ABSTRACT
We previously published on the design and synthesis of novel, potent and selective PPARα antagonists suitable for either i.p. or oral in vivo administration for the potential treatment of cancer. Described herein is SAR for a subsequent program, where we set out to identify selective and potent PPARα/δ dual antagonist molecules. Emerging literature indicates that both PPARα and PPARδ antagonism may be helpful in curbing the proliferation of certain types of cancer. This dual antagonism could also be used to study PPARs in other settings. After testing for selective and dual potency, off-target counter screening, metabolic stability, oral bioavailability and associated toxicity, compound 11, the first reported PPARα/δ dual antagonist was chosen for more advanced preclinical evaluation.
Subject(s)
Antineoplastic Agents/pharmacology , Drug Discovery , Ovarian Neoplasms/drug therapy , PPAR alpha/antagonists & inhibitors , PPAR delta/antagonists & inhibitors , Sulfonamides/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Dogs , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Female , Humans , Mice , Molecular Structure , Neoplasms, Experimental/drug therapy , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , PPAR alpha/metabolism , PPAR delta/metabolism , Rats , Structure-Activity Relationship , Sulfonamides/chemical synthesis , Sulfonamides/chemistryABSTRACT
Peroxisome-proliferator activated receptors (PPAR) are members of the nuclear hormone receptor superfamily which regulate gene transcription. PPARα is a key regulator of lipid homeostasis and a negative regulator of inflammation. Under conditions of metabolic stress such as fasting or glucose deprivation, PPARα is upregulated in order to control gene expression necessary for processing alternate fuel sources (e.g. fatty acid oxidation) and thereby promote maintenance of cell viability. Clinically, PPARα expression is upregulated in diseased tissues such as melanoma, chronic lymphocytic leukemia, ovarian and prostate cancer. This may allow for cellular proliferation and metastasis. Importantly, genetic knockouts of PPARα have been shown to be protected against tumor growth in a variety of syngeneic tumors models. We hypothesized that a potent and selective PPARα antagonist could represent a novel cancer therapy. Early in our discovery research, we identified NXT629 (Bravo et al., 2014). Herein we describe the pharmacology of NXT629 and demonstrate that it is a potent and selective PPARα antagonist. We identify NXT629 as a valuable tool for use in in vivo assessment of PPARα due to its good systemic exposure following intraperitoneal injection. We explore the in vivo pharmacology of NXT629 and demonstrate that it is efficacious in pharmacodynamic models that are driven by PPARα. Finally, we probe the efficacy of NXT629 in disease models where PPARα knockouts have shown to be protected. We believe that PPARα antagonists will be beneficial in diseases such as ovarian cancer and melanoma where PPARα and fatty acid oxidation may be involved.
Subject(s)
Aminopyridines/pharmacology , PPAR alpha/antagonists & inhibitors , Sulfonamides/pharmacology , Aminopyridines/pharmacokinetics , Animals , Cell Line, Tumor , Cell Proliferation/drug effects , Fibroblast Growth Factors/blood , Humans , Mice , Neoplasm Metastasis , Neovascularization, Physiologic/drug effects , Rats , Sulfonamides/pharmacokineticsABSTRACT
Tumor-specific metabolic changes can reveal new therapeutic targets. Our findings implicate a supporting role for fatty acid metabolism in chronic lymphocytic leukemia (CLL) cell survival. Peroxisome proliferator-activated receptor (PPAR)-α, a major transcriptional regulator of fatty acid oxidation, was recently shown to be upregulated in CLL. To evaluate PPARα as a potential therapeutic target, we developed a highly selective, potent small molecule antagonist of PPARα, NXT629. NXT629 inhibited agonist-induced transcription of PPARα-regulated genes, demonstrating target engagement in CLL cells. Furthermore, NXT629 induced apoptosis of CLL cells even in the presence of a protective microenvironment. To mimic the proliferative lymphoid compartment of CLL, we examined the activity of NXT629 on CLL cells that were stimulated to proliferate in vitro. NXT629 reduced the number of leukemia cells undergoing cell division. In addition, in two xenograft mouse models of CLL (one a model for nondividing and one for dividing CLL), NXT629 reduced the number of viable CLL cells in vivo. Overall, these results suggest that fatty acid metabolism promotes survival and proliferation of primary CLL cells and that inhibiting PPARα gene regulation could be a new therapeutic approach to treating CLL.
Subject(s)
Aminopyridines/administration & dosage , Apoptosis/drug effects , Cell Proliferation/drug effects , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , PPAR alpha/genetics , Sulfonamides/administration & dosage , Animals , Cell Line, Tumor , Cell Survival/drug effects , Fatty Acids/metabolism , Gene Expression Regulation, Neoplastic , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Mice , PPAR alpha/antagonists & inhibitors , Transcriptional ActivationABSTRACT
INTRODUCTION: Cancer vaccines have the potential to induce curative anti-tumor immune responses and better adjuvants may improve vaccine efficacy. We have previously shown that Hp91, a peptide derived from the B box domain in high-mobility group box protein 1 (HMGB1), acts as a potent immune adjuvant. METHOD: In this study, Hp91 was tested as part of a therapeutic vaccine against human epidermal growth factor receptor 2 (HER2)-positive breast cancer. RESULTS: Free peptide did not significantly augment immune responses but, when delivered in poly(D,L-lactic-co-glycolic) acid nanoparticles (PLGA-NPs), robust activation of dendritic cells (DCs) and increased activation of HER2-specific T cells was observed in vitro. Vaccination of HER2/neu transgenic mice, a mouse breast cancer model that closely mimics the immune modulation and tolerance in some breast cancer patients, with Hp91-loaded PLGA-NPs enhanced the activation of HER2-specific cytotoxic T lymphocyte (CTL) responses, delayed tumor development, and prolonged survival. CONCLUSIONS: Taken together these findings demonstrate that the delivery of the immunostimulatory peptide Hp91 inside PLGA-NPs enhances the potency of the peptide and efficacy of a breast cancer vaccine.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/immunology , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/immunology , Lactic Acid/immunology , Nanoparticles/administration & dosage , Peptides/immunology , Receptor, ErbB-2/genetics , Receptor, ErbB-2/immunology , Animals , Antigen Presentation/immunology , Breast Neoplasms/mortality , Breast Neoplasms/pathology , Cancer Vaccines/immunology , Dendritic Cells/immunology , Disease Models, Animal , Female , Humans , Immunization , Immunomodulation , Mice , Mice, Transgenic , Peptides/administration & dosage , Peptides/chemistry , Polyglycolic Acid , Polylactic Acid-Polyglycolic Acid Copolymer , T-Lymphocytes, Cytotoxic/immunology , Tumor Burden/genetics , Tumor Burden/immunologyABSTRACT
High mobility group box protein 1 (HMGB1) acts as an endogenous danger molecule that is released from necrotic cells and activated macrophages. We have previously shown that peptide Hp91, whose sequence corresponds to an area within the B-Box domain of HMGB1, activates dendritic cells (DCs) and acts as an adjuvant in vivo. Here we investigated the underlying mechanisms of Hp91-mediated DC activation. Hp91-induced secretion of IL-6 was dependent on clathrin- and dynamin-driven endocytosis of Hp91 and mediated through a MyD88- and TLR4-dependent pathway involving p38 MAPK and NFκB. Endosomal TLR4 has been shown to activate the MyD88-independent interferon pathway. Hp91-induced activation of pIRF3 and IL-6 secretion was reduced in IFNαßR knockout DCs, suggesting an amplification loop via the IFNαßR. These findings elucidate the mechanisms by which Hp91 acts as immunostimulatory peptide and may serve as a guide for the future development of synthetic Th1-type peptide adjuvants for vaccines.
Subject(s)
Adjuvants, Immunologic/pharmacology , Dendritic Cells/drug effects , Dendritic Cells/immunology , HMGB1 Protein/pharmacology , Peptide Fragments/pharmacology , Toll-Like Receptor 4/physiology , Animals , Cells, Cultured , Dendritic Cells/metabolism , Female , HMGB1 Protein/chemistry , HMGB1 Protein/immunology , Humans , Immunotherapy, Adoptive/methods , Mice , Mice, Inbred C57BL , Mice, Knockout , Peptide Fragments/immunology , Protein Structure, Tertiary , Signal Transduction/drug effects , Signal Transduction/genetics , Signal Transduction/immunology , Toll-Like Receptor 4/geneticsABSTRACT
Lenalidomide has demonstrated clinical activity in patients with chronic lymphocytic leukemia (CLL), even though it is not cytotoxic for primary CLL cells in vitro. We examined the direct effect of lenalidomide on CLL-cell proliferation induced by CD154-expressing accessory cells in media containing interleukin-4 and -10. Treatment with lenalidomide significantly inhibited CLL-cell proliferation, an effect that was associated with the p53-independent upregulation of the cyclin-dependent kinase inhibitor, p21(WAF1/Cip1) (p21). Silencing p21 with small interfering RNA impaired the capacity of lenalidomide to inhibit CLL-cell proliferation. Silencing cereblon, a known molecular target of lenalidomide, impaired the capacity of lenalidomide to induce expression of p21, inhibit CD154-induced CLL-cell proliferation, or enhance the degradation of Ikaros family zinc finger proteins 1 and 3. We isolated CLL cells from the blood of patients before and after short-term treatment with low-dose lenalidomide (5 mg per day) and found the leukemia cells were also induced to express p21 in vivo. These results indicate that lenalidomide can directly inhibit proliferation of CLL cells in a cereblon/p21-dependent but p53-independent manner, at concentrations achievable in vivo, potentially contributing to the capacity of this drug to inhibit disease-progression in patients with CLL.
Subject(s)
Angiogenesis Inhibitors/pharmacology , Cell Proliferation/drug effects , Cyclin-Dependent Kinase Inhibitor p21/physiology , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Peptide Hydrolases/physiology , Thalidomide/analogs & derivatives , Adaptor Proteins, Signal Transducing , Gene Expression Regulation, Leukemic/drug effects , HeLa Cells , Humans , Lenalidomide , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Signal Transduction/drug effects , Thalidomide/pharmacology , Tumor Cells, Cultured , Tumor Suppressor Protein p53/physiology , Ubiquitin-Protein LigasesABSTRACT
The discovery and SAR of a novel series of potent and selective PPARα antagonists are herein described. Exploration of replacements for the labile acyl sulfonamide linker led to a biaryl sulfonamide series of which compound 33 proved to be suitable for further profiling in vivo. Compound 33 demonstrated excellent potency, selectivity against other nuclear hormone receptors, and good pharmacokinetics in mouse.
Subject(s)
PPAR alpha/antagonists & inhibitors , Sulfonamides/chemistry , Sulfonamides/pharmacology , Animals , Butyrates/chemistry , Butyrates/pharmacology , Humans , Mice , Molecular Structure , Oxazoles/chemistry , Oxazoles/pharmacology , Phenylurea Compounds/chemistry , Phenylurea Compounds/pharmacology , Propionates/chemistry , Propionates/pharmacology , Structure-Activity Relationship , Triazoles/chemistry , Triazoles/pharmacology , Tyrosine/analogs & derivatives , Tyrosine/chemistry , Tyrosine/pharmacologyABSTRACT
The interplay between the tumor cells and the surrounding stroma creates inflammation, which promotes tumor growth and spread. The inflammation is a hallmark for pancreatic adenocarcinoma (PDAC) and is to high extent driven by IL-1α. IL-1α is expressed and secreted by the tumor cells and exerting its effect on the stroma, i.e. cancer associated fibroblasts (CAF), which in turn produce massive amount of inflammatory and immune regulatory factors. IL-1 induces activation of transcription factors such as nuclear factor-κß (NF-κß), but also activator protein 1 (AP-1) via the small G-protein Ras. Dysregulation of Ras pathways are common in cancer as this oncogene is the most frequently mutated in many cancers. In contrast, the signaling events leading up to the expression of IL-1α by tumor cells are not well elucidated. Our aim was to examine the signaling cascade involved in the induction of IL-1α expression in PDAC. We found p38MAPK, activated by the K-Ras signaling pathway, to be involved in the expression of IL-1α by PDAC as blocking this pathway decreased both the gene and protein expression of IL-1α. Blockage of the P38MAPK signaling in PDAC also dampened the ability of the tumor cell to induce inflammation in CAFs. In addition, the IL-1α autocrine signaling regulated the migratory capacity of PDAC cells. Taken together, the blockage of signaling pathways leading to IL-1α expression and/or neutralization of IL-1α in the PDAC microenvironment should be taken into consideration as possible treatment or complement to existing treatment of this cancer.
Subject(s)
Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Cell Movement/genetics , Interleukin-1alpha/genetics , MAP Kinase Signaling System , Cell Communication/drug effects , Cell Line, Tumor , Extracellular Signal-Regulated MAP Kinases/metabolism , Fibroblasts/metabolism , Gene Expression Regulation, Neoplastic , Humans , Inflammation/genetics , Inflammation/metabolism , Interleukin 1 Receptor Antagonist Protein/pharmacology , MAP Kinase Signaling System/drug effects , Protein Kinase Inhibitors/pharmacology , p38 Mitogen-Activated Protein Kinases/antagonists & inhibitors , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
BACKGROUND: Current therapies to treat cancer, although successful for some patients, have significant side-effects and a high number of patients have disease that is either non-responsive or which develops resistance. Our goal was to design a small peptide that possesses similar functions to an antibody drug conjugate with regard to targeting and killing cancer cells, but that overcomes size restrictions. MATERIALS AND METHODS: We designed a novel cancer-specific killer peptide created by fusion of the toxic peptide (KLAKLAK)2 with the cancer recognition peptide LTVSPWY. RESULTS: This bi-functional peptide showed toxicity to breast cancer, prostate cancer, and neuroblastoma cell lines. Only low toxicity to non-cancer cells, colon cancer, lung cancer, and lymphoma cell lines was observed. In vivo injections of the bi-functional peptide caused tumor growth retardation compared to mice treated with control peptides. The bi-functional peptide caused retardation of MDA-MB-435S tumors in vivo and increased survival to 80% at day 100 after tumor implantation, whereas all control animals died at day 70. Previous reports showed that the recognition moiety LTVSPWY targets the tumor-associated antigen HER2. Here we found that our new peptide TP-Tox also excerts toxic effects on HER2-negative cell lines. Therefore, we searched for the molecular target of the bi-specific peptide using immunoprecipitation and mass spectrometry. Our data suggest a possible interaction with RAS GTPase-activating protein binding protein 1 (G3BP1). CONCLUSION: We designed a bi-functional peptide of 23 amino acids and demonstrated its ability to bind and kill several cancer cell lines in vitro and to strongly increase survival in breast cancer bearing mice in vivo. This novel toxin could be used in future cancer therapies and warrants further pre-clinical and clinical exploration.
Subject(s)
Antineoplastic Agents/pharmacology , Oligopeptides/pharmacology , Amino Acid Sequence , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/toxicity , Cell Line, Tumor , Endocytosis , Female , Humans , Mice , Neoplasms/drug therapy , Neoplasms/pathology , Oligopeptides/chemistry , Oligopeptides/toxicity , Protein Binding , Xenograft Model Antitumor AssaysABSTRACT
Chronic lymphocytic leukemia (CLL) cells interact in the marrow with mesenchymal stromal cells (MSCs), which can enhance CLL-cells' resistance to spontaneous or drug-induced apoptosis. Here we examined the effect of oxygen on the growth and function of MSCs from marrow aspirates of CLL patients. Cultures in ambient oxygen provided for poor recovery and growth of MSCs, which developed features of cell senescence. However, MSCs were propagated readily from the same cells when they were cultured at a physiologic oxygen concentration of 5%. Such MSCs promoted short-term CLL-cell survival in either 5% or ambient O2. However, longer-term CLL-cell survival was enhanced when the cocultures were maintained in 5% O2 versus 21% O2 because of increased MSC proliferation and production of soluble prosurvival factors, such as CXCL12. This study establishes the importance of physiologic oxygen concentration in the propagation and function of MSCs derived from marrow aspirates of CLL patients in vitro.
Subject(s)
Bone Marrow Cells/drug effects , Cell Proliferation/drug effects , Mesenchymal Stem Cells/drug effects , Oxygen/pharmacology , Antigens, CD19/metabolism , Bone Marrow Cells/metabolism , CD5 Antigens/metabolism , Cell Survival/drug effects , Cells, Cultured , Cellular Senescence/drug effects , Chemokine CXCL12/metabolism , Coculture Techniques , Dose-Response Relationship, Drug , Flow Cytometry , Humans , Immunoblotting , Immunophenotyping , Leukemia, Lymphocytic, Chronic, B-Cell/blood , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Mesenchymal Stem Cells/metabolism , Receptors, CXCR4/metabolism , Tumor Cells, CulturedABSTRACT
Human immunodeficiency virus type 1 (HIV-1) infection enhances the expression of inhibitory molecules on T cells, leading to T-cell impairment. The signaling pathways underlying the regulation of inhibitory molecules and subsequent onset of T-cell impairment remain elusive. We showed that both autologous and allogeneic T cells exposed to HIV-pulsed dendritic cells (DCs) upregulated cytotoxic T-lymphocyte antigen (CTLA-4), tumor-necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), lymphocyte-activation gene-3 (LAG3), T-cell immunoglobulin mucin-3 (TIM-3), CD160 and certain suppression-associated transcription factors, such as B-lymphocyte induced maturation protein-1 (BLIMP-1), deltex homolog 1 protein (DTX1) and forkhead box P3 (FOXP3), leading to T-cell suppression. This induction was regulated by p38 mitogen-activated protein kinase/signal transducer and activator of transcription-3 (P38MAPK/STAT3) pathways, because their blockade significantly abrogated expression of all the inhibitory molecules studied and a subsequent recovery in T-cell proliferation. Neither interleukin-6 (IL-6) nor IL-10 nor growth factors known to activate STAT3 signaling events were responsible for STAT3 activation. Involvement of the P38MAPK/STAT3 pathways was evident because these proteins had a higher level of phosphorylation in the HIV-1-primed cells. Furthermore, blockade of viral CD4 binding and fusion significantly reduced the negative effects DCs imposed on primed T cells. In conclusion, HIV-1 interaction with DCs modulated their functionality, causing them to trigger the activation of the P38MAPK/STAT3 pathway in T cells, which was responsible for the upregulation of inhibitory molecules.
Subject(s)
Dendritic Cells/virology , HIV-1/immunology , Lymphocyte Activation/immunology , STAT3 Transcription Factor/metabolism , Signal Transduction/immunology , T-Lymphocytes/immunology , p38 Mitogen-Activated Protein Kinases/metabolism , Cell Proliferation/drug effects , Cytosol/drug effects , Cytosol/metabolism , Dendritic Cells/drug effects , Dendritic Cells/immunology , Dendritic Cells/pathology , Enterotoxins/pharmacology , HIV-1/drug effects , Humans , Immunologic Memory/drug effects , Interleukin-10/metabolism , Interleukin-6/metabolism , Lymphocyte Activation/drug effects , MAP Kinase Signaling System/drug effects , Neutralization Tests , Signal Transduction/drug effects , T-Lymphocytes/drug effects , T-Lymphocytes/enzymology , T-Lymphocytes/pathologyABSTRACT
We have previously shown that sorafenib, a multikinase inhibitor, exhibits cytotoxic effects on chronic lymphocytic leukemia (CLL) cells. Because the cellular microenvironment can protect CLL cells from drug-induced apoptosis, it is important to evaluate the effect of novel drugs in this context. Here we characterized the in vitro cytotoxic effects of sorafenib on CLL cells and the underlying mechanism in the presence of marrow stromal cells (MSCs) and nurselike cells (NLCs). One single dose of 10 µmol/L or the repeated addition of 1 µmol/L sorafenib caused caspase-dependent apoptosis and reduced levels of phosphorylated B-RAF, C-RAF, extracellular signal-regulated kinase (ERK), signal transducer and activator of transcription 3 (STAT3) and myeloid cell leukemia sequence 1 (Mcl-1) in CLL cells in the presence of the microenvironment. We show that the RAF/mitogen-activated protein kinase kinase (MEK)/ERK pathway can modulate Mcl-1 expression and contribute to CLL cell viability, thereby associating so-rafenib cytotoxicity to its impact on RAF and Mcl-1. To evaluate if the other targets of sorafenib can affect CLL cell viability and contribute to sorafenib-mediated cytotoxicity, we tested the sensitivity of CLL cells to several kinase inhibitors specific for these targets. Our data show that RAF and vascular endothelial growth factor receptor (VEGFR) but not KIT, platelet-derived growth factor receptor (PDGFR) and FMS-like tyrosine kinase 3 (FLT3) are critical for CLL cell viability. Taken together, our data suggest that sorafenib exerts its cytotoxic effect likely via inhibition of the VEGFR and RAF/MEK/ERK pathways, both of which can modulate Mcl-1 expression in CLL cells. Furthermore, sorafenib induced apoptosis of CLL cells from fludarabine refractory patients in the presence of NLCs or MSCs. Our results warrant further clinical exploration of sorafenib in CLL.
Subject(s)
Apoptosis/drug effects , Benzenesulfonates/pharmacology , Cell Survival/drug effects , Proto-Oncogene Proteins c-bcl-2/metabolism , Pyridines/pharmacology , raf Kinases/metabolism , Cells, Cultured , Humans , Immunoblotting , Leukemia, Lymphocytic, Chronic, B-Cell , Myeloid Cell Leukemia Sequence 1 Protein , Niacinamide/analogs & derivatives , Phenylurea Compounds , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Sorafenib , Tumor Cells, CulturedABSTRACT
Tumor microenvironment is composed of tumor cells, fibroblasts, and infiltrating immune cells, which all work together and create an inflammatory environment favoring tumor progression. The present study aimed to investigate the role of the desmoplastic stroma in pancreatic ductal adenocarcinoma (PDAC) regarding expression of inflammatory factors and infiltration of immune cells and their impact on the clinical outcome. The PDAC tissues examined expressed significantly increased levels of immunomodulatory and chemotactic factors (IL-6, TGFß, IDO, COX-2, CCL2, and CCL20) and immune cell-specific markers corresponding to macrophages, myeloid, and plasmacytoid dendritic cells (DCs) as compared to controls. Furthermore, short-time survivors had the lowest levels of DC markers. Immunostainings indicated that the different immune cells and inflammatory factors are mainly localized to the desmoplastic stroma. Therapies modulating the inflammatory tumor microenvironment to promote the attraction of DCs and differentiation of monocytes into functional DCs might improve the survival of PDAC patients.
Subject(s)
Carcinoma, Pancreatic Ductal/immunology , Pancreatic Neoplasms/immunology , Tumor Microenvironment/immunology , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/mortality , Case-Control Studies , Cytokines/immunology , Cytokines/metabolism , Dendritic Cells/immunology , Dendritic Cells/metabolism , Humans , Immunophenotyping , Inflammation Mediators/immunology , Inflammation Mediators/metabolism , Macrophages/immunology , Macrophages/metabolism , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/mortality , Receptors, Cell Surface/metabolism , Stromal Cells , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolismABSTRACT
The tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) is dynamic, with an extensive interaction between the stroma and tumor cells. The aim of this study was to delineate the cross talk between PDAC and cancer-associated fibroblasts (CAFs), with a focus on the mechanism creating the chronic inflammatory tumor milieu. We assessed the effects of the cross talk between PDAC and CAF cell lines on the creation and sustenance of the inflammatory tumor microenvironment in pancreatic cancer. The coculture of PDAC and CAF cell lines enhanced the levels of inflammatory factors including IL-1α, IL-6, CXCL8, VEGF-A, CCL20, and COX-2. CAFs were superior to tumor cells regarding the production of most inflammatory factors, and tumor cell-associated IL-1α was established as the initiator of the enhanced production of inflammatory factors through the binding of IL-1α to IL-1 receptor 1 (IL-1R1) expressed predominantly by CAFs. Furthermore, we found a correlation between IL-1α and CXCL8 expression levels in PDAC tissues and correlation between IL-1α expression and the clinical outcome of the patients. This confirmed an important role for the IL-1 signaling cascade in the creation and sustenance of a tumor favorable microenvironment. Neutralization of the IL-1α signaling efficiently diminished the cross talk-induced production of inflammatory factors. These data suggest that the cross talk between PDAC cells and the main stroma cell type, i.e. CAFs, is one essential factor in the formation of the inflammatory tumor environment, and we propose that neutralization of the IL-1α signaling might be a potential therapy for this cancer.
Subject(s)
Carcinoma, Pancreatic Ductal/metabolism , Fibroblasts/metabolism , Inflammation Mediators/metabolism , Interleukin-1alpha/metabolism , Pancreatic Neoplasms/metabolism , Tumor Microenvironment , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/metabolism , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/mortality , Carcinoma, Pancreatic Ductal/pathology , Cell Communication , Cell Line , Coculture Techniques , Fibroblasts/pathology , Gene Expression Profiling , Humans , Interleukin-1alpha/genetics , Interleukin-1alpha/immunology , Neoplasm Staging , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/mortality , Pancreatic Neoplasms/pathology , Receptors, Interleukin-1/genetics , Receptors, Interleukin-1/immunology , Receptors, Interleukin-1/metabolism , Survival Analysis , Tumor Microenvironment/genetics , Tumor Microenvironment/immunologyABSTRACT
Dendritic cell (DC)-based adoptive tumor immunotherapy approaches have shown promising results, but the incidence of tumor regression is low and there is an evident call for identifying culture conditions that produce DCs with a more potent Th1 potential. Routinely, DCs are differentiated in CO(2) incubators under atmospheric oxygen conditions (21% O(2)), which differ from physiological oxygen levels of only 3-5% in tissue, where most DCs reside. We investigated whether differentiation and maturation of DCs under physiological oxygen levels could produce more potent T-cell stimulatory DCs for use in adoptive immunotherapy. We found that immature DCs differentiated under physiological oxygen levels showed a small but significant reduction in their endocytic capacity. The different oxygen levels did not influence their stimuli-induced upregulation of cluster of differentiation 54 (CD54), CD40, CD83, CD86, C-C chemokine receptor type 7 (CCR7), C-X-C chemokine receptor type 4 (CXCR4) and human leukocyte antigen (HLA)-DR or the secretion of interleukin (IL)-6, tumor necrosis factor (TNF)-α and IL-10 in response to lipopolysaccharide (LPS) or a cytokine cocktail. However, DCs differentiated under physiological oxygen level secreted higher levels of IL-12(p70) after exposure to LPS or CD40 ligand. Immature DCs differentiated at physiological oxygen levels caused increased T-cell proliferation, but no differences were observed for mature DCs with regard to T-cell activation. In conclusion, we show that although DCs generated under atmospheric or physiological oxygen conditions are mostly similar in function and phenotype, DCs differentiated under physiological oxygen secrete larger amounts of IL-12(p70). This result could have implications for the use of ex vivo-generated DCs for clinical studies, since DCs differentiated at physiological oxygen could induce increased Th1 responses in vivo.
Subject(s)
Cell Differentiation/drug effects , Dendritic Cells/drug effects , Oxygen/pharmacology , Antigens, CD/immunology , Antigens, CD/metabolism , B7-1 Antigen/immunology , B7-1 Antigen/metabolism , CD40 Antigens/immunology , CD40 Antigens/metabolism , Cell Differentiation/immunology , Cells, Cultured , Coculture Techniques , Dendritic Cells/immunology , Dendritic Cells/physiology , Endocytosis/immunology , Flow Cytometry , HeLa Cells , Humans , Immunoglobulins/immunology , Immunoglobulins/metabolism , Immunohistochemistry , Immunotherapy, Adoptive/methods , Intercellular Adhesion Molecule-1/immunology , Intercellular Adhesion Molecule-1/metabolism , Interleukin-10/immunology , Interleukin-10/metabolism , Interleukin-12/immunology , Interleukin-12/metabolism , Lipopolysaccharides/pharmacology , Lymphocyte Activation/immunology , Lymphocyte Activation/physiology , Membrane Glycoproteins/immunology , Membrane Glycoproteins/metabolism , Neoplasms/immunology , Neoplasms/therapy , Oxygen/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolism , CD83 AntigenABSTRACT
BACKGROUND: The authors investigated thermal injury depth, inflammation, and scarring in human abdominal skin by comparing the histology of incisions made with a standard "cold" scalpel blade, conventional electrosurgery, and the PEAK PlasmaBlade, a novel, low-thermal-injury electrosurgical instrument. METHODS: Approximately 6 and 3 weeks before abdominoplasty, full-thickness incisions were created in the abdominal pannus skin of 20 women, using a scalpel (scalpel), the PlasmaBlade, and a conventional electrosurgical instrument. Fresh (0-week) incisions were made immediately before surgery. After abdominoplasty, harvested incisions were analyzed for scar width, thermal injury depth, burst strength, and inflammatory response. RESULTS: Acute thermal injury depth was reduced 74 percent in PlasmaBlade incisions compared with conventional electrosurgical instrument (p < 0.001). Significant differences in inflammatory response were observed at 3 weeks, with mean CD3 response (T-lymphocytes) 40 percent (p = 0.01) and 21 percent (p ≈ 0.12) higher for the conventional electrosurgical instrument and PlasmaBlade, respectively, compared with the scalpel. CD68 response (monocytes/macrophages) was 52 percent (p = 0.05) and 16 percent (p ≈ 0.35) greater for a conventional electrosurgical instrument and the PlasmaBlade, respectively. PlasmaBlade incisions demonstrated 65 percent (p < 0.001) and 42 percent (p < 0.001) stronger burst strength than a conventional electrosurgical instrument, with equivalence to the scalpel at the 3- and 6-week time points, respectively. Scar width was equivalent for the PlasmaBlade and the scalpel at both time points, and 25 percent (p = 0.01) and 12 percent (p = 0.15) less than for electrosurgery, respectively. CONCLUSIONS: PlasmaBlade incisions demonstrated reduced thermal injury depth, inflammatory response, and scar width in healing skin compared with electrosurgery. These results suggest that the PlasmaBlade may provide clinically meaningful advantages over conventional electrosurgery during human cutaneous wound healing. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, II.(Figure is included in full-text article.).
Subject(s)
Dermatologic Surgical Procedures , Electrosurgery/instrumentation , Wound Healing , Abdomen , Adult , Female , Humans , Plasma Gases/therapeutic use , Skin/pathology , Surgical InstrumentsABSTRACT
OBJECTIVE: To develop an intraoperative method for margin status evaluation during breast conservation therapy (BCT) using an automated analysis of imprint cytology specimens. STUDY DESIGN: Imprint cytology samples were prospectively taken from 47 patients undergoing either BCT or breast reduction surgery. Touch preparations from BCT patients were taken on cut sections through the tumor to generate positive margin controls. For breast reduction patients, slide imprints were taken at cuts through the center of excised tissue. Analysis results from the presented technique were compared against standard pathologic diagnosis. Slides were stained with cytokeratin and Hoechst, imaged with an automated fluorescent microscope, and analyzed with a fast algorithm to automate discrimination between epithelial cells and noncellular debris. RESULTS: The accuracy of the automated analysis was 95% for identifying invasive cancers compared against final pathologic diagnosis. The overall sensitivity was 87% while specificity was 100% (no false positives). This is comparable to the best reported results from manual examination of intraoperative imprint cytology slides while reducing the need for direct input from a cytopathologist. CONCLUSION: This work demonstrates a proof of concept for developing a highly accurate and automated system for the intraoperative evaluation of margin status to guide surgical decisions and lower positive margin rates.
Subject(s)
Breast Neoplasms/diagnosis , Breast Neoplasms/pathology , Diagnostic Imaging/instrumentation , Diagnostic Imaging/methods , Image Processing, Computer-Assisted/methods , Automation , Female , Humans , Prospective Studies , Reproducibility of ResultsABSTRACT
BACKGROUND: Many peptide-based cancer vaccines have been tested in clinical trials with a limited success, mostly due to difficulties associated with peptide stability and delivery, resulting in inefficient antigen presentation. Therefore, the development of suitable and efficient vaccine carrier systems remains a major challenge. METHODS: To address this issue, we have engineered polylactic-co-glycolic acid (PLGA) nanoparticles incorporating: (i) two MHC class I-restricted clinically-relevant peptides, (ii) a MHC class II-binding peptide, and (iii) a non-classical MHC class I-binding peptide. We formulated the nanoparticles utilizing a double emulsion-solvent evaporation technique and characterized their surface morphology, size, zeta potential and peptide content. We also loaded human and murine dendritic cells (DC) with the peptide-containing nanoparticles and determined their ability to present the encapsulated peptide antigens and to induce tumor-specific cytotoxic T lymphocytes (CTL) in vitro. RESULTS: We confirmed that the nanoparticles are not toxic to either mouse or human dendritic cells, and do not have any effect on the DC maturation. We also demonstrated a significantly enhanced presentation of the encapsulated peptides upon internalization of the nanoparticles by DC, and confirmed that the improved peptide presentation is actually associated with more efficient generation of peptide-specific CTL and T helper cell responses. CONCLUSION: Encapsulating antigens in PLGA nanoparticles offers unique advantages such as higher efficiency of antigen loading, prolonged presentation of the antigens, prevention of peptide degradation, specific targeting of antigens to antigen presenting cells, improved shelf life of the antigens, and easy scale up for pharmaceutical production. Therefore, these findings are highly significant to the development of synthetic vaccines, and the induction of CTL for adoptive immunotherapy.
Subject(s)
Antigen Presentation/immunology , Histocompatibility Antigens Class II/immunology , Histocompatibility Antigens Class I/immunology , Immunotherapy/methods , Nanoparticles/chemistry , Neoplasms/therapy , Peptides/immunology , Animals , Biodegradation, Environmental , Cell Differentiation , Cell Line, Tumor , Dendritic Cells/cytology , Dendritic Cells/metabolism , Endocytosis , Humans , Immobilized Proteins/immunology , Lactic Acid/metabolism , Mice , Nanoparticles/ultrastructure , Neoplasms/immunology , Phenotype , Polyglycolic Acid/metabolism , Polylactic Acid-Polyglycolic Acid Copolymer , T-Lymphocytes, Cytotoxic/immunologyABSTRACT
The chemokine CXCL12, via its receptor CXCR4, promotes increased survival of chronic lymphocytic leukemia (CLL) B cells that express high levels of ζ-chain-associated protein (ZAP-70), a receptor tyrosine kinase associated with aggressive disease. In this study, we investigated the underlying molecular mechanisms governing this effect. Although significant differences in the expression or turnover of CXCR4 were not observed between ZAP-70(+) and ZAP-70(-) cell samples, CXCL12 induced greater intracellular Ca(2+) flux and stronger and more prolonged phosphorylation of extracellular signal-regulated kinase (ERK) and mitogen-activated protein kinase/ERK kinase (MEK) in the ZAP-70(+) CLL cells. The CXCL12-induced phosphorylation of ERK and MEK in ZAP-70(+) CLL cells was blocked by sorafenib, a small molecule inhibitor of RAF. Furthermore, ZAP-70(+) CLL cells were more sensitive than ZAP-70(-) CLL cells to the cytotoxic effects of sorafenib in vitro at concentrations that can readily be achieved in vivo. The data suggest that ZAP-70(+) CLL cells may be more responsive to survival factors, like CXCL12, that are elaborated by the leukemia microenvironment, and this sensitivity could be exploited for the development of new treatments for patients with this disease. Moreover, sorafenib may have clinical activity for patients with CLL, particularly those with ZAP-70(+) CLL.
Subject(s)
Benzenesulfonates/pharmacology , Chemokine CXCL12/pharmacology , Pyridines/pharmacology , Signal Transduction/drug effects , raf Kinases/metabolism , Apoptosis/drug effects , Calcium/metabolism , Cell Survival/drug effects , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Extracellular Signal-Regulated MAP Kinases/metabolism , Flow Cytometry , Humans , Immunoblotting , Intracellular Space/drug effects , Intracellular Space/metabolism , Jurkat Cells , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , Mitogen-Activated Protein Kinases/metabolism , Niacinamide/analogs & derivatives , Phenylurea Compounds , Phosphorylation/drug effects , Protein Kinase Inhibitors/pharmacology , Receptors, CXCR4/metabolism , Sorafenib , Time Factors , Tumor Cells, Cultured , ZAP-70 Protein-Tyrosine Kinase/metabolism , raf Kinases/antagonists & inhibitorsABSTRACT
Accumulating evidence indicates that immune impairment in persistent viral infections could lead to T-cell exhaustion. To evaluate the potential contribution of induction of negative costimulatory molecules to impaired T-cell responses, we primed naïve T cells with mature monocyte-derived dendritic cells (MDDCs) pulsed with HIV-1 in vitro. We used quantitative real-time polymerase chain reaction and flow cytometry, respectively, to compare the gene and surface-protein expression profiles of naïve T cells primed with HIV-pulsed or mock-pulsed DCs. We detected elevated expressions of negative costimulatory molecules, including lymphocyte activation gene-3 (LAG-3), CD160, cytolytic T-lymphocyte antigen-4 (CTLA-4), T-cell immunoglobulin mucin-containing domain-3 (TIM-3), programmed death-1 (PD-1) and TRAIL (tumor necrosis-factor-related apoptosis-inducing ligand) in T cells primed by HIV-pulsed DCs. The PD-1(+) T-cell population also coexpressed TIM-3, LAG-3, and CTLA-4. Interestingly, we also found an increase in gene expression of the transcriptional repressors Blimp-1 (B-lymphocyte-induced maturation protein-1) and Foxp3 (forkhead transcription factor) in T-cells primed by HIV-pulsed DCs; Blimp-1 expression was directly proportional to the expression of the negative costimulatory molecules. Furthermore, levels of the effector cytokines interleukin-2, tumor necrosis factor-α and interferon-γ, and perforin and granzyme B were decreased in T-cell populations primed by HIV-pulsed DCs. In conclusion, in vitro priming of naïve T-cells with HIV-pulsed DC leads to expansion of T cells with coexpression of a broad array of negative costimulatory molecules and Blimp-1, with potential deleterious consequences for T-cell responses.