transforming-growth-factor-beta and Carcinoma--Lewis-Lung

Human umbilical endothelial cells (HUVECs) have been proved as an effective whole-cell vaccine inhibiting tumor angiogenesis. However, HUVECs divide a very limited number of passages before entering replicative senescence, which limits its application for clinical situation. Here, we fused HUVECs with human pulmonary adenocarcinoma cell line A549s and investigated the anti-tumor immunity of the hybrids against mice Lewis lung cancer.. HUVECs were fused with A549s using polyethylene glycol and were sorted by flow cytometry. The fusion cells (HUVEC-A549s) were confirmed by testing the expression of telomerase and VE-cadherin, the senescence-associated β-galactosidase activity, and tube formation ability. HUVEC-A549s were then irradiated and injected into the C57BL/6 mice of protective, therapeutic, and metastatic models. The mechanism of the anti-tumor immunity was explored by analyzing mice sera, spleen T lymphocytes, tumor microenvironment, and histological changes.. HUVEC-A549s coexpressed tumor and endothelial markers and maintained the vascular function of tube forming at passage 30 without showing signs of senescence. HUVEC-A549s could induce protective and therapeutic anti-tumor activity for LL(2) model and presented stronger activity against metastasis than HUVECs. Both humoral and cellular immunity were participated in the anti-angiogenic activity, as HUVECs-neutralizing IgG and HUVECs-toxic lymphocytes were increased. Angiogenic mediators (VEGF and TGF-β) and tumor microenvironment cells MDSCs and Tregs were also diminished.. Our findings might provide a novel strategy for HUVECs-related immunotherapy, and this vaccine requires lower culture condition than primary HUVECs while enhancing the anti-tumor immunity.

Topics: Animals; Blotting, Western; Carcinoma, Lewis Lung; Cell Proliferation; Cells, Cultured; Cellular Senescence; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Human Umbilical Vein Endothelial Cells; Humans; Immunity, Cellular; Immunoenzyme Techniques; Mice; Mice, Inbred C57BL; Neovascularization, Pathologic; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes; T-Lymphocytes, Cytotoxic; Transforming Growth Factor beta; Umbilical Veins; Vaccination

Little is known about Yu-Ping-Feng (YPF), a typical Chinese herbal decoction, for its antitumor efficacy in non-small-cell lung cancer (NSCLC). Here, we found that YPF significantly inhibited the growth of Lewis lung cancer, prolonged the survival of tumor-bearing mice, promoted NK cell tumor infiltration, increased the population of NK cells in spleen, and enhanced NK cell-mediated killing activity. The growth suppression of tumors by YPF was significantly reversed by the depletion of NK cells. Furthermore, we found that YPF significantly downregulated the expression of TGF-β, indoleamine 2,3-dioxygenase, and IL-10 in tumor microenvironment. These results demonstrated that YPF has a NK cell-dependent inhibitory effect on Lewis lung cancer.

Topics: Animals; Carcinoma, Lewis Lung; Drugs, Chinese Herbal; Indoleamine-Pyrrole 2,3,-Dioxygenase; Interleukin-10; Killer Cells, Natural; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Transforming Growth Factor beta

Tumor hypoxia induces the expression of growth arrest and DNA damage-inducible protein (GADD34). However, the role of GADD34 in tumor growth remains unclear.. Gadd34 expression was knocked-down through lentivirus-mediated short hairpin RNA (shRNA) in tumor cells, which were subsequently injected subcutaneously into mice. Tumor volumes and myeloid-derived suppressor cells (MDSCs) were monitored. Isolated MDSCs were incubated with tumor supernatant to investigate the impact of GADD34 on cytokine secretion of MDSCs.. We observed that reduction of GADD34 expression significantly suppressed tumor, and resulted in decreased accumulation of MDSCs and T-cells, and inhibition of GADD34 reduced secretion of vascular epithelial growth factor α and transforming growth factor β by MDSCs.. These findings provide a promising strategy for targeting GADD34 activity in order to inhibit tumor growth.

Topics: Animals; Breast Neoplasms; Carcinoma, Lewis Lung; CD11b Antigen; Culture Media, Conditioned; DNA Damage; Female; Flow Cytometry; Humans; Hypoxia; Mice; Mice, Inbred C57BL; Mice, Nude; Myeloid Cells; Myeloid-Derived Suppressor Cells; Neoplasms; Protein Phosphatase 1; Real-Time Polymerase Chain Reaction; RNA, Small Interfering; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Radiotherapy is a widely used treatment for many tumors. Combination therapy using anti-angiogenic agents and radiation has shown promise; however, these combined therapies are reported to have many limitations in clinical trials. Here, we show that radiation transformed tumor endothelial cells (ECs) to fibroblasts, resulting in reduced vascular endothelial growth factor (VEGF) response and increased Snail1, Twist1, Type I collagen, and transforming growth factor (TGF)-β release. Irradiation of radioresistant Lewis lung carcinoma (LLC) tumors greater than 250 mm³ increased collagen levels, particularly in large tumor vessels. Furthermore, concomitant sunitinib therapy did not show a significant difference in tumor inhibition versus radiation alone. Thus, we evaluated multimodal therapy that combined pirfenidone, an inhibitor of TGF-induced collagen production, with radiation and sunitinib treatment. This trimodal therapy significantly reduced tumor growth, as compared to radiation alone. Immunohistochemical analysis revealed that radiation-induced collagen deposition and tumor microvessel density were significantly reduced with trimodal therapy, as compared to radiation alone. These data suggest that combined therapy using pirfenidone may modulate the radiation-altered tumor microenvironment, thereby enhancing the efficacy of radiation therapy and concurrent chemotherapy.

Topics: Angiogenesis Inhibitors; Animals; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Lewis Lung; Cell Line, Tumor; Collagen; Combined Modality Therapy; Indoles; Mice; Microvessels; Pyridones; Pyrroles; Radiation Tolerance; Sunitinib; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A

Transforming growth factor-β (TGF-β) is a potent growth inhibitor in normal epithelial cells. However, a number of malignant tumors produce excessive amounts of TGF-β, which affects the tumor-associated microenvironment by furthering the progression of tumorigenicity. Although it is known that the tumor-associated microenvironment often becomes hypoxic, how hypoxia influences TGF-β signaling in this microenvironment is unknown. We investigated whether TGF-β signaling is influenced by long-term exposure to hypoxia in Lewis lung carcinoma (LLC) cells. When the cells were exposed to hypoxia for more than 10 days, their morphology was remarkably changed to a spindle shape, and TGF-β-induced Smad2 phosphorylation was enhanced. Concomitantly, TGF-β-induced transcriptional activity was augmented under hypoxia, although TGF-β did not influence the activity of a hypoxia-responsive reporter. Consistently, hypoxia influenced the expression of several TGF-β target genes. Interestingly, the expressions of TGF-β type I receptor (TβRI), also termed activin receptor like kinase-5 (ALK5), and TGF-β1 were increased under the hypoxic condition. When we monitored the hypoxia-inducible factor-1 (HIF-1) transcriptional activity by use of green fluorescent protein governed by the hypoxia-responsive element in LLC cells transplanted into mice, TGF-β-induced Smad2 phosphorylation was upregulated in vivo. Our results demonstrate that long-term exposure to hypoxia might alter responsiveness to TGF-β signaling and affected the malignancy of LLC cells.

Topics: Animals; Blotting, Western; Carcinoma, Lewis Lung; Cell Hypoxia; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Immunoprecipitation; Mice; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Transforming Growth Factor beta; Tumor Microenvironment

Umbelliprenin is a member of the 7-prenyloxycoumarins with potential therapeutic properties such as cytotoxic effects on various cancer cells. The present study investigates the effect of umbelliprenin on predominance of Th1 and Th2 responses in Lewis lung cancer (LLC) mouse model. The cytotoxic effect of umbelliprenin was explored on LLC cells and mouse splenocytes by MTT assay. Mice into which LLC had been transplanted were treated with umbelliprenin on alternate days, at 2.5 mg/200 µl intraperitoneally. Foxp3, TNF-α and TGF-β mRNA expressions were assessed in tumor and lung tissues of LLC mice. In addition, IL-10, IFN-γ and IL-4 levels were determined in sera and also in splenocyte culture supernatants at the presence of tumor cell lysate (10 µg/ml) and Con A (3 µg/ml) after 72 h. Results showed the cytotoxic effects of umbelliprenin on LLC cells (IC₅₀ = 51.6 ± 5.4 µM) while no adverse effect was seen at this concentration on normal splenocytes. TNF-α mRNA expression in both lung and tumor tissues was increased. However, Foxp3 and TGF-β expressions were decreased in tumor tissues. Serum level of IFN-γ was elevated in the umbelliprenin treated cancerous mice compared to the control group while IL-10 and IL-4 secretions were reduced. Tumor size was also decreased in umbelliprenin treated group. In summary, umbelliprenin has shown a partially Th1 bias with a reduction of regulatory immune response. Although the mechanism behind this action is not known, it is speculated that upon changing the Th1/Th2 balance in favour of Th1, umbelliprenin induces its antitumor activity.

Topics: Animals; Carcinoma, Lewis Lung; Cell Line, Tumor; Female; Forkhead Transcription Factors; Interferon-gamma; Interleukin-10; Lung Neoplasms; Mice; Neoplasm Proteins; Th1 Cells; Th2 Cells; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; Umbelliferones

There is growing evidence that generation of adenosine from ATP, which is mediated by the CD39/CD73 enzyme pair, predetermines immunosuppressive and proangiogenic properties of myeloid cells. We have previously shown that the deletion of the TGF-β type II receptor gene (Tgfbr2) expression in myeloid cells is associated with decreased tumor growth, suggesting protumorigenic effect of TGF-β signaling. In this study, we tested the hypothesis that TGF-β drives differentiation of myeloid-derived suppressor cells into protumorigenic terminally differentiated myeloid mononuclear cells (TDMMCs) characterized by high levels of cell-surface CD39/CD73 expression. We found that TDMMCs represent a major cell subpopulation expressing high levels of both CD39 and CD73 in the tumor microenvironment. In tumors isolated from mice with spontaneous tumor formation of mammary gland and conditional deletion of the type II TGF-β receptor in mammary epithelium, an increased level of TGF-β protein was associated with further increase in number of CD39(+)CD73(+) TDMMCs compared with MMTV-PyMT/TGFβRII(WT) control tumors with intact TGF-β signaling. Using genetic and pharmacological approaches, we demonstrated that the TGF-β signaling mediates maturation of myeloid-derived suppressor cells into TDMMCs with high levels of cell surface CD39/CD73 expression and adenosine-generating capacity. Disruption of TGF-β signaling in myeloid cells resulted in decreased accumulation of TDMMCs, expressing CD39 and CD73, and was accompanied by increased infiltration of T lymphocytes, reduced density of blood vessels, and diminished progression of both Lewis lung carcinoma and spontaneous mammary carcinomas. We propose that TGF-β signaling can directly induce the generation of CD39(+)CD73(+) TDMMCs, thus contributing to the immunosuppressive, proangiogenic, and tumor-promoting effects of this pleiotropic effector in the tumor microenvironment.

Topics: 5'-Nucleotidase; Animals; Antigens, CD; Apyrase; Bone Marrow Cells; Carcinoma, Lewis Lung; Cell Differentiation; Cell Line, Tumor; Cell Movement; Female; Mammary Glands, Animal; Mammary Neoplasms, Animal; Mice; Mice, Inbred C57BL; Mice, Knockout; Myeloid Cells; Protein Serine-Threonine Kinases; Receptor, Transforming Growth Factor-beta Type II; Receptors, Transforming Growth Factor beta; Signal Transduction; T-Lymphocytes; Transforming Growth Factor beta; Tumor Microenvironment; Vascular Endothelial Growth Factor A

TGFBI, a transforming growth factor β-induced extracellular matrix protein, circulates at a level of ~300ng/ml in humans and modulates several integrin-mediated cellular functions. The protein contains an N-terminal EMI domain, four consecutive FAS1 domains, and the RGD motif. Each FAS1 domain and the RGD motif have been known to interact with avb3 integrin. Here, we found that the binding affinity (Kd) of TGFBI for αvβ3 integrin was approximately 3.8×10(-8)M, a value ~2300-fold higher than that of a single FAS1 domain, and demonstrated that this greater affinity was due to the cooperative action of the four FAS1 domains and the RGD motif. Moreover, TGFBI exhibited more potent anti-angiogenic and anti-tumorigenic activities, even at a 100-fold lower molar dose than the reported effective dose of the FAS1 domain. Finally, our data showed that TGFBI specifically targeted the tumor vasculature and accumulated at the tumor site. Collectively, our results support the theory that TGFBI acts as a potent endogenous anti-tumor and anti-angiogenic molecule by targeting αvβ3 integrin, and highlights the importance of physiological circulating TGFBI levels in inhibiting tumor growth.

Topics: Animals; Antibodies, Monoclonal; Antibody Formation; Blotting, Western; Carcinoma, Lewis Lung; Cell Proliferation; Endothelium, Vascular; Extracellular Matrix Proteins; fas Receptor; Humans; Immunoenzyme Techniques; Integrin alphaVbeta3; Male; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Nude; Neovascularization, Pathologic; Oligopeptides; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins; Signal Transduction; Transforming Growth Factor beta; Tumor Cells, Cultured

The interactions of tumor cells with stromal fibroblasts influence tumor biology, but the exact mechanisms involved are still unclear. In the present study, we evaluated the effects of a human lung fibroblast cell line, TIG-3, on Lewis lung carcinoma (LLC) cells both in vitro and in vivo.. LLC and TIG-3 cells were co-cultured/co-implanted in vitro and in vivo. Cell invasion was assayed. Local tumor growth, as well as lung metastasis, were evaluated after subcutaneous cell co-implantation into NOD/SCID/γ-null (NOG) mice. LLC, and TIG-3 cells were pre-treated with either SB431542, a small molecule TGF-β receptor antagonist, or siRNA for transforming growth factor (TGF)-β before co-culture or co-implantation, and the effects of pre-treatments were compared both in cell culture and in mice.. Subcutaneous LLC tumor growth (L group) in NOG mice was significantly increased by co-implantation of TIG-3 cells (L+T group) at four weeks. The number of macroscopic lung metastases was also significantly increased in the L+T group in comparison to the L group. In vitro cell invasion was significantly increased in the L+T group in comparison to the L group. In vitro expression of phosphorylated-SMAD3 was significantly increased in the L+T group in comparison to the L group. Furthermore, pre-treatment with either SB431542 or siRNA for TGF-β reduced the invasiveness both in culture and in mice.. This study suggested that in vitro as well as in vivo progression of LLC was facilitated by co-culture/co-implantation with TIG-3 cells, and that this process was at least in part dependent on TGF-β-mediated interactions.

Topics: Animals; Blotting, Western; Carcinoma, Lewis Lung; Cell Line; Cell Line, Tumor; Coculture Techniques; Disease Progression; Humans; Mice; Mice, Inbred NOD; Mice, SCID; Neoplasm Invasiveness; Neoplasm Transplantation; Transforming Growth Factor beta

The effects of TGF-β on dendritic cells (DCs) on the tumor microenvironment are not well understood. We report, here, the establishment of an in vitro lung cancer microenvironment by co-incubation of seminaphtharhodafluor (SNARF) labeled Lewis lung cancer (LLC) cells, carboxyfluorescein succinimidyl ester (CFSE) labeled fibroblasts and 4-chloromethyl-7-hydroxycoumarin (CMHC) labeled DCs. Raw 264.7, EL4 and NCI-H446 cells were able to synthesize TGF-β which was determined by flow cyto-metry and western blotting, respectively. Furthermore, TGF-β efficiently increased regulatory T-cell (Treg) expansion and upregulated DC B7H1 and GITRL expression. TGF-β and the co-incubation of LLC cells, fibroblasts with DCs could augment the expression of B7H1 and GITRL molecules of DCs. The data presented here indicate that the B7H1 and GITRL molecules may play an important role in TGF-β-induced Treg expansion of lung cancer microenvironment.

Topics: Animals; B7-H1 Antigen; Carcinoma, Lewis Lung; Cell Communication; Cell Differentiation; Cell Line, Tumor; Dendritic Cells; Female; Fibroblasts; Flow Cytometry; Mice; Mice, Inbred BALB C; Recombinant Proteins; T-Lymphocytes, Regulatory; Transforming Growth Factor beta; Tumor Microenvironment; Tumor Necrosis Factors; Up-Regulation

Tumor-associated macrophages (TAMs) constitute a major component of the immune cell infiltrate observed in the tumor microenvironment (TME). Factors present in the TME, including tumor growth factor-β (TGF-β), allow tumors to circumvent host-mediated immune responses to promote tumor progression. However, the molecular mechanism(s) involved are not clear. Toll-like receptors (TLRs) are important mediators of innate immune responses by immune cells, whose activation triggers the production of molecules required for anti-tumoral responses. Interleukin (IL) receptor-associated kinase (IRAK)-M is an inactive serine/threonine kinase, predominantly expressed in macrophages and is a potent negative regulator of TLR signaling. In this study, we show that TAMs express significantly higher levels of IRAK-M compared with peritoneal macrophages in a syngeneic mouse model of lung cancer. Subcutaneous implantation of Lewis lung carcinoma cells in IRAK-M(-/-) mice resulted in a five-fold reduction in tumor growth as compared with tumors in wild-type (WT) animals. Furthermore, compared with WT TAMs, TAMs isolated from IRAK-M(-/-) mice displayed features of a classically activated (M1) rather than alternatively activated (M2) phenotype, as manifest by greater expression of IL-12, interferon-γ (IFN-γ) and inducible nitric oxide synthase. Human lung cancer cells induced IRAK-M expression in human peripheral blood mononuclear cells (PBMCs) when co-cultured together. Tumor cell-induced expression of IRAK-M was dependent on the activation of TGF-β pathway. Similarly, treatment of human PBMCs or mouse macrophage cell line, RAW 264.4, with TGF-β, induced IRAK-M expression. Interestingly, IRAK-M gene expression in 439 human lung adenocarcinoma tumors correlated with poor survival in patients with lung cancer. Together, our data demonstrates that TGF-β-dependent induction of IRAK-M expression is an important, clinically relevant mechanism by which tumors may circumvent anti-tumor responses of macrophages.

Topics: Animals; Blotting, Western; Carcinoma, Lewis Lung; Cell Line; Cell Line, Tumor; Cells, Cultured; Coculture Techniques; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Interferon-gamma; Interleukin-1 Receptor-Associated Kinases; Interleukin-12; Lung Neoplasms; Macrophages; Macrophages, Peritoneal; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Monocytes; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Survival Analysis; Transforming Growth Factor beta

A main goal of cancer immunology research is the formation of Ag-specific memory T cell immunity capable of activation upon tumor re-encounter. The requirements necessary to overcome the inhibitory signals present in the tumor microenvironment and form such memory T cell responses are unknown. In contrast to previous studies targeting tumors expressing highly immunogenic model Ags, we demonstrate that alleviating tumor-induced suppression along with vaccination against authentic Ags during the perioperative period provides long-lasting protection against a highly suppressive and poorly immunogenic melanoma. In this study, we employed DNA vaccination with an immunologically optimized mouse melanoma-shared Ag, Trp1ee/ng, combined with systemic TGF-β blockade during the perioperative period of primary tumor resection, to confer protection against B16 melanoma, and against JBRH, an independently derived melanoma unrelated to B16. Importantly, we demonstrate that correlative to memory responses, perioperative immunotherapy increases the formation of tumor-infiltrating and tumor-reactive CD8(+) T cells expressing low levels of the transcription factor T-bet, defined as memory precursor effector cells. We show that conditions for an immunologically fertile environment are met when TGF-β blockade and vaccination are applied during the perioperative period of primary tumor resection. These findings address limitations of current CD8(+) T cell immunotherapies against cancer by generating effective CD8(+) T cell memory recall responses.

Topics: Animals; Cancer Vaccines; Carcinoma, Lewis Lung; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Drug Therapy, Combination; Immunization, Secondary; Immunologic Memory; Immunotherapy, Adoptive; Lymphocytes, Tumor-Infiltrating; Male; Melanoma, Experimental; Membrane Glycoproteins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Oxidoreductases; Perioperative Period; Stem Cells; Transforming Growth Factor beta; Vaccines, DNA

Motility of endothelial cells is a requirement for the vascularization of solid malignancies. While tumors have been shown to produce a host of angiogenic factors, including TGF-β, the mechanisms by which such factors regulate endothelial cell motility have not yet been defined. Thus, the role of the serine/threonine phosphatase PP-1 in regulating endothelial cell motility and cytoskeletal architecture was studied. The present study demonstrated that TGF-β stimulation of motility is dependent on PP-1. Likewise, TGF-β was shown to up-regulate paxillin expression through a process that was PP-1 dependent. The interplay between PP-1 and TGF-β was further observed by the induction of cell rounding and the loss of paxillin-actin co precipitations upon PP-1 inhibition and the compensation for these effects by TGF-β. Studies initiated to determine how PP-1 might regulate motility showed its role in maintaining cytoskeletal organization and its capacity to directly dephosphorylate the focal adhesion scaffolding protein paxillin. These studies suggest that the interplay between TGF-β and PP-1 regulates the motility of endothelial cells that is critical to the process of angiogenesis.

Topics: Actins; Animals; Carcinoma, Lewis Lung; Cell Movement; Culture Media, Conditioned; Cytoskeleton; Endothelial Cells; Focal Adhesions; Furans; Humans; Lipids; Mice; Paxillin; Phosphorylation; Protein Phosphatase 1; Transforming Growth Factor beta; Up-Regulation

Thyroid transcription factor-1 (TTF-1) is expressed in lung cancer, but its functional roles remain unexplored. TTF-1 gene amplification has been discovered in a part of lung adenocarcinomas, and its action as a lineage-specific oncogene is highlighted. Epithelial-to-mesenchymal transition (EMT) is a crucial event for cancer cells to acquire invasive and metastatic phenotypes and can be elicited by transforming growth factor-beta (TGF-beta). Mesenchymal-to-epithelial transition (MET) is the inverse process of EMT; however, signals that induce MET are largely unknown. Here, we report a novel functional aspect of TTF-1 that inhibits TGF-beta-mediated EMT and restores epithelial phenotype in lung adenocarcinoma cells. This effect was accompanied by down-regulation of TGF-beta target genes, including presumed regulators of EMT, such as Snail and Slug. Moreover, silencing of TTF-1 enhanced TGF-beta-mediated EMT. Thus, TTF-1 can exert a tumor-suppressive effect with abrogation of cellular response to TGF-beta and attenuated invasive capacity. We further revealed that TTF-1 down-regulates TGF-beta2 production in A549 cells and that TGF-beta conversely decreases endogenous TTF-1 expression, suggesting that enhancement of autocrine TGF-beta signaling accelerates the decrease of TTF-1 expression and vice versa. These findings delineate potential links between TTF-1 and TGF-beta signaling in lung cancer progression through regulation of EMT and MET and suggest that modulation of TTF-1 expression can be a novel therapeutic strategy for treatment of lung adenocarcinoma.

Topics: Adenocarcinoma; Animals; Cadherins; Carcinoma, Lewis Lung; Cell Line, Tumor; Cell Movement; Cloning, Molecular; DNA-Binding Proteins; Epithelial Cells; Humans; Lung Neoplasms; Matrix Metalloproteinase 2; Mesoderm; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Signal Transduction; Transcription Factors; Transforming Growth Factor beta

This study reveals that the IL-15 rapidly released into serum upon IL-12 injection into tumor-bearing mice is critical for the subsequent leukocytic infiltration of the tumor and tumor-bearing tissue. The increase in serum IL-15 occurs within 2 h after IL-12 injection concomitantly with a decrease in cytoplasmic IL-15 in tumor-associated Mphi (TAM). Injection of anti-IL-15 one hour prior to IL-12 abrogates subsequent leukocytic infiltration into the tumor and prevents the IL-12-induced reduction of primary tumor mass and the clearance of metastases. Administration of anti-IL-15 18 h after IL-12 did not have a detectable impact on IL-12-induced leukocytic infiltration of the tumor. Deletion of NK cells had no impact on the IL-12-induced change in the functional phenotype of TAM or on the subsequent initiation of leukocytic infiltration of the tumor. In concert with our previous studies demonstrating that IL-12 reduces tumor-supportive activities of TAM, the current study supports the hypothesis that functional re-programming of TAM not only undermines Mphi support for tumor growth but also contributes to a critical step in the initiation of anti-tumor immune responses. In this context, the functional plasticity and pro-immunogenic potential of TAM may constitute a significant and unappreciated target in existing cytokine therapies.

Topics: Animals; Antibodies; Carcinoma, Lewis Lung; Cell Line, Tumor; Cytokines; Cytoplasm; Enzyme-Linked Immunosorbent Assay; Female; Flow Cytometry; Gene Expression Regulation, Neoplastic; Interleukin-12; Interleukin-15; Leukocytes; Lung; Macrophages; Mice; Mice, Inbred C57BL; Reverse Transcriptase Polymerase Chain Reaction; Transforming Growth Factor beta; Tumor Burden

CD4+CD25+ regulatory T cells (Treg) that suppress T cell-mediated immune responses may also regulate other arms of an effective immune response. In particular, in this study we show that Treg directly inhibit NKG2D-mediated NK cell cytotoxicity in vitro and in vivo, effectively suppressing NK cell-mediated tumor rejection. In vitro, Treg were shown to inhibit NKG2D-mediated cytolysis largely by a TGF-beta-dependent mechanism and independently of IL-10. Adoptively transferred Treg suppressed NK cell antimetastatic function in RAG-1-deficient mice. Depletion of Treg before NK cell activation via NKG2D and the activating IL-12 cytokine, dramatically enhanced NK cell-mediated suppression of tumor growth and metastases. Our data illustrate at least one mechanism by which Treg can suppress NK cell antitumor activity and highlight the effectiveness of combining Treg inhibition with subsequent NK cell activation to promote strong innate antitumor immunity.

Topics: Animals; Carcinoma, Lewis Lung; Cell Communication; Cell Line, Tumor; Immunotherapy, Adoptive; Interleukin-12; Killer Cells, Natural; Ligands; Lung Neoplasms; Lymphoma; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Neoplasm Transplantation; Neoplasms, Experimental; NK Cell Lectin-Like Receptor Subfamily K; Receptors, Immunologic; Receptors, Natural Killer Cell; Spleen; T-Lymphocytes, Regulatory; Transforming Growth Factor beta

The modifying effects of a Kunitz trypsin inhibitor (KTI) and a Bowman-Birk trypsin inhibitor (BBI), purified from soybean trypsin inhibitor, as dietary supplements on experimental and spontaneous pulmonary metastasis of murine Lewis lung carcinoma 3LL cells as well as peritoneal disseminated metastasis model in human ovarian cancer HRA cells were investigated in i.v., s.c. and i.p. injection models in mice. Seven groups of female C57BL/6 or nude mice were fed a basal diet (control group) or the basal diet supplemented with KTI or BBI (5, 15, or 50 g/kg). Here we show that, in an in vivo spontaneous metastasis assay, the diet supplementation with KTI (15 and 50 g/kg), but not with BBI, for 28 days immediately after s.c. tumor cell inoculation significantly inhibited the formation of lung metastasis in C57BL/6 mice in a dose-dependent manner. The inhibition of lung metastasis was not due to direct antitumor effects of KTI. In an in vivo experimental metastasis assay, the diet supplementation with KTI or BBI for 21 days after i.v. tumor cell inoculation did not reduce the number of lung tumor colonies. In addition, KTI (15 or 50 g/kg) treatment in a peritoneal disseminated metastasis model of HRA cells resulted in a 40% reduction in total tumor burden when compared with control animals. Immunoblot analysis revealed that KTI specifically reduced expression of uPA protein as well as phosphorylation of MAP kinase and PI3 kinase proteins in the cells stimulated with agonists (G-CSF for 3LL cells or TGF-beta1 for HRA cells). These results suggest that dietary supplementation of KTI more efficiently regulates the mechanism involved in the entry into vascular circulation of tumor cells (intravasation) than in extravasation during the metastatic process. KTI treatment may also be beneficial for ovarian cancer patients with or at risk for peritoneal disseminated metastasis; it greatly reduces tumor burden in part by inhibiting phosphorylation of MAP kinase and PI3 kinase, leading to suppression of uPA expression.

Topics: Animals; Carcinoma, Lewis Lung; Cell Line, Tumor; Dietary Supplements; Enzyme Inhibitors; Female; Gene Expression Regulation, Neoplastic; Granulocyte Colony-Stimulating Factor; Humans; Lung Neoplasms; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Models, Animal; Ovarian Neoplasms; Peritoneal Neoplasms; Phosphatidylinositol 3-Kinases; Phosphorylation; Transforming Growth Factor beta; Transforming Growth Factor beta1; Trypsin Inhibitor, Bowman-Birk Soybean; Trypsin Inhibitor, Kunitz Soybean; Trypsin Inhibitors; Urokinase-Type Plasminogen Activator

Tumor vascularization is a complex process that requires structural reorganization and increased motility by endothelial cells. Studies were conducted to identify the tumor-derived mediators and signaling pathways that lead to this increased endothelial cell motility. Using the Lewis lung carcinoma (LLC) tumor model, these studies showed that prostaglandin E2 (PGE2) and transforming growth factor-beta (TGFbeta) were the mediators that were responsible for the migration-stimulatory activity produced by the tumor cells. The response of endothelial cells to these tumor-derived motility-stimulatory factors involved a decline in the activity of the serine/threonine phosphatase PP-2A. Inhibition PP-2A either pharmacologically or genetically increased endothelial cell migration. Concurrent with the decline in PP-2A activity as a result of exposure to PGE2/TGFbeta was a loss of PP-2A co-precipitation with the inositol phosphatase PTEN and an increase in the PTEN serine phosphorylation level. Since hyperphosphorylation has been shown to inhibit the ability of PTEN to act as an antagonist to phosphatidylinositide 3-kinase (PI3K), the role of PI3K in PGE2/TGFbeta-stimulated migration was examined. These studies showed that the increased endothelial cell motility that resulted from PGE2/TGFbeta inhibition of PP-2A was dependent on PI3K.

Topics: Animals; Carcinoma, Lewis Lung; Cell Line; Cell Movement; Dinoprostone; Endothelial Cells; Humans; Mice; Neovascularization, Pathologic; Phosphatidylinositol 3-Kinases; Phosphoprotein Phosphatases; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Phosphatase 2; PTEN Phosphohydrolase; Transforming Growth Factor beta; Tumor Suppressor Proteins

Thrombospondin-1 (TSP-1) is a potent inhibitor of tumor growth and angiogenesis. The antiangiogenic activity of TSP-1 has been mapped to the procollagen homology region and the type 1 repeats (TSR) using synthetic peptides. To elucidate the molecular mechanisms that are involved in the inhibition of tumor growth by the TSRs, we have expressed recombinant versions of these motifs and have assayed their ability to inhibit the growth of experimental B16F10 melanomas and Lewis lung carcinomas. Recombinant proteins that contain all three TSRs (3TSR) or the second TSR with (TSR2+RFK) or without (TSR2) the transforming growth factor-beta (TGFbeta) activating sequence (RFK) have been expressed in Drosophila S2 cells. In addition, recombinant proteins with mutations in either the RFK sequence (TSR2+QFK) or the WSHWSPW sequence [TSR2 (W/T)] of the second TSR have been prepared. Similar to platelet TSP-1, these proteins are potent inhibitors of endothelial cell migration, and 3TSR of human TSP-1 (3TSR/hTSP-1) and TSR2+RFK activate TGFbeta. An 81% inhibition of B16F10 tumor growth is observed at 2.5 mg (135 nmol)/kg/day of the recombinant 3TSR/hTSP-1. A comparable level of inhibition is observed with 2.5 mg (360 nmol)/kg/day of TSR2+RFK. By contrast, 3TSR of mouse TSP-2 (3TSR/mTSP-2), TSR2+QFK, and TSR2 are significantly less effective. TSR2+RFK and TSR2 reduce tumor vessel density, but TSR2+RFK has a greater effect on B16F10 tumor cell apoptosis and proliferation. Concurrent treatment of B16F10 tumor-bearing mice with TSR2+RFK and either a soluble form of the TGFbeta receptor or an antibody to active TGFbeta reduces the inhibition of B16F10 tumor growth to levels that are comparable with those of TSR2 and TSR2+QFK. By contrast, the presence of the TGFbeta-activating sequence does not increase the level of inhibition of Lewis lung carcinoma experimental tumor growth. These data indicate that the TSRs inhibit tumor growth by inhibition of angiogenesis and regulation of tumor cell growth and apoptosis. The regulation of tumor cell growth and apoptosis is TGFbeta dependent, whereas the inhibition of angiogenesis is not.

Topics: Animals; Antineoplastic Agents; Apoptosis; Carcinoma, Lewis Lung; Cell Division; Cell Movement; Endothelium, Vascular; Growth Inhibitors; Humans; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neovascularization, Pathologic; Peptide Fragments; Recombinant Proteins; Thrombospondin 1; Transforming Growth Factor beta

In this study, we show that Delta-9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, suppresses host immune reactivity against lung cancer. In two different weakly immunogenic murine lung cancer models, intermittent administration of THC (5 mg/kg, four times/wk i.p. for 4 wk) led to accelerated growth of tumor implants compared with treatment with diluent alone. In contrast to our findings in immunocompetent mice, THC did not affect tumor growth in tumor-bearing SCID mice. The immune inhibitory cytokines, IL-10 and TGF-beta, were augmented, while IFN-gamma was down-regulated at both the tumor site and in the spleens of THC-treated mice. Administration of either anti-IL-10- or anti-TGF-beta-neutralizing Abs prevented the THC-induced enhancement in tumor growth. Both APC and T cells from THC-treated mice showed limited capacities to generate alloreactivity. Furthermore, lymphocytes from THC-treated mice transferred the effect to normal mice, resulting in accelerated tumor growth similar to that seen in the THC-treated mice. THC decreased tumor immunogenicity, as indicated by the limited capacity for tumor-immunized, THC-treated mice to withstand tumor rechallenge. In vivo administration of a specific antagonist of the CB2 cannabinoid receptor also blocked the effects of THC. Our findings suggest the THC promotes tumor growth by inhibiting antitumor immunity by a CB2 receptor-mediated, cytokine-dependent pathway.

Topics: Adoptive Transfer; Animals; Antibodies, Monoclonal; Antigen-Presenting Cells; Carcinoma, Lewis Lung; Cell Division; Cytokines; Dronabinol; Growth Inhibitors; Immunity, Innate; Immunosuppressive Agents; Injections, Intraperitoneal; Interleukin-10; Lymphocyte Culture Test, Mixed; Lymphocyte Subsets; Lymphocyte Transfusion; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, SCID; Neoplasm Transplantation; Receptors, Cannabinoid; Receptors, Drug; Severe Combined Immunodeficiency; T-Lymphocytes; Transforming Growth Factor beta; Tumor Cells, Cultured

Production of high levels of granulocyte-macrophage CSF (GM-CSF) by LLC-LN7 tumors results in myelopoietic stimulation and an increase in cells having natural suppressor (NS) activity. Prior studies showed these NS cells could be isolated from the bone marrow of tumor-bearing mice with an Ab (ER-MP12) that recognized GM-progenitor cells. The present study showed these cells to also be in the spleen, lymph node, and tumor, and that treatment of tumor-bearing mice with low doses of IFN-gamma plus TNF-alpha reduced the frequency of E-MP12+ cells. Studies focused on characterizing the intratumoral ER-MP12+ cells and the mechanism by which they suppress T cell proliferation. When isolated and seeded in soft agar with CSF-containing LLC-LN7 supernatants, the ER-MP12+ cells grew into colonies, most of which contained both granulocytic and monocytic cells. Tumor-derived ER-MP12+ cells and their culture supernatants were suppressive to T cell proliferation. Among the factors produced by ER-MP12+ cells were TGF-beta, nitric oxide (NO), IL-10, and prostaglandin E2 (PGE2). However, it was TGF-beta and NO that mediated the suppression of T cell proliferation by ER-MP12+ cells. Intratumoral ER-MP12+ cells could be maintained as suppressive blastlike cells for at least 4 days in cultures containing CSFs, but adding IFN-gamma plus TNF-alpha to these cultures caused their differentiation mainly into nonsuppressive TNF-alpha-secreting monocytic cells. These results show that intratumoral ER-MP12+ cells having homology to GM-progenitor cells suppress T cell function by producing TGF-beta and NO. IFN-gamma/TNF-alpha treatment stimulates their differentiation and shift from production of TGF-beta and NO to production of TNF-alpha.

Topics: Animals; Carcinoma, Lewis Lung; Cell Differentiation; Granulocytes; Hematopoietic Stem Cells; Immunity, Innate; Interferon-gamma; Lymphocyte Activation; Macrophages; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Nitric Oxide; Transforming Growth Factor beta; Tumor Cells, Cultured; Tumor Necrosis Factor-alpha