RESUMO
Genome-wide association studies (GWAS) have identified more than a hundred single nucleotide variants (SNV) associated with the risk of gastroesophageal cancer (GEC). The majority of the identified SNVs map to noncoding regions of the genome. Uncovering the causal SNVs and genes they modulate could help improve GEC prevention and treatment. Herein, we used HiChIP against histone 3 lysine 27 acetylation (H3K27ac) to simultaneously annotate active promoters and enhancers, identify the interactions between them, and detect nucleosome-free regions (NFR) harboring potential causal SNVs in a single assay. The application of H3K27ac HiChIP in GEC relevant models identified 61 potential functional SNVs that reside in NFRs and interact with 49 genes at 17 loci. The approach led to a 67% reduction in the number of SNVs in linkage disequilibrium at these 17 loci, and at 7 loci, a single putative causal SNV was identified. One SNV, rs147518036, located within the promoter of the UDP-glucuronate decarboxylase 1 (UXS1) gene, seemed to underlie the GEC risk association captured by the rs75460256 index SNV. The rs147518036 SNV creates a GABPA DNA recognition motif, resulting in increased promoter activity, and CRISPR-mediated inhibition of the UXS1 promoter reduced the viability of the GEC cells. These findings provide a framework that simplifies the identification of potentially functional regulatory SNVs and target genes underlying risk-associated loci. In addition, the study implicates increased expression of the enzyme UXS1 and activation of its metabolic pathway as a predisposition to gastric cancer, which highlights potential therapeutic avenues to treat this disease. Significance: Epigenomic footprinting using a histone posttranslational modification targeted 3D genomics methodology elucidates functional noncoding sequence variants and their target genes at cancer risk loci.
Assuntos
Epigenômica , Neoplasias Esofágicas , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Estudo de Associação Genômica Ampla/métodos , Epigenômica/métodos , Histonas/genética , Histonas/metabolismo , Linhagem Celular TumoralRESUMO
Lymph nodes (LNs) are frequently the first sites of metastasis. Currently, the only prognostic LN assessment is determining metastatic status. However, there is evidence suggesting that LN metastasis is facilitated by the formation of a pre-metastatic niche induced by tumour derived extracellular vehicles (EVs). Therefore, it is important to detect and modify the LN environmental changes. Earlier work has demonstrated that neutrophil extracellular traps (NETs) can sequester and promote distant metastasis. Here, we first confirmed that LN NETs are associated with reduced patient survival. Next, we demonstrated that NETs deposition precedes LN metastasis and NETs inhibition diminishes LN metastases in animal models. Furthermore, we discovered that EVs are essential to the formation of LN NETs. Finally, we showed that lymphatic endothelial cells secrete CXCL8/2 in response to EVs inducing NETs formation and the promotion of LN metastasis. Our findings reveal the role of EV-induced NETs in LN metastasis and provide potential immunotherapeutic vulnerabilities that may occur early in the metastatic cascade.
Assuntos
Armadilhas Extracelulares , Vesículas Extracelulares , Animais , Metástase Linfática/patologia , Células Endoteliais , Linfonodos/patologiaRESUMO
BACKGROUND: Adenocarcinoma of the proximal stomach is the fastest rising malignancy in North America. It is commonly associated with peritoneal accumulation of malignant ascites (MA), a fluid containing cancer and inflammatory cells and soluble proteins. Peritoneal metastasis (PM) is the most common site of gastric cancer (GC) progression after curative-intent surgery and is the leading cause of death among GC patients. METHODS/RESULTS: Using a panel of gastric adenocarcinoma cell lines (human: MKN 45, SNU-5; murine: NCC-S1M), we demonstrate that prior incubation of GC cells with MA results in a significant (> 1.7-fold) increase in the number of cells capable of adhering to human peritoneal mesothelial cells (HPMC) (p < 0.05). We then corroborate these findings using an ex vivo PM model and show that MA also significantly enhances the ability of GC cells to adhere to strips of human peritoneum (p < 0.05). Using a multiplex ELISA, we identify MIF and VEGF as consistently elevated across MA samples from GC patients (p < 0.05). We demonstrate that agents that block the effects of MIF or VEGF abrogate the ability of MA to stimulate the adhesion of GC cells to adhere to human peritoneum and promote both ex vivo and in vivo metastases. CONCLUSION: Agents targeting MIF or VEGF may be relevant to the treatment or prevention of PM in GC patients.
Assuntos
Adenocarcinoma , Neoplasias Peritoneais , Neoplasias Gástricas , Humanos , Animais , Camundongos , Neoplasias Peritoneais/secundário , Ascite/patologia , Neoplasias Gástricas/patologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Linhagem Celular TumoralRESUMO
Surgical resection, the cornerstone of curative intent treatment for gastric adenocarcinoma, is associated with a high rate of infection-related post-operative complications, leading to an increased incidence of metastasis to the peritoneum. However, the mechanisms underlying this process are poorly understood. Lipopolysaccharide (LPS), an antigen from Gram-negative bacteria, represents a potential mechanism via induction of local and systemic inflammation through activation of Toll-like receptor (TLR). Here, we use both a novel ex vivo model of peritoneal metastasis and in vivo animal models to assess gastric cancer cell adhesion to peritoneum both before and after inhibition of the TLR4 pathway. We demonstrate that activation of TLR4 by either LPS or Gram-negative bacteria (E. coli) significantly increases the adherence of gastric cancer cells to human peritoneal mesothelial cells, and that this increased adherence is abrogated by inhibition of the TLR4 signal cascade and downstream TAK1 and MEK1/2 pathways. We also demonstrate that the influence of LPS on adherence extends to peritoneal tissue and metastatic spread. Furthermore, we show that loss of TLR4 at the site of metastasis reduces tumor cell adhesion, implicating the TLR4 signaling cascade in potentiating metastatic adhesion and peritoneal spread. These results identify potential therapeutic targets for the clinical management of patients undergoing resection for gastric cancer.
Assuntos
Adenocarcinoma , Neoplasias Peritoneais , Neoplasias Gástricas , Animais , Escherichia coli/metabolismo , Humanos , Lipopolissacarídeos/farmacologia , Peritônio , Receptor 4 Toll-Like/metabolismoRESUMO
Neutrophils promote tumor growth and metastasis at multiple stages of cancer progression. One mechanism through which this occurs is via release of neutrophil extracellular traps (NETs). We have previously shown that NETs trap tumor cells in both the liver and the lung, increasing their adhesion and metastasis following postoperative complications. Multiple studies have since shown that NETs play a role in tumor progression and metastasis. NETs are composed of nuclear DNA-derived web-like structures decorated with neutrophil-derived proteins. However, it is unknown which, if any, of these NET-affiliated proteins is responsible for inducing the metastatic phenotype. In this study, we identify the NET-associated carcinoembryonic Ag cell adhesion molecule 1 (CEACAM1) as an essential element for this interaction. Indeed, blocking CEACAM1 on NETs, or knocking it out in a murine model, leads to a significant decrease in colon carcinoma cell adhesion, migration and metastasis. Thus, this work identifies NET-associated CEACAM1 as a putative therapeutic target to prevent the metastatic progression of colon carcinoma.
Assuntos
Antígenos CD/metabolismo , Antígeno Carcinoembrionário/metabolismo , Moléculas de Adesão Celular/metabolismo , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Armadilhas Extracelulares/imunologia , Armadilhas Extracelulares/metabolismo , Neutrófilos/imunologia , Células A549 , Animais , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Células HT29 , Humanos , Camundongos , Neutrófilos/patologiaRESUMO
While emerging data suggest nucleotide oligomerization domain receptor 1 (NOD1), a cytoplasmic pattern recognition receptor, may play an important and complementary role in the immune response to bacterial infection, its role in cancer metastasis is entirely unknown. Hence, we sought to determine the effects of NOD1 on metastasis. NOD1 expression in paired human primary colon cancer, human and murine colon cancer cells were determined using immunohistochemistry and immunoblotting (WB). Clinical significance of NOD1 was assessed using TCGA survival data. A series of in vitro and in vivo functional assays, including adhesion, migration, and metastasis, was conducted to assess the effect of NOD1. C12-iE-DAP, a highly selective NOD1 ligand derived from gram-negative bacteria, was used to activate NOD1. ML130, a specific NOD1 inhibitor, was used to block C12-iE-DAP stimulation. Stable knockdown (KD) of NOD1 in human colon cancer cells (HT29) was constructed with shRNA lentiviral transduction and the functional assays were thus repeated. Lastly, the predominant signaling pathway of NOD1-activation was identified using WB and functional assays in the presence of specific kinase inhibitors. Our data demonstrate that NOD1 is highly expressed in human colorectal cancer (CRC) and human and murine CRC cell lines. Clinically, we demonstrate that this increased NOD1 expression negatively impacts survival in patients with CRC. Subsequently, we identify NOD1 activation by C12-iE-DAP augments CRC cell adhesion, migration and metastasis. These effects are predominantly mediated via the p38 mitogen activated protein kinase (MAPK) pathway. This is the first study implicating NOD1 in cancer metastasis, and thus identifying this receptor as a putative therapeutic target.
Assuntos
Adenocarcinoma/metabolismo , Neoplasias do Colo/metabolismo , Proteína Adaptadora de Sinalização NOD1/fisiologia , Adenocarcinoma/patologia , Animais , Adesão Celular , Linhagem Celular , Movimento Celular , Neoplasias do Colo/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
INTRODUCTION: Surgery is essential for cure of early-stage non-small cell lung cancer (NSCLC). Rates of postoperative bacterial pneumonias, however, remain high, and clinical data suggests that post-operative infectious complications confer an increased risk for metastasis. Toll-like receptors (TLRs) mediate the inflammatory response to infection by recognizing evolutionarily conserved bacterial structures at the surface of numerous pulmonary cell types; yet, little is known about how host TLR activation influences NSCLC metastasis. TLR4 recognizes gram-negative bacterium lipopolysaccharide activating the innate immune system. METHODS: C57BL/6 and TLR4 knockout murine airways were inoculated with Escherichia coli or lipopolysaccharide. Hepatic metastasis assays and intravital microscopy were performed. Bronchoepithelial conditioned media was generated through coincubation of bronchoepithelial cells with TLR4 activating Escherichia coli or lipopolysaccharide. Subsequently, H59 NSCLC were stimulated with conditioned media and subject to various adhesion assays. RESULTS: We demonstrate that gram-negative Escherichia coli pneumonia augments the formation of murine H59 NSCLC liver metastases in C57BL/6 mice through TLR4 activation. Additionally, infected C57BL/6 mice demonstrate increased H59 NSCLC in vivo hepatic sinusoidal adhesion compared with negative controls, a response that is significantly diminished in TLR4 knockout mice. Similarly, intratracheal injection of purified TLR4 activating lipopolysaccharide increases in vivo adhesion of H59 cells to murine hepatic sinusoids. Furthermore, H59 cells incubated with bronchoepithelial conditioned medium show increased cell adhesion to in vitro extracellular matrix proteins and in vivo hepatic sinusoids through a mechanism dependent on bronchoepithelial TLR4 activation and interleukin-6 secretion. CONCLUSION: TLR4 is a viable therapeutic target for NSCLC metastasis augmented by gram-negative pneumonia.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/microbiologia , Infecções por Escherichia coli/patologia , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/microbiologia , Pneumonia Bacteriana/patologia , Receptor 4 Toll-Like/metabolismo , Animais , Brônquios/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular , Células Epiteliais/patologia , Escherichia coli/isolamento & purificação , Infecções por Escherichia coli/metabolismo , Infecções por Escherichia coli/microbiologia , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/microbiologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Pneumonia Bacteriana/metabolismo , Pneumonia Bacteriana/microbiologiaRESUMO
Targeting the dynamic tumor immune microenvironment (TIME) can provide effective therapeutic strategies for cancer. Neutrophils are the predominant leukocyte population in mice and humans, and mounting evidence implicates these cells during tumor growth and metastasis. Neutrophil extracellular traps (NETs) are networks of extracellular neutrophil DNA fibers that are capable of binding tumor cells to support metastatic progression. Here we demonstrate for the first time that circulating NET levels are elevated in advanced esophageal, gastric and lung cancer patients compared to healthy controls. Using pre-clinical murine models of lung and colon cancer in combination with intravital video microscopy, we show that NETs functionally regulate disease progression and that blocking NETosis through multiple strategies significantly inhibits spontaneous metastasis to the lung and liver. Further, we visualize how inhibiting tumor-induced NETs decreases cancer cell adhesion to liver sinusoids following intrasplenic injection - a mechanism previously thought to be driven primarily by exogenous stimuli. Thus, in addition to neutrophil abundance, the functional contribution of NETosis within the TIME has critical translational relevance and represents a promising target to impede metastatic dissemination.
Assuntos
Apoptose/imunologia , Armadilhas Extracelulares/metabolismo , Metástase Neoplásica/imunologia , Neoplasias/patologia , Neutrófilos/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Adesão Celular/imunologia , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Progressão da Doença , Armadilhas Extracelulares/efeitos dos fármacos , Feminino , Humanos , Microscopia Intravital , Masculino , Camundongos , Pessoa de Meia-Idade , Metástase Neoplásica/prevenção & controle , Neoplasias/sangue , Neoplasias/tratamento farmacológico , Neoplasias/imunologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Cultura Primária de Células , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/imunologia , Adulto JovemRESUMO
Surgical resection of early stage nonsmall cell lung cancer (NSCLC) is necessary for cure. However, rates of postoperative bacterial pneumonias remain high and may confer an increased risk for metastasis. Toll-like receptors (TLRs) mediate the inflammatory cascade by recognizing microbial products at the surface of numerous cell types in the lung; however, little is known about how host TLRs influence NSCLC metastasis. TLR2 recognizes gram-positive bacterial cell wall components activating innate immunity. We demonstrate that lower respiratory tract infection with Streptococcus pneumonia augments the formation of murine H59 NSCLC liver metastases in C57BL/6 mice through host TLR2 activation. Infected mice demonstrate increased H59 and human A549 NSCLC adhesion to hepatic sinusoids in vivo compared with noninfected controls, a response that is significantly diminished in TLR2 knock-out mice. Intra-tracheal injection of purified TLR2 ligand lipoteichoic acid into mice similarly augments in vivo adhesion of H59 cells to hepatic sinusoids. Additionally, H59 and A549 NSCLC cells incubated with bronchoepithelial conditioned media show increased cell adhesion to extracellular matrix components in vitro and hepatic sinusoids in vivo in a manner that is dependent on bronchoepithelial TLR2 activation and interleukin-6 secretion. TLR2 is therefore a potential therapeutic target for gram-positive pneumonia-driven NSCLC metastasis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/patologia , Pneumonia/complicações , Streptococcus pneumoniae/patogenicidade , Receptor 2 Toll-Like/metabolismo , Animais , Apoptose , Carcinoma Pulmonar de Células não Pequenas/etiologia , Adesão Celular , Proliferação de Células , Humanos , Lipopolissacarídeos/farmacologia , Neoplasias Hepáticas/etiologia , Neoplasias Pulmonares/etiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pneumonia/microbiologia , Receptor 2 Toll-Like/genética , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Despite advances in cancer treatment, metastasis remains today the main cause of cancer death. Local control through complete surgical resection of the primary tumor continues to be a key principle in cancer treatment. However, surgical interventions themselves lead to adverse oncologic outcomes and are associated with significantly increased rates of metastasis. Neutrophils through release of neutrophil extracellular traps (NETs) in response to infections were shown to be able to capture circulating cancer cells, and in doing so, support the development of metastatic disease. To be able to intervene on this process, understanding the exact molecular nature of these mechanisms is crucial. We therefore hypothesize and demonstrate that ß1-integrin is an important factor mediating the interactions between circulating tumor cells and NETs. We show that ß1-integrin expression on both cancer cells and NETs is important for the adhesion of circulating tumor cells to NETs both in vitro and in vivo. Using a murine model of intra-abdominal sepsis to mimic the postoperative inflammatory environment, we show that ß1-integrin expression is upregulated in the context of inflammation in vivo. Ultimately, we show that this increased early cancer cell adhesion to NETs in vivo and this effect is abrogated when mice are administered DNAse 1. Our data therefore sheds light on the first molecular mechanism by which NETs can trap circulating tumor cells (CTCs), broadening our understanding of this process.
Assuntos
Armadilhas Extracelulares/metabolismo , Inflamação/patologia , Integrina beta1/metabolismo , Neoplasias Pulmonares/patologia , Células Neoplásicas Circulantes/patologia , Neutrófilos/patologia , Animais , Western Blotting , Adesão Celular , Proliferação de Células , Modelos Animais de Doenças , Citometria de Fluxo , Imunofluorescência , Humanos , Inflamação/metabolismo , Integrina beta1/química , Integrina beta1/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Neoplásicas Circulantes/metabolismo , Infiltração de Neutrófilos , RNA Interferente Pequeno/genética , Células Tumorais CultivadasRESUMO
Neutrophil Extracellular Traps (NETs) have been recently identified as part of the neutrophil's antimicrobial armamentarium. Apart from their role in fighting infections, recent research has demonstrated that they may be involved in many other disease processes, including cancer progression. Isolating purified NETs is a crucial element to allow the study of these functions. In this video, we demonstrate a simplified method of cell free NET isolation from human whole blood using readily available reagents. Isolated NETs can then be used for immunofluorescence staining, blotting or various functional assays. This enables an assessment of their biologic properties in the absence of the potential confounding effects of neutrophils themselves. A density gradient separation technique is employed to isolate neutrophils from healthy donor whole blood. Isolated neutrophils are then stimulated by phorbol 12-myristate 13-acetate (PMA) to induce NETosis. Activated neutrophils are then discarded, and a cell-free NET stock is obtained. We then demonstrate how isolated NETs can be used in an adhesion assay with A549 human lung cancer cells. The NET stock is used to coat the wells of a 96 well cell culture plate O/N, and after ensuring an adequate NET monolayer formation on the bottom of the wells, CFSE labeled A549 cells are added. Adherent cells are quantified using a Nikon TE300 fluorescent microscope. In some wells, 1000U DNAse1 is added 10 min before counting to degrade NETs.
Assuntos
Armadilhas Extracelulares , Neutrófilos/citologia , Humanos , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Surgery is required for the curative treatment of lung cancer but is associated with high rates of postoperative pneumonias predominantly caused by gram negative bacteria. Recent evidence suggests that these severe infectious complications may decrease long term survival after hospital discharge via cancer recurrence, but the mechanism is unclear. Lung cancer cells have recently been demonstrated to express Toll-like receptors (TLR) that mediate pathogen recognition. We hypothesized that incubation of non-small cell lung cancer (NSCLC) cells with heat-inactivated Escherichia coli can augment cancer cell adhesion, migration and metastasis via TLR4 signaling. Incubation of murine and human NSCLC cells with E. coli increased in vitro cell adhesion to collagen I, collagen IV and fibronectin, and enhanced in vitro migration. Using hepatic intravital microscopy, we demonstrated that NSCLC cells have increased in vivo adhesion to hepatic sinusoids after coincubation with gram negative bacteria. These enhanced cell adhesion and migration phenotypes following incubation with E. coli were attenuated at three levels: inhibition of TLR4 (Eritoran), p38 MAPK (BIRB0796) and ERK1/2 phosphorylation (PD184352). Incubation of murine NSCLC cells in vitro with E. coli prior to intrasplenic injection significantly augmented formation of in vivo hepatic metastases 2 weeks later. This increase was abrogated by NSCLC TLR4 blockade using Eritoran. TLR4 represents a potential therapeutic target to help prevent severe postoperative infection driven cancer metastasis.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/secundário , Escherichia coli/patogenicidade , Neoplasias Pulmonares/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptor 4 Toll-Like/fisiologia , Animais , Adesão Celular , Linhagem Celular Tumoral , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , FosforilaçãoRESUMO
The majority of patients with cancer undergo at least one surgical procedure as part of their treatment. Severe postsurgical infection is associated with adverse oncologic outcomes; however, the mechanisms underlying this phenomenon are unclear. Emerging evidence suggests that neutrophils, which function as the first line of defense during infections, facilitate cancer progression. Neutrophil extracellular traps (NETs) are extracellular neutrophil-derived DNA webs released in response to inflammatory cues that trap and kill invading pathogens. The role of NETs in cancer progression is entirely unknown. We report that circulating tumor cells become trapped within NETs in vitro under static and dynamic conditions. In a murine model of infection using cecal ligation and puncture, we demonstrated microvascular NET deposition and consequent trapping of circulating lung carcinoma cells within DNA webs. NET trapping was associated with increased formation of hepatic micrometastases at 48 hours and gross metastatic disease burden at 2 weeks following tumor cell injection. These effects were abrogated by NET inhibition with DNAse or a neutrophil elastase inhibitor. These findings implicate NETs in the process of cancer metastasis in the context of systemic infection and identify NETs as potential therapeutic targets.
RESUMO
BACKGROUND: Esophageal cancer is an aggressive malignancy, and emerging data suggest that postoperative infections may promote cancer progression. Systemic exposure to lipopolysaccharide (LPS), a Gram-negative bacterial antigen involved in such infections, has been shown to increase cancer cell adhesion to the hepatic sinusoids in vivo. We investigated the direct impact of LPS on the migratory ability of esophageal cancer cells via the LPS receptor toll-like receptor 4 (TLR4). METHODS: Human esophageal squamous carcinoma cell lines and immortalized normal esophageal mucosa cells were tested for TLR4 surface expression by reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. TLR4 signaling in response to LPS stimulation was tested in these cells by measuring p38 MAP kinase phosphorylation on Western blot. The impact of TLR4 signaling was measured by static adhesion assays in vitro and on early in vivo migration by intravital microscopy of the liver. RESULTS: Upon LPS stimulation, phosphorylation of p38 was detected in the human esophageal cancer cells HKESC-2. Also, LPS-stimulated HKESC-2 cells showed a twofold increased adhesion to fibronectin and to hepatic sinusoidal endothelium. These effects were abolished by TLR4 inhibition using the small-molecule inhibitor eritoran. Adhesion to fibronectin and hepatic sinusoidal endothelium was also diminished by blockade of p38 phosphorylation and inhibitors of selectin-selectin ligand binding. CONCLUSION: LPS can increase the migratory ability of human esophageal cancer cells by increasing their adhesive properties through TLR4 signaling and selectin ligands. TLR4, p38, and selectin blockade may therefore prove to be a new therapeutic strategy for this aggressive malignancy.
Assuntos
Neoplasias Esofágicas/patologia , Lipopolissacarídeos/farmacologia , Selectinas/fisiologia , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/fisiologia , Animais , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Fibronectinas/metabolismo , Humanos , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptor 4 Toll-Like/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
Although circulating neutrophils are associated with distant metastasis and poor outcome in a number of epithelial malignancies, it remains unclear whether neutrophils play an active causal role in the metastatic cascade. Using in vivo models of metastasis, we found that neutrophils promote cancer cell adhesion within liver sinusoids and, thereby, influence metastasis. Neutrophil depletion before cancer cell inoculation resulted in a decreased number of gross metastases in an intrasplenic model of liver metastasis. This effect was reversed when inflamed neutrophils were co-inoculated with cancer cells. In addition, early adhesion within liver sinusoids was inhibited in the absence of neutrophils and partially restored with a short perfusion of isolated activated neutrophils. Intravital microscopy showed that cancer cells adhered directly on top of arrested neutrophils, indicating that neutrophils may act as a bridge to facilitate interactions between cancer cells and the liver parenchyma. The adhesion of lipopolysaccharide-activated neutrophils to cancer cells was mediated by neutrophil Mac-1/ICAM-1. Our findings, therefore, show a novel role for neutrophils in the early adhesive steps of liver metastasis.
Assuntos
Neoplasias Hepáticas Experimentais/secundário , Antígeno de Macrófago 1/metabolismo , Células Neoplásicas Circulantes/patologia , Neutrófilos/patologia , Animais , Carcinoma Pulmonar de Lewis/sangue , Carcinoma Pulmonar de Lewis/imunologia , Carcinoma Pulmonar de Lewis/metabolismo , Carcinoma Pulmonar de Lewis/patologia , Agregação Celular/fisiologia , Comunicação Celular/fisiologia , Linhagem Celular Tumoral , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Lipopolissacarídeos/farmacologia , Neoplasias Hepáticas Experimentais/sangue , Neoplasias Hepáticas Experimentais/imunologia , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Confocal , Ativação de Neutrófilo/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologiaRESUMO
Infectious complications resulting from resection of colorectal cancer (CRC) elevates the risk of cancer recurrence and metastasis, but the reason for this risk relationship is unknown. Defining the mechanisms responsible may offer opportunities to improve outcomes in a majority of patients whose tumors are resected as part of their therapy. The complex formed between Toll receptor TLR4 and myeloid differentiation factor MD2 defines a major cell surface receptor for lipopolysaccharide (LPS), a gram-negative bacterial antigen that has been implicated in infectious complications after CRC resection. As the TLR4/MD2 complex is expressed on CRC cells, we hypothesized that LPS may promote liver metastasis in CRC by stimulating TLR4 signaling. In support of this hypothesis, we report here that LPS enhances liver metastasis of human CRC cells that express TLR4/MD2 after intrasplenic graft of immunocompromised nude mice. Compared with TLR4 nonexpressing, nonmetastatic CRC cells, we observed increased in vitro adherence to different extracellular matrices and human umbilical vein endothelial cells (HUVEC). Furthermore, we observed an increased likelihood of in vivo capture within hepatic sinusoids after LPS treatment. No differences were apparent in phosphorylation of p38 and MAPK isoforms, but in metastatic CRC cells expressing surface TLR4 treatment with LPS increased Ser473 phosphorylation of AKT kinase. We showed that enhanced adherence elicited by LPS in these cells could be blocked at three different levels, using Eritoran (TLR4 small molecule antagonist), PI-103 (PI3K inhibitor), or anti-ß1 integrin blocking antibodies. Taken together, the results indicate that stimulation of the TLR4/MD2 complex by LPS activates PI3K/AKT signaling and promotes downstream ß1 integrin function, thereby increasing the adhesiveness and metastatic capacity of CRC cells. Our findings suggest that inhibiting LPS-induced TLR4 signaling could improve therapeutic outcomes by preventing cancer metastasis during the perioperative period of CRC resection.
Assuntos
Antígenos de Bactérias/farmacologia , Neoplasias Colorretais/patologia , Lipopolissacarídeos/farmacologia , Neoplasias Hepáticas/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Western Blotting , Adesão Celular/efeitos dos fármacos , Separação Celular , Neoplasias Colorretais/metabolismo , Citometria de Fluxo , Células HT29 , Humanos , Integrina beta1/metabolismo , Neoplasias Hepáticas/secundário , Antígeno 96 de Linfócito/metabolismo , Camundongos , Camundongos Nus , Microscopia de Fluorescência , Metástase Neoplásica/patologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Increased soluble L-selectin levels have been shown to attenuate local inflammation-mediated microvascular leakage, and failure to generate high levels has been associated with increased risk of acute respiratory distress syndrome in septic patients. We hypothesized that failure to shed L-selectin in systemic inflammation would result in increased local inflammation-induced leukocyte adherence and microvascular leakage. METHODS: Using intraperitoneal lipopolysaccharide (LPS) or control bicarbonate buffered saline (BBS) and intrascrotal TNFalpha or BBS, mice were randomized to systemic inflammation (LPSip + BBSis), local inflammation (BBSip + TNFis), both (LPSip + TNFis), or control (BBSip+BBSis). Furthermore, mice received intraperitoneal L-selectin Sheddase inhibitor (Ro31-9790) or control vector. With intravital microscopy on cremaster muscle, we measured leukocyte-endothelial cell interactions and microvascular leakage (permeability index). Surface L-selectin was measured by flow cytometry (MCF). RESULTS: Without Ro31-9790, systemic inflammation attenuated increases induced by local inflammation in leukocyte adherence and vascular leakage. Ro31-9790 significantly increased adherence and leakage in systemic and systemic + local inflammation. L-selectin was shed progressively by increasing degrees of inflammation. Ro31-9790 limited this shedding of L-selectin. CONCLUSION: In systemic inflammation, L-selectin shedding is required to limit local inflammation-mediated leukocyte adherence and microvascular leakage. Failure to shed L-selectin may increase leukocyte-mediated end-organ injury in septic patients.
Assuntos
Permeabilidade Capilar/imunologia , Selectina L/fisiologia , Neutrófilos/fisiologia , Sepse/fisiopatologia , Animais , Adesão Celular , Células Endoteliais/fisiologia , Fluoresceína-5-Isotiocianato , Ácidos Hidroxâmicos/farmacologia , Inflamação/tratamento farmacológico , Selectina L/efeitos dos fármacos , Selectina L/metabolismo , Lipopolissacarídeos , Masculino , Camundongos , Distribuição Aleatória , Escroto/irrigação sanguínea , Sepse/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Given that leukocyte delivery to remote sites is diminished in states of systemic inflammation, such as sepsis, and activated leukocytes may be responsible for endothelial injury leading to vascular leakage, we hypothesized that intra-abdominal sepsis would diminish microvascular leakage at remote sites by altering leukocyte-endothelial interactions. METHODS: Using a murine intravital microscopy model, we examined leukocyte-endothelial interactions and vascular leakage at a peripheral site in the presence of local and/or systemic inflammation. Forty mice were randomized to 1 of 4 study groups: local infection (orchitis), systemic infection (intra-abdominal sepsis by cecal ligation and puncture), local and systemic infection, and control. Postcapillary venules of the cremaster muscle were examined by bright light and fluorescence intravital microscopy. Microvascular leakage was determined after intravenous administration of fluorescent albumin. RESULTS: Systemic infection attenuated the increases in both leukocyte adherence and local infection-induced microvascular permeability. Neutrophil cell-surface expression of L-selectin, as determined by flow cytometry, diminished with both local and systemic infection, whereas expression of CD11b increased with systemic, but not local, infection. CONCLUSIONS: These data suggest that systemic (intra-abdominal) sepsis diminishes local inflammation-mediated vascular leakage by attenuating leukocyte adherence.
Assuntos
Abdome/irrigação sanguínea , Neutrófilos/imunologia , Orquite/imunologia , Sepse/imunologia , Sepse/fisiopatologia , Animais , Permeabilidade Capilar/imunologia , Adesão Celular/imunologia , Endotélio Vascular/citologia , Endotélio Vascular/imunologia , Migração e Rolagem de Leucócitos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos , Microcirculação/fisiologia , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/imunologia , Orquite/fisiopatologiaRESUMO
BACKGROUND: Hypertonic saline (HTS) attenuates polymorphonuclear neutrophil (PMN)-mediated tissue injury after hemorrhagic shock. We hypothesized that HTS resuscitation reduces early in vivo endothelial cell (EC)-PMN interactions and late lung PMN sequestration in a two-hit model of hemorrhagic shock followed by mimicked infection. METHODS: Thirty-two mice were hemorrhaged (40 mm Hg) for 60 minutes and then given intratracheal lipopolysaccharide (10 microg) 1 hour after resuscitation with shed blood and either HTS (4 mL/kg 7.5% NaCl) or Ringer's lactate (RL) (twice shed blood volume). Eleven controls were not manipulated. Cremaster intravital microscopy quantified 5-hour EC-PMN adherence, myeloperoxidase assay assessed lung PMN content (2 1/2 and 24 hours), and lung histology determined 24-hour PMN transmigration. RESULTS: Compared with RL, HTS animals displayed 55% less 5-hour EC-PMN adherence (p = 0.01), 61% lower 24-hour lung myeloperoxidase ( p= 0.007), and 57% lower mean 24-hour lung histologic score ( p= 0.027). CONCLUSION: Compared with RL, HTS resuscitation attenuates early EC-PMN adhesion and late lung PMN accumulation in hemorrhagic shock followed by inflammation. HTS resuscitation may attenuate PMN-mediated organ damage.
Assuntos
Modelos Animais de Doenças , Endotélio Vascular/imunologia , Hidratação/métodos , Infiltração de Neutrófilos/imunologia , Neutrófilos/imunologia , Síndrome do Desconforto Respiratório/etiologia , Ressuscitação/métodos , Solução Salina Hipertônica/uso terapêutico , Sepse/complicações , Sepse/tratamento farmacológico , Choque Hemorrágico/complicações , Choque Hemorrágico/tratamento farmacológico , Animais , Avaliação Pré-Clínica de Medicamentos , Endotélio Vascular/efeitos dos fármacos , Escherichia coli , Inflamação , Soluções Isotônicas/farmacologia , Soluções Isotônicas/uso terapêutico , Lipopolissacarídeos/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos , Neutrófilos/efeitos dos fármacos , Peroxidase/análise , Distribuição Aleatória , Síndrome do Desconforto Respiratório/enzimologia , Síndrome do Desconforto Respiratório/patologia , Síndrome do Desconforto Respiratório/prevenção & controle , Lactato de Ringer , Solução Salina Hipertônica/farmacologia , Sepse/imunologia , Choque Hemorrágico/imunologia , Método Simples-Cego , Fatores de TempoRESUMO
OBJECTIVE: To evaluate the in vivo effects of hypertonic saline (HTS) resuscitation on the interactions of endothelial cells (ECs) and polymorphonuclear neutrophils (PMNs) and vascular permeability after hemorrhagic shock. SUMMARY BACKGROUND DATA: The PMN has been implicated in the pathogenesis of EC damage and organ injury following hemorrhagic shock. Compared to Ringer's lactate (RL), HTS resuscitation diminishes PMN and EC adhesion molecule expression and organ sequestration of PMNs. METHODS: In a murine model of hemorrhagic shock (50 mmHg for 45 minutes followed by resuscitation) using intravital microscopy on cremaster muscle, the authors studied PMN-EC interactions and vascular leakage (epifluorescence after 50 mg/kg fluorescent albumin) in three resuscitation groups: HTS (shed blood + 4 cc/kg 7.5% HTS, n = 12), RL (shed blood + RL [2x shed blood volume], n = 12), and sham (no hemorrhage or resuscitation, n = 9). EC ICAM-1 expression was evaluated by immunohistochemistry. Data, presented as mean +/- SEM, were evaluated by analysis of variance with Bonferroni correction. RESULTS: There were no differences between groups in flow mechanics. Compared to RL, HTS animals (t = 90 minutes) displayed diminished PMN rolling and PMN adhesion to EC at time intervals beyond t = 0. There were no differences between the sham and HTS groups. Vascular leakage was 45% lower in HTS than in RL-resuscitated animals. Cremaster EC ICAM-1 expression was similar in the two groups. CONCLUSIONS: Using HTS instead of RL to resuscitate hemorrhagic shock diminishes vascular permeability in vivo by altering PMN-EC interactions. HTS could serve as a novel means of immunomodulation in hemorrhagic shock victims, potentially reducing PMN-mediated tissue injury.