RESUMEN
Neutrophils are expanded and abundant in cancer-bearing hosts. Under the influence of CXCR1 and CXCR2 chemokine receptor agonists and other chemotactic factors produced by tumors, neutrophils, and granulocytic myeloid-derived suppressor cells (MDSCs) from cancer patients extrude their neutrophil extracellular traps (NETs). In our hands, CXCR1 and CXCR2 agonists proved to be the major mediators of cancer-promoted NETosis. NETs wrap and coat tumor cells and shield them from cytotoxicity, as mediated by CD8+ T cells and natural killer (NK) cells, by obstructing contact between immune cells and the surrounding target cells. Tumor cells protected from cytotoxicity by NETs underlie successful cancer metastases in mice and the immunotherapeutic synergy of protein arginine deiminase 4 (PAD4) inhibitors, which curtail NETosis with immune checkpoint inhibitors. Intravital microscopy provides evidence of neutrophil NETs interfering cytolytic cytotoxic T lymphocytes (CTLs) and NK cell contacts with tumor cells.
Asunto(s)
Trampas Extracelulares/metabolismo , Neoplasias Experimentales/terapia , Receptores de Quimiocina/agonistas , Receptores de Interleucina-8A/agonistas , Receptores de Interleucina-8B/agonistas , Animales , Línea Celular Tumoral , Citotoxicidad Inmunológica/inmunología , Células HT29 , Humanos , Microscopía Intravital/métodos , Células Asesinas Naturales/inmunología , Ligandos , Ratones , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/metabolismo , Receptores de Quimiocina/inmunología , Receptores de Quimiocina/metabolismo , Receptores de Interleucina-8A/inmunología , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/inmunología , Receptores de Interleucina-8B/metabolismo , Linfocitos T Citotóxicos/inmunologíaRESUMEN
In humans, IL-8 (CXCL8) is a key chemokine for chemotaxis of polymorphonuclear leukocytes and monocytes/macrophages when acting on CXCR1 and CXCR2. CXCL8 activity on neutrophils includes chemotaxis and eliciting the extrusion of neutrophil extracellular traps (NETs). In this study, we show that concentrations of IL-8 that induce NETosis surpass in at least one order of magnitude those required to elicit chemoattraction in human neutrophils. IL-8-induced NETosis was less dependent on G-proteins than migration, while extracellular Ca+2 chelation similarly inhibited both processes. Reactive oxygen species (ROS) were more important for NETosis than for chemotaxis as evidenced by neutralization with N-acetyl -cysteine. Interestingly, selective blockade with anti-CXCR1 mAb inhibited NETosis much more readily than chemotaxis, while pharmacological inhibition of both CXCR1 and CXCR2, or selective inhibition for CXCR2 alone, similarly inhibited both functions. Together, these results propose a model according to which low concentrations of IL-8 in a gradient attract neutrophils to the inflammatory foci, while high receptor-saturating concentrations of IL-8 give rise to NETosis once leukocytes reach the core of the inflammatory insult.
Asunto(s)
Quimiotaxis/inmunología , Trampas Extracelulares/inmunología , Interleucina-8/inmunología , Neutrófilos/inmunología , Acetilcisteína/metabolismo , Humanos , Especies Reactivas de Oxígeno/metabolismo , Receptores de Interleucina-8A/antagonistas & inhibidores , Receptores de Interleucina-8A/metabolismo , Receptores de Interleucina-8B/antagonistas & inhibidores , Receptores de Interleucina-8B/metabolismo , Transducción de Señal/inmunologíaRESUMEN
Phenolic compounds might modulate adiposity. Here, we report our observation that polyphenols and phenolic acids inhibit adipogenesis in 3T3-L1 with different intensity depending on the family and the stage of differentiation. While quercetin and resveratrol inhibited lipid accumulation along the whole process of differentiation, apigenin and myricetin were active during the early and latest stages, but not intermediate, contrary to hesperidin. The activity of phenolic acids was limited to the early stages of the differentiation process, except p-coumaric and ellagic acids. This anti-adipogenic effect was accompanied by down-regulation of Scd1 and Lpl. Molecular docking analysis revealed that the inhibitory activity of these phenolic compounds over the early stages of adipogenesis exhibits a significant correlation (r = 0.7034; p = 0.005) with their binding affinity to the ligand-binding domain of PPARγ. Results show that polyphenols and phenolic acids would interact with specific residues of the receptor, which could determine their potential anti-adipogenic activity during the early stages of the differentiation. Residues Phe264, His266, Ile281, Cys285 and Met348 are the most frequently involved in these interactions, which might suggest a crucial role for these amino acids modulating the activity of the receptor. These data contribute to elucidate the possible mechanisms of phenolic compounds in the control of adipogenesis.
Asunto(s)
Adipogénesis/efectos de los fármacos , Metabolismo de los Lípidos/efectos de los fármacos , PPAR gamma/genética , Fenoles/farmacología , Células 3T3-L1 , Adipogénesis/genética , Animales , Apigenina/química , Apigenina/farmacología , Diferenciación Celular/efectos de los fármacos , Flavonoides/química , Flavonoides/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Hesperidina/química , Hesperidina/farmacología , Ratones , Simulación del Acoplamiento Molecular , PPAR gamma/metabolismo , Fenoles/química , Quercetina/química , Quercetina/farmacología , Resveratrol/química , Resveratrol/farmacología , Estearoil-CoA Desaturasa/genéticaRESUMEN
BACKGROUND: Despite the discovery of the p.V617F in JAK2, the molecular pathogenesis of some chronic myeloproliferative neoplasms remains unclear. Although very rare, different studies have identified CBL (Cas-Br-Murine ecotropic retroviral transforming sequence) mutations in V617FJAK2-negative patients, mainly located in the RING finger domain. In order to determine the frequency of CBL mutations in these diseases, we studied different regions of all CBL family genes (CBL, CBLB and CBLC) in a selected group of patients with myeloproliferative neoplasms. We also included V617FJAK2-positive patients to check whether mutations in CBL and JAK2 are mutually exclusive events. DESIGN AND METHODS: Using denaturing high performance liquid chromatography, we screened for mutations in CBL, CBLB and CBLC in a group of 172 V617FJAK2-negative and 232 V617FJAK2-positive patients with myeloproliferative neoplasms not selected for loss of heterozygosity. The effect on cell proliferation of the mutations detected was analyzed on a 32D(FLT3) cell model. RESULTS: An initial screening of all coding exons of CBL, CBLB and CBLC in 44 V617FJAK2-negative samples revealed two new CBL mutations (p.C416W in the RING finger domain and p.A678V in the proline-rich domain). Analyses performed on 128 additional V617FJAK2-negative and 232 V617FJAK2-positive samples detected three CBL changes (p.T402HfsX29, p.P417R and p.S675C in two cases) in four V617FJAK2-positive patients. None of these mutations was found in 200 control samples. Cell proliferation assays showed that all of the mutations promoted hypersensitivity to interleukin-3 in 32D(FLT3) cells. CONCLUSIONS: Although mutations described to date have been found in the RING finger domain and in the linker region of CBL, we found a similar frequency of mutations in the proline-rich domain. In addition, we found CBL mutations in both V617FJAK2-positive (4/232; 1.7%) and negative (2/172; 1.2%) patients and all of them promoted hypersensitivity to interleukin-3.
Asunto(s)
Janus Quinasa 2/genética , Mutación , Trastornos Mieloproliferativos/genética , Proteínas Proto-Oncogénicas c-cbl/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Secuencia Conservada , Exones , Proteínas de Fusión bcr-abl/deficiencia , Proteínas de Fusión bcr-abl/genética , Expresión Génica , Orden Génico , Humanos , Interleucina-3/farmacología , Janus Quinasa 2/metabolismo , Ratones , Datos de Secuencia Molecular , Trastornos Mieloproliferativos/metabolismo , Proteínas Proto-Oncogénicas c-cbl/metabolismoRESUMEN
Rationale: The CEA-CD3 T cell bispecific antibody cibisatamab (CEA-TCB) is currently undergoing clinical trials. Here we study its performance against three-dimensional tumor organoids in cocultures with T cells as compared to a higher affinity CEACAM5-CD3 (CEACAM5-TCB) bispecific antibody using time-lapse confocal microscopy. Methods: Pre-labelled spheroids derived from colon cancer cell lines and primary organoids derived from four colorectal cancer surgical specimens, which expressed different graded levels of CEA, were exposed in cocultures to T lymphocytes. Cocultures were treated with CEA-CD3 T-cell engagers and were followed by live confocal microscopy. Caspase 3 activation detected in real-time was used as an indicator of tumor cell death. Co-cultures were also set up with autologous tumor-associated fibroblasts to test the co-stimulatory effect of a fibroblast activated protein (FAP)- targeted 4-1BBL bispecific antibody fusion protein currently undergoing clinical trials. Results: Tumor-cell killing of 3D colon carcinoma cultures was dependent on the levels of surface CEA expression, in such a way that the lower affinity agent (CEA-TCB) did not mediate killing by human preactivated T cells below a certain CEA expression threshold, while the high affinity construct (CEACAM5-TCB) remained active on the low CEA expressing organoids. Modelling heterogeneity in the levels of CEA expression by coculturing CEA high and low organoids showed measurable but weak bystander killing. Cocultures of tumor organoids, autologous fibroblasts and T cells allowed to observe a costimulatory effect of anti-FAP-4-1BBL both to release IFNγ and to attain more efficacious tumor cell killing. Conclusion: Three-dimensional tumor cocultures with T cells using live confocal microscopy provide suitable models to test the requirements for colon-cancer redirected killing as elicited by CEA-targeted T-cell engagers undergoing clinical trials and treatment allow combinations to be tested in a relevant preclinical system.
Asunto(s)
Anticuerpos Biespecíficos , Antígeno Carcinoembrionario , Neoplasias del Colon , Linfocitos T , Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/farmacología , Complejo CD3/inmunología , Antígeno Carcinoembrionario/inmunología , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/inmunología , Neoplasias del Colon/metabolismo , Humanos , Activación de Linfocitos , Organoides/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
Interleukin-8 (CXCL8) produced in the tumor microenvironment correlates with poor response to checkpoint inhibitors and is known to chemoattract and activate immunosuppressive myeloid leukocytes. In human cancer, IL8 mRNA levels correlate with IL1B and TNF transcripts. Both cytokines induced IL-8 functional expression from a broad variety of human cancer cell lines, primary colon carcinoma organoids, and fresh human tumor explants. Although IL8 is absent from the mouse genome, a similar murine axis in which TNFα and IL-1ß upregulate CXCL1 and CXCL2 in tumor cells was revealed. Furthermore, intratumoral injection of TNFα and IL-1ß induced IL-8 release from human malignant cells xenografted in immunodeficient mice. In all these cases, the clinically used TNFα blockers infliximab and etanercept or the IL-1ß inhibitor anakinra was able to interfere with this pathogenic cytokine loop. Finally, in paired plasma samples of patients with cancer undergoing TNFα blockade with infliximab in a clinical trial, reductions of circulating IL-8 were substantiated. SIGNIFICANCE: IL-8 attracts immunosuppressive protumor myeloid cells to the tumor microenvironment, and IL-8 levels correlate with poor response to checkpoint inhibitors. TNFα and IL-1ß are identified as major inducers of IL-8 expression on malignant cells across cancer types and models in a manner that is druggable with clinically available neutralizing agents. This article is highlighted in the In This Issue feature, p. 2007.
Asunto(s)
Citocinas , Factor de Necrosis Tumoral alfa , Animales , Citocinas/metabolismo , Humanos , Infliximab/farmacología , Infliximab/uso terapéutico , Interleucina-1beta/metabolismo , Interleucina-8/genética , Ratones , Microambiente Tumoral , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Activation-induced cell death (AICD) is a complex immunoregulatory mechanism that causes the demise of a fraction of T-lymphocytes upon antigen-driven activation. In the present study we investigated the direct role of TNF in AICD of CD8 T lymphocytes. Methods: Human peripheral mononuclear cells were isolated from healthy donors and fresh tumor-infiltrating lymphocytes were obtained from cancer patients undergoing surgery. T cells were activated with anti-CD3/CD28 mAbs or with a pool of virus peptides, in combination with clinical-grade TNF blocking agents. Results: A portion of CD8 T cells undergoes apoptosis upon CD3/CD28 activation in a manner that is partially prevented by the clinically used anti-TNF agents infliximab and etanercept. TNF-mediated AICD was also observed upon activation of virus-specific CD8 T cells and tumor-infiltrating CD8 T lymphocytes. The mechanism of TNF-driven T cell death involves TNFR2 and production of mitochondrial oxygen free radicals which damage DNA. Conclusion: The use of TNF blocking agents reduces oxidative stress, hyperpolarization of mitochondria, and the generation of DNA damage in CD8 T celss undergoing activation. The fact that TNF mediates AICD in human tumor-reactive CD8 T cells suggests that the use of TNF-blocking agents can be exploited in immunotherapy strategies.
Asunto(s)
Apoptosis/efectos de los fármacos , Linfocitos T CD8-positivos/citología , Etanercept/farmacología , Infliximab/farmacología , Inhibidores del Factor de Necrosis Tumoral/farmacología , Anciano , Anciano de 80 o más Años , Linfocitos T CD8-positivos/efectos de los fármacos , Femenino , Voluntarios Sanos , Humanos , Inmunoterapia , Leucocitos Mononucleares/citología , Activación de Linfocitos/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/citología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Masculino , Persona de Mediana Edad , Mitocondrias/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Estrés Oxidativo/efectos de los fármacosRESUMEN
Few therapies are currently available for patients with KRAS-driven cancers, highlighting the need to identify new molecular targets that modulate central downstream effector pathways. Here we found that the microRNA (miRNA) cluster including miR181ab1 is a key modulator of KRAS-driven oncogenesis. Ablation of Mir181ab1 in genetically engineered mouse models of Kras-driven lung and pancreatic cancer was deleterious to tumor initiation and progression. Expression of both resident miRNAs in the Mir181ab1 cluster, miR181a1 and miR181b1, was necessary to rescue the Mir181ab1-loss phenotype, underscoring their nonredundant role. In human cancer cells, depletion of miR181ab1 impaired proliferation and 3D growth, whereas overexpression provided a proliferative advantage. Lastly, we unveiled miR181ab1-regulated genes responsible for this phenotype. These studies identified what we believe to be a previously unknown role for miR181ab1 as a potential therapeutic target in 2 highly aggressive and difficult to treat KRAS-mutated cancers.
Asunto(s)
Carcinogénesis/metabolismo , Neoplasias Pulmonares/metabolismo , MicroARNs/metabolismo , Familia de Multigenes , Neoplasias Experimentales/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ARN Neoplásico/metabolismo , Animales , Carcinogénesis/genética , Línea Celular Tumoral , Proliferación Celular , Humanos , Neoplasias Pulmonares/genética , Ratones , Ratones Noqueados , MicroARNs/genética , Neoplasias Experimentales/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , ARN Neoplásico/genéticaRESUMEN
We developed a new targeted cationic nanoparticulate system composed of poly(D,L-lactic-co-glycolic acid) (PLGA), 1,2-dioleoyl-3-(trimethylammonium) propane (DOTAP) and asialofetuin (AF), and found it to be a highly effective formulation for gene delivery to liver tumor cells. The nanoparticles (NP) were prepared by a modified solvent evaporation process that used two protocols in order to encapsulate (NP1 particles) or adsorb (NP2 particles) plasmid DNA. The final particles are in the nanoscale range. pDNA loaded in PLGA/DOTAP/AF particles with high loading efficiency showed a positive surface charge. Targeted asialofetuin-nanoparticles (AF-NP) carrying genes encoding for luciferase and interleukin-12 (IL-12) resulted in increased transfection efficiencies compared to free DNA and to plain (non-targeted) systems, even in the presence of 60% fetal bovine serum (FBS). The results of transfections performed on HeLa cells, defective in asialoglycoprotein receptors (ASGPr-), confirmed the receptor-mediated endocytosis mechanism. In summary, this is the first time that asialoglycoprotein receptor targeting by PLGA/DOTAP/DNA nanoparticles carrying the therapeutic gene IL-12 has been shown to be efficient in gene delivery to liver cancer cells in the presence of a very high concentration of serum, and this could be a potential system for in vivo application.