krn-7000 and Neoplasm-Metastasis

Invariant natural killer T (iNKT) cells have adjuvant activity due to their ability to produce large amounts of IFN-gamma, which activates other cells in innate and acquired systems, and orchestrates protective immunity. Based on these adjuvant mechanisms, we developed iNKT cell-targeted adjuvant therapy and carried out a phase I/IIa trial on advanced lung cancer patients. The patient group with increased numbers of IFN-gamma-producing cells showed prolonged survival with a median survival time of 31.9 months. Sixty percent of the patients in this group survived for more than 2 years with only a primary treatment and without tumor progression and metastasis.

Topics: Adaptive Immunity; Adjuvants, Immunologic; Animals; Cancer Vaccines; Clinical Trials as Topic; Galactosylceramides; Humans; Immunity, Innate; Immunotherapy; Interferon-gamma; Lung Neoplasms; Natural Killer T-Cells; Neoplasm Metastasis

Patients with colorectal cancer frequently develop liver metastases after, and perhaps as a consequence of, lifesaving surgical resection of the primary tumor. This creates a potential opportunity for prophylactic metastatic treatment with novel immunostimulatory molecules. Here, we used state-of-the-art intravital imaging of an experimental liver metastasis model to visualize the early behavior and function of invariant natural killer T (iNKT) cells stimulated with α-galactosylceramide (α-GalCer). Intravenous α-GalCer prior to tumor cell seeding in the liver significantly inhibited tumor growth. However, some seeding tumor cells survived. A multiple dosing regimen reduced tumor burden and prolonged the life of mice, whereas tumors returned within 5 days after a single dose of α-GalCer. With multiple doses of α-GalCer, iNKT cells increased in number and granularity (as did NK cells). As a result, the total number of contacts and time in contact with tumors increased substantially. In the absence of iNKT cells, the beneficial effect of α-GalCer was lost. Robust cytokine production dissipated over time. Repeated therapy, even after cytokine dissipation, led to reduced tumor burden and prolonged survival. Serial transplantation of tumors exposed to α-GalCer-activated iNKT cells did not induce greater resistance, suggesting no obvious epigenetic or genetic immunoediting in tumors exposed to activated iNKT cells. Very few tumor cells expressed CD1d in this model, and as such, adding monomers of CD1d-α-GalCer further reduced tumor growth. The data suggest early and repeated stimulation of iNKT cells with α-GalCer could have direct therapeutic benefit for patients with colorectal cancer who develop metastatic liver disease.

Topics: Animals; Antigens, CD1d; Cell Line, Tumor; Colorectal Neoplasms; Disease Models, Animal; Galactosylceramides; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Natural Killer T-Cells; Neoplasm Metastasis

The nuclear receptors liver X receptor α (LXRα) and LXRβ are lipid sensors that regulate lipid metabolism and immunity. Natural killer T (NKT) cells, a T cell subset expressing surface markers of both natural killer cells and T lymphocytes and involved in antitumor immunity, are another abundant immune cell type in the liver. The potential function of the metabolic regulators LXRα/β in hepatic NKT cells remains unknown. In this study, we examined the role of LXRα and LXRβ in NKT cells using mice deficient for LXRα and/or LXRβ, and found that hepatic invariant NKT (iNKT) cells are drastically decreased in LXRα/β-KO mice. Cytokine production stimulated by the iNKT cell activator α-galactosylceramide was impaired in LXRα/β-KO hepatic mononuclear cells and in LXRα/β-KO mice. iNKT cell-mediated antitumor effect was also disturbed in LXRα/β-KO mice. LXRα/β-KO mice transplanted with wild-type bone marrow showed decreased iNKT cells in the liver and spleen. The thymus of LXRα/β-KO mice showed a decreased population of iNKT cells. In conclusion, LXRα and LXRβ are essential for NKT cell-mediated immunity, such as cytokine production and hepatic antitumor activity, and are involved in NKT cell development in immune tissues, such as the thymus.

Topics: Animals; Antineoplastic Agents; Bone Marrow; Bone Marrow Transplantation; Cytokines; Galactosylceramides; Killer Cells, Natural; Leukocytes, Mononuclear; Liver; Liver Neoplasms; Liver X Receptors; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Neoplasm Metastasis; Spleen; Thymocytes

Invariant NKT (

Topics: Animals; Antigens, Neoplasm; Cell Line, Tumor; Fingolimod Hydrochloride; Galactosylceramides; Immunotherapy; Interleukin-12; Interleukin-18; Killer Cells, Natural; Melanoma, Experimental; Mice; Mice, Knockout; Natural Killer T-Cells; Neoplasm Metastasis; Sphingosine-1-Phosphate Receptors; Thymoma

α-galactosylceramide (GalCer), which is a natural killer T (NKT) cell ligand, has been reported to exert therapeutic effects against cancer in humans and mice. Toll-like receptor (TLR) agonists systemically or locally boost antitumor efficacy in mouse cancer models. In our previous study, the co-administration of GalCer and a TLR agonist synergistically enhanced interferon-γ (IFN-γ) production in mouse splenocytes in vitro and in vivo. The increased IFN-γ production promoted a tumor antigen-specific Th1 response. Therefore, co-treatment with GalCer and a TLR agonist is expected to exert an enhanced antitumor effect. In the present study, we examined the effect of GalCer and lipopolysaccharide (LPS) combination therapy in a mouse lung-metastasis model. GalCer and LPS combination therapy markedly decreased the number of lung metastatic tumor nodes. Co-treatment with GalCer and LPS enhanced the mRNA expression of CXCL9 and CXCL10 in mediastinal lymph nodes (MLNs) and increased the number of CD8+ cells in the MLNs. Furthermore, the depletion of CD8+ T cells canceled the antitumor effect of GalCer and LPS combination therapy. Thus, GalCer and LPS combination therapy significantly enhanced tumor antigen-specific immune responses and suppressed tumor growth in a mouse lung-metastasis model.

Topics: Animals; Antigens, Neoplasm; Antineoplastic Combined Chemotherapy Protocols; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Chemokine CXCL10; Chemokine CXCL9; Disease Models, Animal; Flow Cytometry; Galactosylceramides; Interferon-gamma; Ligands; Lipopolysaccharides; Lung Neoplasms; Male; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Spleen; Toll-Like Receptors

Immune suppression in the tumour microenvironment remains a major limitation to successful immunotherapy of cancer. In the current study, we analysed whether the natural killer T cell-activating glycolipid α-galactosylceramide could overcome immune suppression and improve vaccination against metastatic breast cancer.. Mice with metastatic breast cancer (4T1 model) were therapeutically treated with a Listeria monocytogenes-based vaccine expressing tumour-associated antigen Mage-b followed by α-galactosylceramide as separate agents, or as a complex of α-galactosylceramide stably incorporated into Listeria-Mage-b. Effects on metastases, tumour weight, toxicity and immune responses were determined.. Sequential treatments of mice with established 4T1 breast carcinomas using Listeria-Mage-b followed by α-galactosylceramide as a separate agent was highly effective at reducing metastases, but was accompanied by severe liver toxicity. In contrast, combined therapy using Listeria-Mage-b modified by incorporation of α-galactosylceramide resulted in nearly complete elimination of metastases without toxicity. This was associated with a significant increase in the percentage of natural killer T cells in the spleen, and an increase in natural killer cell activity and in T cell responses to Mage-b.. Our results suggest that direct incorporation of α-galactosylceramide into a live bacterial vaccine vector is a promising non-toxic new approach for the treatment of metastatic breast cancer.

Topics: Animals; Antigens, Neoplasm; Apoptosis; Blotting, Western; Cancer Vaccines; Cell Adhesion; Cell Cycle; Cell Movement; Cell Proliferation; Female; Flow Cytometry; Galactosylceramides; Immunoenzyme Techniques; Immunotherapy; Killer Cells, Natural; Listeria monocytogenes; Lymphocyte Activation; Mammary Neoplasms, Experimental; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Metastasis; Neoplasm Proteins; Real-Time Polymerase Chain Reaction; Recombination, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes; Tumor Cells, Cultured; Vaccination

Vitamin A compounds are promising for cancer prevention and reducing risk of recurrence. Herein we have evaluated the combination of all-trans-retinoic acid (RA), a vitamin A metabolite, and alpha-galactosylceramide (αGalCer), a lipid immune activator, in Balb/C mice inoculated with syngeneic 4T1 breast tumor cells on reduction in breast tumor growth and lung metastasis. In Balb/c inoculated with the syngenic 4T1 primary tumor, and administered dendritic cells treated with RA + αGalCer, the size of the primary tumor and the number of lung metastatic foci were reduced. When 4T1 cells were introduced into the circulation as a model of hematogenous spread of tumor cells and RA and αCalCer were administered directly to mice without dendritic cells, lung metastatic foci were reduced 70% (P < 0.05), whereas each agent alone resulted in an intermediate decrease. Concomitantly, the expression of matrix metalloproteinases (MMP), membrane type-1 (MT1)-MMP and MMP3, were reduced by RA + αGalCer in lung. MMP3 protein was also reduced in plasma and culture supernatants from RA + αGalCer-treated 4T1 cells. Together, our results provide new evidence that a nutritional-immunological combination of RA + αGalCer may be promising for preventing or slowing the growth of metastatic foci, and suggest reduced MMP production as a possible mechanism.

Topics: Animals; Cell Proliferation; Female; Galactosylceramides; Lung Neoplasms; Mammary Neoplasms, Experimental; Matrix Metalloproteinase 14; Matrix Metalloproteinase 3; Mice; Mice, Inbred BALB C; Neoplasm Metastasis; Neoplasm Transplantation; Tretinoin

Invariant natural killer T (iNKT) cells are known to have marked immunomodulatory capacity due to their ability to produce copious amounts of effector cytokines. Here, we report the structure and function of a novel class of aromatic α-galactosylceramide structurally related glycolipids with marked Th1 bias in both mice and men, leading to superior tumour protection in vivo. The strength of the Th1 response correlates well with enhanced lipid binding to CD1d as a result of an induced fit mechanism that binds the aromatic substitution as a third anchor, in addition to the two lipid chains. This induced fit is in contrast to another Th1-biasing glycolipid, α-C-GalCer, whose CD1d binding follows a conventional key-lock principle. These findings highlight the previously unexploited flexibility of CD1d in accommodating galactose-modified glycolipids and broaden the range of glycolipids that can stimulate iNKT cells. We speculate that glycolipids can be designed that induce a similar fit, thereby leading to superior and more sustained iNKT cell responses in vivo.

Topics: Animals; Antigens, CD1d; Galactosylceramides; Mice; Natural Killer T-Cells; Neoplasm Metastasis; Neoplasms; Protein Binding

Alpha-Galactosylceramide (alpha-GalCer) is a potent CD1d ligand that activates natural killer like T-cells (NKT), leading to the production of helper T (Th) 1 and Th2 cytokines that mediate various immunemodulatory and antitumor effects. Here, we determined whether the administration of adenovirus-vector-encoding mouse interleukin-2 (AdmIL-2) can augment the antitumor effect of alpha-GalCer on subcutaneous and metastatic tumors in mice. Mice were intraperitoneally injected with alpha-GalCer on days 7, 10 and 13 after tumor inoculation, with or without intratumoral injection of AdmIL-2 on day 7. alpha-GalCer treatment increased the serum levels of interferon-gamma, while intratumoral injection of AdmIL-2 elevated serum IL-2 levels. A combination of alpha-GalCer and AdmIL-2 (alpha-GalCer/AdmIL-2) inhibited the in vivo tumor growth and improved the survival of tumor-bearing mice, as compared to the use of a single agent. Experiments on spontaneous metastasis models revealed that alpha-GalCer/AdmIL-2 reduced lung metastasis and prolonged survival, as compared to control groups. In addition, the splenic and liver mononuclear cells from mice treated with alpha-GalCer/AdmIL-2 showed enhanced cytolytic activity against NK-sensitive YAC-1 and NK-resistant 3LL tumors. Moreover, alpha-GalCer/AdmIL-2 treatment expanded the absolute numbers of lung and liver NK, NKT and T-cells as well as the TNF-related apoptosis-inducing ligand (TRAIL) expression of these cells. This study shows the efficacy of alpha-GalCer/AdmIL-2 immunomodulatory therapy, and provides a cellular mechanism on how it exerts the antitumor effects.

Topics: Adenoviridae; Animals; Carcinoma, Lewis Lung; Cell Line, Tumor; Cell Proliferation; Cytotoxicity Tests, Immunologic; Cytotoxicity, Immunologic; Female; Flow Cytometry; Galactosylceramides; Gene Transfer Techniques; Injections, Intraperitoneal; Interferon-gamma; Interleukin-2; Killer Cells, Natural; Mice; Mice, Inbred C57BL; Natural Killer T-Cells; Neoplasm Metastasis; Survival Analysis; T-Lymphocytes; TNF-Related Apoptosis-Inducing Ligand; Tumor Burden

The CD1d reactive glycolipid, alpha-galactosylceramide (alpha-GalCer), potently activates T cell receptor-alpha type I invariant NKT cells that secondarily stimulate the proliferation and activation of other leukocytes, including NK cells. Here we report a rational approach to improving the antitumor activity of alpha-GalCer by using delayed interleukin (IL)-21 treatment to mature the alpha-GalCer-expanded pool of NK cells into highly cytotoxic effector cells. In a series of experimental and spontaneous metastases models in mice, we demonstrate far superior antitumor activity of the alpha-GalCer/IL-21 combination above either agent alone. Superior antitumor activity was critically dependent upon the increased perforin-mediated cytolytic activity of NK cells. Transfer of alpha-GalCer-pulsed dendritic cells (DCs) followed by systemic IL-21 caused an even more significant reduction in established (day 8) metastatic burden and prolonged survival. In addition, this combination prevented chemical carcinogenesis more effectively. Combinations of IL-21 with other NK cell-activating cytokines, such as IL-2 and IL-12, were much less effective in the same experimental metastases models, and these cytokines did not substitute effectively for IL-21 in combination with alpha-GalCer. Overall, the data suggest that NK cell antitumor function can be enhanced greatly by strategies that are designed to expand and differentiate NK cells via DC activation of NKT cells.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Cytotoxicity Tests, Immunologic; Dendritic Cells; Galactosylceramides; Immunotherapy; Interferon-gamma; Interleukins; Killer Cells, Natural; Lymphocyte Activation; Membrane Glycoproteins; Mice; Mice, Knockout; Neoplasm Metastasis; Neoplasms; Perforin; Pore Forming Cytotoxic Proteins

NKT cells, a novel murine lymphoid lineage bearing an invariant T cell receptor encoded by V alpha 14 and J alpha 281 gene segments, recognize a specific ligand glycolipid, alpha-galactosylceramide (alpha-GalCer) in a CD1d-dependent manner. Recent research has revealed that activated V alpha 14 NKT cells have dramatic antitumor effects against a wide variety of tumor cell lines in vivo and in vitro. Here, we demonstrate strong in vivo antitumor effects brought about by treatment with alpha-GalCer-pulsed dendritic cells in comparison with in vitro-activated V alpha 14 NKT cells. Furthermore, we show a significant expansion of endogenous V alpha 14 NKT cells in the lung following the administration of alpha-GalCer-pulsed dendritic cells. The feasibility of immunotherapy with alpha-GalCer-pulsed dendritic cells is discussed.

Topics: Animals; Antigens, CD1; Antigens, CD1d; Antineoplastic Agents; Cell Culture Techniques; Cell Line, Tumor; Cell Lineage; Cells, Cultured; Chromium Radioisotopes; Dendritic Cells; Galactosylceramides; Immunotherapy; Leukocytes, Mononuclear; Ligands; Lung; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neoplasm Metastasis; Reverse Transcriptase Polymerase Chain Reaction; RNA

Liver metastasis of primary tumors is clinically a major problem. We examined the antitumor activity of KRN7000, an alpha-galactosylceramide, in mice with liver metastasis of the B16 melanoma. KRN7000 significantly inhibited tumor growth in the liver, and its potency was similar to that of interleukin-12. The KRN7000 administration resulted in a high percentage of cured mice, which acquired tumor-specific immunity. To study what kinds of antitumor effector cells participated in killing tumor cells, we then performed immunohistological analysis of tumor-infiltrating cells, and found that KRN7000 induced marked invasion of NK1.1+ cells, CD8+ cells, and F4/80+ cells (macrophages) into B16 tumor nodules. In addition, it appeared that KRN7000-treated, liver-associated macrophages possessed strong lytic activity against tumor cells. These results suggest that NK cells, NK1.1+ T (NKT) cells, cytotoxic T lymphocytes, and macrophages play an important role in killing tumor cells in the liver, and that KRN7000 may be useful for the treatment of cancer liver metastasis.

Topics: Animals; Antigens; Antigens, Differentiation; Antigens, Ly; Antigens, Surface; Antineoplastic Agents; CD8 Antigens; Female; Galactosylceramides; Immunohistochemistry; Interleukin-12; Lectins, C-Type; Liver Neoplasms; Macrophages; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Metastasis; Neoplasm Transplantation; NK Cell Lectin-Like Receptor Subfamily B; Proteins; Recombinant Proteins; Time Factors; Tumor Cells, Cultured