Immunotherapy approach with recombinant survivin adjuvanted with alum and MIP suppresses tumor growth in murine model of breast cancer

Survivin has received attention as a potential target for cancer immunotherapy because of its crucial role in oncogenesis. We undertook this study to evaluate the immunotherapeutic potential of combination of recombinant survivin along with adjuvant alum and immune modulator Mycobacterium indicus pranii (MIP). In vivo efficacy of the combination was studied in an invasive murine breast cancer model. Recombinant survivin protein was purified from Escherichia coli based expression system and characterized by western blotting. Purified survivin protein was combined with alum and MIP and was used for immunization of Balb/c mice. Antigen-primed animals were then challenged with syngeneic mammary tumor cells known as 4T-1. Balb/c mice spontaneously develop tumor when inoculated with 4T-1 cells. Antigen and adjuvant combination was immunogenic and significantly suppressed tumor growth in mice immunized with combination of recombinant survivin (10?µg), alum, and MIP. This is the first report that describes a combination immunotherapy approach using recombinant survivin, alum, and MIP in highly metastatic murine breast cancer model and holds promise for development of new biotherapeutics for cancer.     

Stromelysin-3 suppresses tumor cell apoptosis in a murine model

Stromelysin-3 (STR-3) is a matrix metalloproteinase with a unique pattern of expression and substrate specificity. During embryogenesis and remodeling of normal adult tissues, STR-3 is produced by stromal cells in direct contact with epithelial cells undergoing regional apoptosis and selective cell survival. STR-3 is also overexpressed by interdigitating stromal cells in primary epithelial malignancies. Although STR-3 does not degrade classic extracellular matrix components, the enzyme promotes the establishment of local tumors in nude mice by as yet undefined mechanisms. STR-3 is induced when malignant epithelial cells come into contact with surrounding stromal elements; the active stromal cell-derived 45 kDa enzyme is subsequently processed to a 35 kDa protein without enzymatic activity. We have generated MCF-7 transfectants expressing wild type or catalytically inactive 45 kDa STR-3 (STR-3wt and STR-3cat-) or secreted 35 kDa STR-3 (35 kDa STR-3sec) and evaluated their implantation and survival in nude mice. Tumors developed significantly more rapidly in animals receiving STR-3wt, rather than vector-only, STR-3cat- or 35 kDa STR-3sec transfectants. Most importantly, STR-3wt tumors had a significantly lower percentage of apoptotic cells than tumors derived from vector-only, STR-3cat- or 35 kDa STR-3sec transfectants. Taken together, these studies suggest that the active STR-3 enzyme may increase tumor take by suppressing tumor cell apoptosis and that 45 kDa to 35 kDa STR-3 processing limits STR-3 activity at the tumor/stromal interface. Because STR-3 is secreted as an active enzyme rather than a proform, subsequent 45 kDa to 35 kDa STR-3 processing may represent a novel mechanism for regulating enzymatic activity.     

Berberine inhibits human tongue squamous carcinoma cancer tumor growth in a murine xenograft model

Our primary studies showed that berberine induced apoptosis in human tongue cancer SCC-4 cells in vitro. But there is no report to show berberine inhibited SCC-4 cancer cells in vivo on a murine xenograft animal model. SCC-4 tumor cells were implanted into mice and groups of mice were treated with vehicle, berberine (10 mg/kg of body weight) and doxorubicin (4 mg/kg of body weight). The tested agents were injected once per four days intraperitoneally (i.p.), with treatment starting 4 weeks prior to cells inoculation. Treatment with 4 mg/kg of doxorubicin or with 10 mg/kg of berberine resulted in a reduction in tumor incidence. Tumor size in xenograft mice treated with 10 mg/kg berberine was significantly smaller than that in the control group. Our findings indicated that berbeirne inhibits tumor growth in a xenograft animal model. Therefore, berberine may represent a tongue cancer preventive agent and can be used in clinic.     

Triparanol suppresses human tumor growth in vitro and in vivo

Despite the improved contemporary multidisciplinary regimens treating cancer, majority of cancer patients still suffer from adverse effects and relapse, therefore posing a significant challenge to uncover more efficacious molecular therapeutics targeting signaling pathways central to tumorigenesis. Here, our study have demonstrated that Triparanol, a cholesterol synthesis inhibitor, can block proliferation and induce apoptosis in multiple human cancer cells including lung, breast, liver, pancreatic, prostate cancer and melanoma cells, and growth inhibition can be rescued by exogenous addition of cholesterol. Remarkably, we have proved Triparanol can significantly repress Hedgehog pathway signaling in these human cancer cells. Furthermore, study in a mouse xenograft model of human lung cancer has validated that Triparanol can impede tumor growth in vivo. We have therefore uncovered Triparanol as potential new cancer therapeutic in treating multiple types of human cancers with deregulated Hedgehog signaling.     

Enhanced anti-tumor effects achieved in a murine tumor model using combination therapy of recombinant human manganese superoxide dismutase and adriamycin

Manganese superoxide dismutase (MnSOD) is the only primary antioxidant enzyme in mitochondria that scavenges superoxide radicals. Overexpressing MnSOD in cancer cells by cDNA transfection suppresses tumor formation and reverses malignant growth. In this study, we examined the effect of recombinant human manganese superoxide dismutase (rhMnSOD) alone and in combination with adriamycin (ADR) against solid tumors of sarcoma 180 in Institute of Cancer Research (ICR) mice. Administration of rhMnSOD alone and in combination with ADR significantly inhibited tumor growth in a dose-dependent manner. The use of rhMnSOD in combination with ADR enhanced ADR’s anti-tumor potency without increasing toxicity. Histopathological examination provided evidence of the anti-tumor effect. In addition, we found lymphocyte infiltration of the tumors, with an increase in both CD4- and CD8-positive cells in the treated tumors. The expression of CD4 and CD8 was up-regulated with increasing dose of rhMnSOD, and the combination treatment with ADR further enhanced this up-regulation. Collectively, these data indicate that rhMnSOD may exhibit an anti-tumor effect by stimulating the immune system and promoting the recruitment of lymphocytes into the tumor to kill tumor cells. Thus MnSOD may constitute a potential new therapeutic agent to be exploited as an adjuvant in cancer therapy.     

Semaphorin 3A suppresses tumor growth and metastasis in mice melanoma model

Background

Recent understanding on cancer therapy indicated that targeting metastatic signature or angiogenic switch could be a promising and rational approach to combat cancer. Advancement in cancer research has demonstrated the potential role of various tumor suppressor proteins in inhibition of cancer progression. Current studies have shown that axonal sprouting inhibitor, semaphorin 3A (Sema 3A) acts as a potent suppressor of tumor angiogenesis in various cancer models. However, the function of Sema 3A in regulation of melanoma progression is not well studied, and yet to be the subject of intense investigation.

Methodology/Principal Findings

In this study, using multiple in vitro and in vivo approaches we have demonstrated that Sema 3A acts as a potent tumor suppressor in vitro and in vivo mice (C57BL/6) models. Mouse melanoma (B16F10) cells overexpressed with Sema 3A resulted in significant inhibition of cell motility, invasiveness and proliferation as well as suppression of in vivo tumor growth, angiogenesis and metastasis in mice models. Moreover, we have observed that Sema 3A overexpressed melanoma clone showed increased sensitivity towards curcumin and Dacarbazine, anti-cancer agents.

Conclusions

Our results demonstrate, at least in part, the functional approach underlying Sema 3A mediated inhibition of tumorigenesis and angiogenesis and a clear understanding of such a process may facilitate the development of novel therapeutic strategy for the treatment of cancer.     

Zinc depletion suppresses tumor growth in mice

The hypothesis that in tumor-bearing animals an increase of host hepatic zinc metallothionein (Zn-MT) causes a restriction of zinc in the tumor tissue was studied. Three types of tumors were induced in laboratory mice by cell transplant. Tumor growth appears to be inhibited under zinc-deficient conditions, even in cases where zinc deficiency was started after tumor cell transplant. The survival times of tumor-bearing mice were prolonged by administration of cadmium chloride, which induces the synthesis of a combined zinc-cadmium metallothionein derivative in the host liver, but not in the tumor tissue, leading to an increase of hepatic zinc in the treated animals. The uptake of⁶⁵Zn by the liver of Cd-treated, tumor bearing mice was significantly higher than that of controls whereas uptake of⁶⁵Zn by tumor cells was significantly higher in controls than in the treated animals. These results suggest that restriction of zinc intake suppresses tumor growth.     

Lycopene suppresses ovalbumin-induced airway inflammation in a murine model of asthma

In this study, we attempt to determine whether lycopene regulates inflammatory mediators in the ovalbumin (OVA)-induced murine asthma model. To address this, mice were sensitized and challenged with OVA, and then treated with lycopene before the last OVA challenge. Administration of lycopene significantly alleviated the OVA-induced airway hyperresponsiveness to inhaled methacholine. Administration of lycopene also resulted in a significant inhibition of the infiltration of inflammatory immunocytes into the bronchoalveolar lavage, and attenuated the gelatinolytic activity of matrix metalloproteinase-9 and the expression of eosinophil peroxidase. Additionally, lycopene reduced the increased levels of GATA-3 mRNA level and IL-4 expression in OVA-challenged mice. However, it increased T-bet mRNA level and IFN-γ expression in lycopene-challenged mice. These findings provide new insight into the immunopharmacological role of lycopene in terms of its effects in a murine model of asthma.     

Stable expression of a secretable deletion mutant of recombinant human thrombomodulin in mammalian cells

Thrombomodulin is an endothelial cell membrane protein which plays a central regulatory role in the protein C anticoagulant pathway. The human thrombomodulin intronless gene was isolated from a genomic DNA library and used to isolate the coding region. A mammalian expression vector, phd-TMD1, encoding all the extracellular domains of human thrombomodulin but lacking the transmembrane and cytoplasmic domains was constructed. Stable phd-TMD 1 transformants, in both hamster AV12-644 and human 293 cells, expressed functionally active recombinant thrombomodulin as a secreted, soluble product. Soluble thrombomodulin was secreted as two major proteins of 105 kDa and 75 kDa, both of which were purified to homogeneity. The kinetic properties for protein C activation of the two proteins were very different: the Kd for thrombin, Km for protein C, and Ca2+ optima were 3.0 nM, 1.5 microM, and 1-3 mM for the 105-kDa protein and 16 nM, 2.3 microM, and 0.2-0.5 mM for the 75-kDa protein. In clotting and platelet activation assays, the 105-kDa protein was a much more potent anticoagulant than the 75-kDa protein. Both forms of the protein had the amino-terminal sequence Ala19-Pro-Ala-Glu-Pro-Gln. Amino acid composition analysis indicated that both forms of the protein had the same amino acid content which was consistent with the predicted protein comprising residues Ala19 to Ser515. The difference in size appeared to be due to glycosylation as both forms were of similar size following chemical deglycosylation. These studies suggest that (1) secretable thrombomodulin derivatives can be used to study structure-function relationships of the extracellular domains of this important regulatory protein, (2) the extent of glycosylation has profound effects on the kinetic and anticoagulant properties of human thrombomodulin, and (3) soluble recombinant human thrombomodulins may be developed as clinically significant therapeutic anticoagulants.     

Expression of the human fast-twitch skeletal muscle troponin I cDNA in a human ovarian carcinoma suppresses tumor growth

To explore the efficiency and mechanism of ovarian carcinoma gene therapy with the human fast-twitch skeletal muscle troponin I gene (Tnl-fast), Tnl-fast cDNA was transferred into human ovarian adenocarcinoma cell-line SK-OV-3. In vitro, the cell growth and cell cycle of Tnl-fast-, vector-, and mock-transfected cells were determined by MTT and flow cytometry assay, respectively. The conditioned media of Tnl-fast-, vector-, and mock-transfected SK-OV-3 cells were collected, and the cell proliferation inhibiting rates of human umbilical cord venous endothelial cells (HUVECs) by the three conditioned media were assayed. All the three cell lines were implanted into node mice, and the tumor growth, cell apoptosis, angiogenesis, and expression of Tnl-fast were observed or analyzed, respectively. In vitro, expression of Tnl-fast protein had no inhibiting effect on the growth of the dominant and stable transfectant cells, but endothelium, when compared with vector-transfected cells and nontransfected parental SK-OV-3 cells. Implantation of stable clone expressing Tnl-fast in the female BALB/c nude mice inhibits primary tumor growth by an average of 73%. The nude mice grafts expressing Tnl-fast exhibit a significant decrease of microvascular density, a higher rate of tumor cells apoptosis and a comparable proliferation rate as control. Our study, to our knowledge, shows the slowed down growth of the primary ovarian carcinoma, suggested that grafts were self-inhibitory by halting angiogenesis. Our data might also provide a novel useful strategy for cancer therapy by antiangiogenic gene therapy with a specific angiogenesis inhibitor Tnl-fast.     

Potential use of tuftsin in treatment of candida peritonitis in a murine model

A major complication of continuous ambulatory peritoneal dialysis (CAPD) is peritonitis caused by Candida albicans. Increasing the activity of the peritoneal macrophages, the predominant cell type found in the peritoneal cavity, may be a promising treatment for this infection. Tuftsin was found to increase thioglycollate-elicited mouse peritoneal macrophage activity. 2x10(-7) M tuftsin enhanced two-fold cell association with radiolabelled candida, superoxide aniom production, and killing activity. Thus, a model consisting of mice undergoing peritoneal dialysis was developed in order to study the use of tuftsin as a therapeutic drug against peritoneal candidiasis. Administration of tuftsin (50 micrograms/mouse) before candidiasis induction with a lethal dose of candida (7x10(8) candida per mouse) improved mouse survival up to 70%, compared with 10% in the control group. The potential of tuftsin as a treatment for candidiasis was shown when the infection was induced with a sublethal dose of candida. Daily intraperitoneal injections of tuftsin (50 micrograms) to the sublethally infected mice caused a significant decrease in the number of candida recovered from the peritoneal cavity and from the blood (from 700 +/- 190 to 110 +/- 26 CFU/ml and from 100 +/- 26 CFU/ml to 17 +/- 8 CFU/ml, respectively). In addition, a larger number of peritoneal macrophages with greater phagocytic and killing activity were found in the tuftsin-treated mice. The effect of tuftsin may promote its potential use in the therapy of peritonitis in patients undergoing chronic peritoneal dialysis.     

Picropodophyllin inhibits tumor growth of human nasopharyngeal carcinoma in a mouse model

Insulin-like growth factor-1 receptor (IGF-1R) is a cell membrane receptor with tyrosine kinase activity and plays important roles in cell transformation, tumor growth, tumor invasion, and metastasis. Picropodophyllin (PPP) is a selective IGF-1R inhibitor and shows promising antitumor effects for several human cancers. However, its antitumor effects in nasopharyngeal carcinoma (NPC) remain unclear. The purpose of this study is to investigate the antitumor activity of PPP in NPC using in vitro cell culture and in vivo animal model. We found that PPP dose-dependently decreased the IGF-induced phosphorylation and activity of IGF-1R and consequently reduced the phosphorylation of Akt, one downstream target of IGF-1R. In addition, PPP inhibited NPC cell proliferation in vitro. The half maximal inhibitory concentration (IC50) of PPP for NPC cell line CNE-2 was ?1 μM at 24 h after treatment and ?0.5 μM at 48 h after treatment, respectively. Moreover, administration of PPP by intraperitoneal injection significantly suppressed the tumor growth of xenografted NPC in nude mice. Taken together, these results suggest targeting IGF-1R by PPP may represent a new strategy for treatment of NPCs with positive IGF-1R expression.     

Inhibition of yes‐associated protein suppresses brain metastasis of human lung adenocarcinoma in a murine model

Yes‐associated protein (YAP) is a main mediator of the Hippo pathway and promotes cancer development and progression in human lung cancer. We sought to determine whether inhibition of YAP suppresses metastasis of human lung adenocarcinoma in a murine model. We found that metastatic NSCLC cell lines H2030‐BrM3(K‐ras^G12C mutation) and PC9‐BrM3 (EGFR^Δexon19 mutation) had a significantly decreased p‐YAP(S127)/YAP ratio compared to parental H2030 (K‐ras^G12C mutation) and PC9 (EGFR^Δexon19 mutation) cells (P < .05). H2030‐BrM3 cells had significantly increased YAP mRNA and expression of Hippo downstream genes CTGF and CYR61 compared to parental H2030 cells (P < .05). Inhibition of YAP by short hairpin RNA (shRNA) and small interfering RNA (siRNA) significantly decreased mRNA expression in downstream genes CTGF and CYR61 in H2030‐BrM3 cells (P < .05). In addition, inhibiting YAP by YAP shRNA significantly decreased migration and invasion abilities of H2030‐BrM3 cells (P < .05). We are first to show that mice inoculated with YAP shRNA‐transfected H2030‐BrM3 cells had significantly decreased metastatic tumour burden and survived longer than control mice (P < .05). Collectively, our results suggest that YAP plays an important role in promoting lung adenocarcinoma brain metastasis and that direct inhibition of YAP by shRNA suppresses H2030‐BrM3 cell brain metastasis in a murine model.     

The murine CC chemokine, 6C-kine, inhibits tumor growth and angiogenesis in a human lung cancer SCID mouse model

The recently described CC chemokine, 6C-kine, is unique in that it contains -six rather than the usual four conserved cysteines typical of this family. Furthermore, murine 6C-kine binds to one of the CXC chemokine receptors CXCR3, in addition to its other known receptor CCR7. We have shown that two other ligands of CXCR3, IP-10 and MIG, are potent inhibitors of tumor growth in severe combined immunodeficiency (SCID) mice. We postulated that murine 6C-kine may also inhibit tumor growth via inhibition of angiogenesis in this model. SCID mice (n=6 per group) inoculated with A549 human lung cancer cells were treated with either 6C-kine (100 ng intra-tumor injection every other day) or control protein for 8 weeks. Tumors from murine 6C-kine-treated mice (288 ± 26 mm³) were significantly smaller than tumors from control treated mice (788 ± 156 mm³, P=0.005). Additionally, murine 6C-kine reduced metastases compared with controls (0.5 ± 0.3 vs 3.0 ± 1.2 metastases per animal, P=0.05). Tumor vascularity (as assessed by vessel density counting) was reduced in murine 6C-kine-treated mice compared with controls. Murine 6C-kine had no direct effect on proliferation of A549 cells, and there were no differences in the infiltration of leukocyte sub-populations, assessed by flow cytometry, in the treatment groups. Interestingly, human 6C-kine, unlike murine 6C-kine, does not bind CXCR3 and had no anti-tumor effect in the same model. These data suggest that murine 6C-kine has anti-tumor effects independent of its leukocyte-recruiting activity. Furthermore, while not confirmatory, these data lend further support to the fact that CXCR3 may be the receptor for angiostatic CXC chemokines. Received: 15 June 2000 / Accepted: 18 August 2000     

A recombinant influenza virus with a CTLA4-specific scFv inhibits tumor growth in a mouse model

Oncolytic viruses (OV) have shown excellent safety and efficacy in preclinical and clinical studies. Influenza A virus (IAV) is considered a promising oncolytic virus. In this report, we generated a recombinant influenza virus expressing an immune checkpoint blockade agent targeting CTLA4. Using reverse genetics, a recombinant influenza virus, termed rFlu-CTLA4, encoding the heavy chain of a CTLA4 antibody on the PB1 segment and the light chain of the CTLA4 antibody on the PA segment was produced. RFlu-CTLA4 could replicate to high titers, and antibodies were produced in the allantoic fluid of infected eggs. Furthermore, the selective cytotoxicity of the virus was higher in various hepatocellular carcinoma cancer cell lines than in the normal cell line L02 in vitro, as indicated by MTS assays. More importantly, in a subcutaneous H22 mouse hepatocarcinoma model, intratumoral injections of rFlu-CTLA4 inhibited the growth of treated tumors and increased the overall survival of mice compared with injections of the PR8 virus. Taken together, these results warrant further exploration of this novel recombinant influenza virus for its potential use as a single or combination agent for cancer immunotherapy.     

Depletion of CXCR2 inhibits tumor growth and angiogenesis in a murine model of lung cancer

The Glu-Leu-Arg(+) (ELR(+)) CXC chemokines are potent promoters of angiogenesis and have been demonstrated to induce a significant portion of nonsmall cell lung cancer-derived angiogenic activity and support tumorigenesis. ELR(+) CXC chemokines share a common chemokine receptor, CXCR2. We hypothesized that CXCR2 mediates the proangiogenic effects of ELR(+) CXC chemokines during tumorigenesis. To test this postulate, we used syngeneic murine Lewis lung cancer (LLC; 3LL, H-2(b)) heterotopic and orthotopic tumor model systems in C57BL/6 mice replete (CXCR2(+/+)) and deficient in CXCR2 (CXCR2(-/-)). We first demonstrated a correlation of the expression of endogenous ELR(+) CXC chemokines with tumor growth and metastatic potential of LLC tumors. Next, we found that LLC primary tumors were significantly reduced in growth in CXCR2(-/-) mice. Moreover, we found a marked reduction in the spontaneous metastases of heterotopic tumors to the lungs of CXCR2(-/-) mice. Morphometric analysis of the primary tumors in CXCR2(-/-) mice demonstrated increased necrosis and reduced vascular density. These findings were further confirmed in CXCR2(+/+) mice using specific neutralizing Abs to CXCR2. The results of these studies support the notion that CXCR2 mediates the angiogenic activity of ELR(+) CXC chemokines in a preclinical model of lung cancer.     

Depletion of beta-arrestin-2 promotes tumor growth and angiogenesis in a murine model of lung cancer

Arrestins are adaptor/scaffold proteins that complex with activated and phosphorylated G protein-coupled receptor to terminate G protein activation and signal transduction. These complexes also mediate downstream signaling, independently of G protein activation. We have previously shown that beta-arrestin-2 (betaarr2) depletion promotes CXCR2-mediated cellular signaling, including angiogenesis and excisional wound closure. This study was designed to investigate the role of betaarr2 in tumorigenesis using a murine model of lung cancer. To that end, heterotopic murine Lewis lung cancer and tail vein metastasis tumor model systems in betaarr2-deficient mice (betaarr2(-/-)) and control littermates (betaarr2(+/+)) were used. betaarr2(-/-) mice exhibited a significant increase in Lewis lung cancer tumor growth and metastasis relative to betaarr2(+/+) mice. This correlated with decreased number of tumor-infiltrating lymphocytes but with elevated levels of the ELR(+) chemokines (CXCL1/keratinocyte-derived chemokine and CXCL2/MIP-2), vascular endothelial growth factor, and microvessel density. NF-kappaB activity was also enhanced in betaarr2(-/-) mice, whereas hypoxia-inducible factor-1alpha expression was decreased. Inhibition of CXCR2 or NF-kappaB reduced tumor growth in both betaarr2(-/-) and betaarr2(+/+) mice. NF-kappaB inhibition also decreased ELR(+) chemokines and vascular endothelial growth factor expression. Altogether, the data suggest that betaarr2 modulates tumorigenesis by regulating inflammation and angiogenesis through activation of CXCR2 and NF-kappaB.     

Knockdown of Atg7 suppresses Tumorigenesis in a murine model of liver cancer

Hepatocellular Carcinoma (HCC) is the most common type of primary liver cancer in adults and a leading cause of cancer-related deaths worldwide. Studies have shown that autophagy is significantly involved in carcinogenesis, in particular, driven by activated RAS signaling. Autophagy related 7 (Atg7) is a critical component for the formation of autophagosome and required for autophagy processes. We investigated the role of autophagy in RAS-driven tumorigenesis in the liver, via the knockdown of Atg7 in the model. Transposon vectors encoding short hairpin RNAs targeting Atg7 (Atg7 shRNA) were constructed. Inhibition of autophagy via Atg7 knockdown was tested in Hep3B cells cultured in nutrient-starved medium. Formation of autophagosome was suppressed in nutrient-starved Hep3B cells expressing Atg7 shRNA, demonstrating that it efficiently inhibited autophagy in HCC cells. Transposons encoding Atg7 shRNA were mixed with those expressing HRAS^G12V and p53 shRNA, and subsequently used for hydrodynamic injection to 5-week-old C57BL/6 mice. Tumorigenesis in livers induced by HRAS^G12V and p53 shRNA was significantly suppressed by Atg7 knockdown. The inhibition of autophagy led to a decreased proliferation of cancer cells, as determined by Ki-67 staining. Our data indicate that knockdown of Atg7 led to a significant decrease in tumorigenesis in a murine HCC model induced by activated RAS. Inhibition of autophagosome formation is expected to be a therapeutic option for liver cancer.     

TNF-alpha-treated DC exacerbates disease in a murine tumor metastasis model

Due to the pivotal role that dendritic cells (DC) play in eliciting functional anti-tumor T cell responses, immunotherapeutic approaches utilizing DC-based vaccines have readily been exploited. It has been argued that, in the setting of immunotherapy, mature DC will be more efficient at T cell priming and, therefore, required for effective vaccination. As TNF-alpha is commonly used as a DC maturation factor, we have examined the efficacy of treatment with DC matured with TNF-alpha (DC-TNF) in a murine model of melanoma. We have now shown that treatment with DC-TNF leads to an increase in the number of lung metastases as compared to mice treated with immature DC. No differences in the number of CD4⁺CD25⁺ T-regulatory cells were measured in the lungs of DC-TNF-treated mice. On examination of the infiltrating lymphocytes, an enhanced secretion of IL-10 and a higher percentage of CD4⁺IL-10⁺ T cells were measured in the lungs of DC-TNF-treated mice. However, treatment with DC-TNF did not enhance the number of melanoma lesions in the lungs of IL-10 knockout mice or in mice depleted of CD4⁺ T cells. Together, these studies indicate that treatment of melanoma-bearing mice with DC treated with TNF-alpha can induce IL-10 production by resident cells at the tumor site, leading to immune tolerance and exacerbation of disease.