSVtk gene became sensitive to low concentrations of GCV. Analysis by RT-PCR showed HSVtk expression in transduced B16BL6tk⁺ cells. Apoptotic cell death was found in B16BL6tk⁺ cells treated with GCV (20 μM). The sensitivity of B16BL6tk⁺ cells to GCV was also examined in vivo. Tumors inoculated subcutaneously into C57BL6 mice regressed rapidly when treated with GCV (50 mg/kg twice a day) and disappeared completely after 14 days treatment. The mice remained in remission for 5 months. A bystander effect through which nontransduced B16BL6 cells were also inhibited by GCV administration when cocultured with B16BL6tk⁺ cells was expected. However, only slight killing of nontransduced cells was observed in vitro. Analysis of the bystander effect in vivo showed complete regression of tumors inoculated with a mixture of cells mostly consisting of B16BL6tk⁺ cells. A distant bystander effect was also examined. There was no regression of wild-type tumors raised at a distant site from primary B16BL6tk⁺ tumors. The failure of a more effective bystander effect indicates the need for further investigation of the possible use of combined gene therapy to treat melanoma. Received: 30 May 2001 / Revised: 19 July 2001 / Accepted: 30 August 2001     

Comparison of a treatment strategy combining CCI-779 plus DTIC versus DTIC monotreatment in human melanoma in SCID mice

This study compares the antineoplastic potential of a novel treatment strategy combining cell cycle inhibitor-779 (CCI-779) plus dacarbazine (DTIC) versus DTIC monotreatment, the current chemotherapeutic mainstay in combating metastatic melanoma. A controlled four-group parallel study design comprising 24-40 mice per tumor cell line was used in a severe combined immunodeficiency (SCID)-mouse xenotransplantation model. SCID mice were injected with 518A2, Mel-JUSO, or 607B human melanoma cells. After they developed tumors, mice received daily CCI-779 or solvent over 14 days. From treatment day 4-8 mice were additionally injected with DTIC or saline. Treatment with CCI-779 plus DTIC was superior to single agent DTIC in two out of three cell lines (P<0.05). The tumor weight reduction was 44+/-17 and 61+/-6% compared with DTIC monotreatment in Mel-JUSO and 607B melanomas, respectively (P<0.05). In contrast, in 518A2 xenotransplants, CCI-779 plus DTIC treatment was as effective as DTIC monotreatment. CCI-779 monotherapy exerted no statistically significant antitumor effect. Collectively, these data indicate that CCI-779 has the potential to increase the chemotherapeutic efficacy, as the combination of CCI-779 plus DTIC proved to be more efficacious compared to DTIC monotherapy in two out of three melanoma cell lines in vivo.     

Gene transfer of secreted-type modified interleukin-18 gene to B16F10 melanoma cells suppresses in vivo tumor growth through inhibition of tumor vessel formation

Interleukin-18 is a novel cytokine identified as a strong inducer of interferon-gamma. Interleukin-18 has been shown to have similar bioactivities to interleukin-12 and to have antitumor efficacy in experimental models. In this study, we investigated whether the introduction of the interleukin-18 gene to B16F10 melanoma cells can induce antitumor response or not. Before the transfection, we modified the interleukin-18 gene to enable transfected tumor cells to secrete bioactive interleukin-18, because interleukin-18 does not have a signal sequence and requires processing by the interleukin-1 converting enzyme to attain the mature form. We found that B16 melanoma cells transduced with hybrid cDNA consisting of the interferon-beta signal sequence and mature interleukin-18 sequence, but not native interleukin-18, secreted a large amount of interleukin-18 and exhibited retarded tumor growth when injected in syngeneic mice. The antitumor effect was mostly abrogated by administration of anti-interferon-gamma antibody, but was not affected by in vivo depletion of CD8+ T cells or natural killer cells. Histologic analysis revealed that vascularization was markedly reduced and that necrosis was extensively induced in interleukin-18-secreting B16F10 melanoma (B16/IL18) tissues, whereas abundant tumor vessel formation was observed in B16/IL18 tissues of interferon-gamma-neutralized mice. We also found that chemokines, interferon-inducible protein-10 and monokine induced by interferon-gamma, were produced in B16/IL18 tissues and that the expression of both chemokines was dependent on that of interferon-gamma in the tumor tissues. Further, we showed that B16 melanoma cells secreted both chemokines in response to interferon-gamma. In addition, the expression of angiogenin, an angiogenic factor of melanoma, in B16 melanoma cells was reduced by interferon-gamma treatment. These results indicate that gene transfer of secreted-type interleukin-18 to B16F10 melanoma cells is a useful method of triggering an antitumor response without any systemic adverse effects and that the antitumor efficacy is mainly mediated by antiangiogenic activity, which is possibly involved in at least two dynamic changes induced by interferon-gamma inside B16 melanoma cells: the upregulation of antiangiogenic chemokines, interferon-inducible protein-10 and monokine induced by interferon-gamma, and the downregulation of angiogenic factor, angiogenin.     

Interleukin-18 and the costimulatory molecule B7-1 have a synergistic anti-tumor effect on murine melanoma; implication of combined immunotherapy for poorly immunogenic malignancy

Interleukin-18 has been described recently as a cytokine secreted primarily by Kupffer cells. Furthermore, it has been shown that it has significant anti-tumor effects, which are mediated by T cells and natural killer cells, in a manner similar to interleukin-12. Here, we report the evaluation of the effects of the systemic administration of interleukin-18 in combination with B7-1 (CD80) expressed on tumor cells [interleukin-18 + B7-1] on the growth of murine B16 melanoma in vivo. After the subcutaneous inoculation of B16 melanoma, B16 tumors grew progressively in immunocompetent syngeneic C57BL/6 mice. Mice treated with either interleukin-18 or immunized with B7-1-transduced B16 did not demonstrate significant anti-tumor effect. The combination of the two treatments, however, resulted in dramatic suppression of melanoma formation, tumor growth, and a significant improvement in survival. Inhibitory effects of [interleukin-18 + B7-1] on lung metastasis in mice were also detected. Additionally, mice treated with [interleukin-18 + B7-1] showed an increase of natural killer cytotoxicity and interferon-gamma production in vivo. Unlike [interleukin-18 + B7-1], [interleukin-12 + B7-1] did not have a strong anti-tumor effect against B16 melanoma. Histologic characterization after the [interleukin-18 + B7-1] treatment confirmed the infiltration of natural killer cells into the tumor, suggesting that natural killer cells may be involved in the [interleukin-18 + B7-1]-induced anti-tumor effect. This finding was confirmed by showing that depletion of NK1.1+ cells before immunization inhibits the [interleukin-18 + B7-1]-induced anti-tumor effect. Depletion of CD3+ cells in vivo also decreased the anti-tumor effect of [interleukin-18 + B7-1], suggesting the importance of CD3+ T cells. Collectively, combination with interleukin-18 and B7-1 expression has synergistic anti-tumor effects against B16 murine melanoma.     

Peritumoral CpG oligodeoxynucleotide treatment inhibits tumor growth and metastasis of B16F10 melanoma cells

Although melanoma mostly affects the skin, it is notorious for its propensity to easily develop metastasis. Metastatic melanoma is highly resistant to a variety of therapies. We examined the anti-metastatic potential of peritumoral monotherapy against murine cutaneous B16F10 melanoma with synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs. We demonstrated that repeated peritumoral injections of CpG ODN significantly reduced skin tumor size. Peritumoral CpG ODN-treatment of skin tumors prevented the development of pulmonary B16F10 colonies. Adoptive transfer of splenocytes obtained from CpG ODN-treated mice markedly reduced the number of previously established pulmonary colonies in recipient na?ve mice. T-lymphocyte depletion studies indicated that the anti-metastatic effect was dependent on both CD4+ and CD8+ T cells. These results suggest that CpG ODN are promising as a preventive and therapeutic anti-metastatic measure against melanoma.