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A subset of individuals exposed to Mycobacterium tuberculosis (Mtb) that we refer to as 'resisters' (RSTR) show evidence of IFN-γ- T cell responses to Mtb-specific antigens despite serially negative results on clinical testing. Here we found that Mtb-specific T cells in RSTR were clonally expanded, confirming the priming of adaptive immune responses following Mtb exposure. RSTR CD4+ T cells showed enrichment of TH17 and regulatory T cell-like functional programs compared to Mtb-specific T cells from individuals with latent Mtb infection. Using public datasets, we showed that these TH17 cell-like functional programs were associated with lack of progression to active tuberculosis among South African adolescents with latent Mtb infection and with bacterial control in nonhuman primates. Our findings suggested that RSTR may successfully control Mtb following exposure and immune priming and established a set of T cell biomarkers to facilitate further study of this clinical phenotype.
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Linfocitos T CD4-Positivos , Mycobacterium tuberculosis , Tuberculosis , Mycobacterium tuberculosis/inmunología , Humanos , Animales , Adolescente , Tuberculosis/inmunología , Tuberculosis/microbiología , Linfocitos T CD4-Positivos/inmunología , Células Th17/inmunología , Femenino , Macaca mulatta , Masculino , Fenotipo , Interferón gamma/metabolismo , Interferón gamma/inmunología , Antígenos Bacterianos/inmunología , Tuberculosis Latente/inmunología , Tuberculosis Latente/microbiología , Sudáfrica , Adulto Joven , Linfocitos T Reguladores/inmunología , AdultoRESUMEN
In vitro cancer cultures, including three-dimensional organoids, typically contain exclusively neoplastic epithelium but require artificial reconstitution to recapitulate the tumor microenvironment (TME). The co-culture of primary tumor epithelia with endogenous, syngeneic tumor-infiltrating lymphocytes (TILs) as a cohesive unit has been particularly elusive. Here, an air-liquid interface (ALI) method propagated patient-derived organoids (PDOs) from >100 human biopsies or mouse tumors in syngeneic immunocompetent hosts as tumor epithelia with native embedded immune cells (T, B, NK, macrophages). Robust droplet-based, single-cell simultaneous determination of gene expression and immune repertoire indicated that PDO TILs accurately preserved the original tumor T cell receptor (TCR) spectrum. Crucially, human and murine PDOs successfully modeled immune checkpoint blockade (ICB) with anti-PD-1- and/or anti-PD-L1 expanding and activating tumor antigen-specific TILs and eliciting tumor cytotoxicity. Organoid-based propagation of primary tumor epithelium en bloc with endogenous immune stroma should enable immuno-oncology investigations within the TME and facilitate personalized immunotherapy testing.
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Modelos Inmunológicos , Neoplasias Experimentales/inmunología , Organoides/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Microambiente Tumoral/inmunología , Animales , Antígeno B7-H1/inmunología , Técnicas de Cocultivo , Femenino , Humanos , Inmunoterapia , Masculino , Ratones , Ratones Endogámicos BALB C , Proteínas de Neoplasias/inmunología , Neoplasias Experimentales/patología , Neoplasias Experimentales/terapia , Organoides/patologíaRESUMEN
Metastases are the main cause of cancer deaths, but the mechanisms underlying metastatic progression remain poorly understood. We isolated pure populations of cancer cells from primary tumors and metastases from a genetically engineered mouse model of human small cell lung cancer (SCLC) to investigate the mechanisms that drive the metastatic spread of this lethal cancer. Genome-wide characterization of chromatin accessibility revealed the opening of large numbers of distal regulatory elements across the genome during metastatic progression. These changes correlate with copy number amplification of the Nfib locus, and differentially accessible sites were highly enriched for Nfib transcription factor binding sites. Nfib is necessary and sufficient to increase chromatin accessibility at a large subset of the intergenic regions. Nfib promotes pro-metastatic neuronal gene expression programs and drives the metastatic ability of SCLC cells. The identification of widespread chromatin changes during SCLC progression reveals an unexpected global reprogramming during metastatic progression.
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Neoplasias Pulmonares/patología , Factores de Transcripción NFI/metabolismo , Metástasis de la Neoplasia/patología , Carcinoma Pulmonar de Células Pequeñas/patología , Secuencias de Aminoácidos , Animales , Línea Celular Tumoral , Células Cultivadas , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Ratones , Factores de Transcripción NFI/genética , Regiones Promotoras Genéticas , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Regulación hacia ArribaRESUMEN
To identify disease-relevant T cell receptors (TCRs) with shared antigen specificity, we analyzed 778,938 TCRß chain sequences from 178 non-small cell lung cancer patients using the GLIPH2 (grouping of lymphocyte interactions with paratope hotspots 2) algorithm. We identified over 66,000 shared specificity groups, of which 435 were clonally expanded and enriched in tumors compared to adjacent lung. The antigenic epitopes of one such tumor-enriched specificity group were identified using a yeast peptide-HLA A∗02:01 display library. These included a peptide from the epithelial protein TMEM161A, which is overexpressed in tumors and cross-reactive epitopes from Epstein-Barr virus and E. coli. Our findings suggest that this cross-reactivity may underlie the presence of virus-specific T cells in tumor infiltrates and that pathogen cross-reactivity may be a feature of multiple cancers. The approach and analytical pipelines generated in this work, as well as the specificity groups defined here, present a resource for understanding the T cell response in cancer.
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Carcinoma de Pulmón de Células no Pequeñas/inmunología , Mapeo Epitopo/métodos , Epítopos de Linfocito T/genética , Neoplasias Pulmonares/inmunología , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Linfocitos T/inmunología , Algoritmos , Presentación de Antígeno , Antígenos de Neoplasias/metabolismo , Células Cultivadas , Reacciones Cruzadas , Epítopos de Linfocito T/metabolismo , Antígeno HLA-A2/metabolismo , Humanos , Unión Proteica , Especificidad del Receptor de Antígeno de Linfocitos TRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer.
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Adenocarcinoma/fisiopatología , Carcinoma Ductal Pancreático/fisiopatología , Modelos Animales de Enfermedad , Adenocarcinoma/genética , Animales , Carcinoma Ductal Pancreático/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Regulación Neoplásica de la Expresión Génica , Vectores Genéticos/genética , Genoma/genética , Humanos , Lentivirus/genética , Ratones , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
Phenotype-based small-molecule screening is a powerful method to identify molecules that regulate cellular functions. However, such screens are generally performed in vitro under conditions that do not necessarily model complex physiological conditions or disease states. Here, we use molecular cell barcoding to enable direct in vivo phenotypic screening of small-molecule libraries. The multiplexed nature of this approach allows rapid in vivo analysis of hundreds to thousands of compounds. Using this platform, we screened >700 covalent inhibitors directed toward hydrolases for their effect on pancreatic cancer metastatic seeding. We identified multiple hits and confirmed the relevant target of one compound as the lipase ABHD6. Pharmacological and genetic studies confirmed the role of this enzyme as a regulator of metastatic fitness. Our results highlight the applicability of this multiplexed screening platform for investigating complex processes in vivo.
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Evaluación Preclínica de Medicamentos/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Imagen Molecular/métodos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundario , Ratones , Ratones SCID , Monoacilglicerol Lipasas/antagonistas & inhibidores , Monoacilglicerol Lipasas/genética , Trasplante de Neoplasias , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologíaRESUMEN
The activation of innate and adaptive immunity is always balanced by inhibitory signalling mechanisms to maintain tissue integrity. We have identified the E3 ligase c-Cbl--known for its roles in regulating lymphocyte signalling--as a modulator of dendritic cell activation. In c-Cbl-deficient dendritic cells, Toll-like receptor-induced expression of proinflammatory factors, such as interleukin-12, is increased, correlating with a greater potency of dendritic-cell-based vaccines against established tumours. This proinflammatory phenotype is accompanied by an increase in nuclear factor (NF)-κB activity. In addition, c-Cbl deficiency reduces both p50 and p105 levels, which have been shown to modulate the stimulatory function of NF-κB. Our data indicate that c-Cbl has a crucial, RING-domain-dependent role in regulating dendritic cell maturation, probably by facilitating the regulatory function of p105 and/or p50.
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Células Dendríticas/inmunología , Subunidad p50 de NF-kappa B/metabolismo , Proteínas Proto-Oncogénicas c-cbl/genética , Proteínas Proto-Oncogénicas c-cbl/metabolismo , Animales , Antígenos Nucleares/biosíntesis , Antígenos Nucleares/metabolismo , Proteínas Cromosómicas no Histona/biosíntesis , Proteínas Cromosómicas no Histona/metabolismo , Células Dendríticas/metabolismo , Femenino , Interleucina-12/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal/inmunología , Receptores Toll-Like/metabolismoRESUMEN
OBJECTIVES: Pathologists have routinely observed distinct histologic patterns of growth in early-stage lung adenocarcinoma (LUAD), which have been suggested to be associated with prognosis. Herein, we investigated the relationship between LUAD patterns of growth, as defined by the updated international association for the study of lung cancer (IASLC) grading criteria, and differences in the tumor immune microenvironment to identify predictors of response to immunotherapy. METHODS: 174 resected stage I-III LUAD tumors were classified by histologic pattern of growth (i.e. solid, micropapillary, acinar, papillary, and lepidic) and then grouped as well differentiated, moderately differentiated, and poorly differentiated. Comprehensive multiplatform analysis including whole exome sequencing, gene expression profiling, immunohistochemistry, CIBERSORT, and T-cell receptor sequencing was performed and groups were compared for differences in genomic drivers, immune cell infiltrate, clonality, and survival. Finally, multivariate analysis was performed adjusting for pathologic stage and smoking status. RESULTS: Poorly differentiated tumors demonstrated a strong association with smoking relative to moderately differentiated or well differentiated tumors. However, unlike in prior reports, poorly differentiated tumors were not associated with a worse survival after curative-intent resection. Genomic analysis revealed that poorly differentiated tumors are associated with high tumor mutation burden but showed no association with oncogenic drivers. Immune analyses revealed that poorly differentiated tumors are associated with increased T-cell clonality, expression of PD-L1, and infiltration by cytotoxic CD8 T-cells, activated CD4 T-cells, and pro-inflammatory (M1) macrophages. Finally, multivariate analysis controlling for stage and smoking status confirmed independence of immune differences between IASLC grade groups. CONCLUSIONS: Poorly differentiated tumors, as defined by the updated IASLC grading criteria, are associated with a distinct immunogenic tumor microenvironment that predicts for therapeutic response to immune agents, including checkpoint inhibitors, and should be included in the clinical trial design of immunotherapy studies in early-stage lung adenocarcinoma.
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Adenocarcinoma del Pulmón , Adenocarcinoma , Neoplasias Pulmonares , Adenocarcinoma/patología , Adenocarcinoma del Pulmón/genética , Antígeno B7-H1 , Biomarcadores de Tumor/genética , Humanos , Neoplasias Pulmonares/patología , Pronóstico , Microambiente Tumoral/genéticaRESUMEN
In this work, we find that CD8+ T cells expressing inhibitory killer cell immunoglobulin-like receptors (KIRs) are the human equivalent of Ly49+CD8+ regulatory T cells in mice and are increased in the blood and inflamed tissues of patients with a variety of autoimmune diseases. Moreover, these CD8+ T cells efficiently eliminated pathogenic gliadin-specific CD4+ T cells from the leukocytes of celiac disease patients in vitro. We also find elevated levels of KIR+CD8+ T cells, but not CD4+ regulatory T cells, in COVID-19 patients, correlating with disease severity and vasculitis. Selective ablation of Ly49+CD8+ T cells in virus-infected mice led to autoimmunity after infection. Our results indicate that in both species, these regulatory CD8+ T cells act specifically to suppress pathogenic T cells in autoimmune and infectious diseases.
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Enfermedades Autoinmunes , COVID-19 , Animales , Linfocitos T CD8-positivos , Humanos , Ratones , Receptores KIR , Linfocitos T ReguladoresRESUMEN
Previous reports show that Ly49 + CD8 + T cells can suppress autoimmunity in mouse models of autoimmune diseases. Here we find a markedly increased frequency of CD8 + T cells expressing inhibitory Killer cell Immunoglobulin like Receptors (KIR), the human equivalent of the Ly49 family, in the blood and inflamed tissues of various autoimmune diseases. Moreover, KIR + CD8 + T cells can efficiently eliminate pathogenic gliadin-specific CD4 + T cells from Celiac disease (CeD) patients' leukocytes in vitro . Furthermore, we observe elevated levels of KIR + CD8 + T cells, but not CD4 + regulatory T cells, in COVID-19 and influenza-infected patients, and this correlates with disease severity and vasculitis in COVID-19. Expanded KIR + CD8 + T cells from these different diseases display shared phenotypes and similar T cell receptor sequences. These results characterize a regulatory CD8 + T cell subset in humans, broadly active in both autoimmune and infectious diseases, which we hypothesize functions to control self-reactive or otherwise pathogenic T cells. ONE-SENTENCE SUMMARY: Here we identified KIR + CD8 + T cells as a regulatory CD8 + T cell subset in humans that suppresses self-reactive or otherwise pathogenic CD4 + T cells.
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An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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Immunotherapy targeting T cells is increasingly utilized to treat solid tumors including non-small cell lung cancer (NSCLC). This requires a better understanding of the T cells in the lungs of patients with NSCLC. Here, we report T cell repertoire analysis in a cohort of 236 early-stage NSCLC patients. T cell repertoire attributes are associated with clinicopathologic features, mutational and immune landscape. A considerable proportion of the most prevalent T cells in tumors are also prevalent in the uninvolved tumor-adjacent lungs and appear specific to shared background mutations or viral infections. Patients with higher T cell repertoire homology between the tumor and uninvolved tumor-adjacent lung, suggesting a less tumor-focused T cell response, exhibit inferior survival. These findings indicate that a concise understanding of antigens and T cells in NSCLC is needed to improve therapeutic efficacy and reduce toxicity with immunotherapy, particularly adoptive T cell therapy.
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Carcinoma de Pulmón de Células no Pequeñas/inmunología , Neoplasias Pulmonares/inmunología , Linfocitos T/inmunología , Adulto , Anciano , Carcinoma de Pulmón de Células no Pequeñas/patología , Células Clonales , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Humanos , Pulmón/patología , Neoplasias Pulmonares/patología , Activación de Linfocitos/inmunología , Recuento de Linfocitos , Masculino , Persona de Mediana Edad , Mutación/genética , Análisis de SupervivenciaRESUMEN
Individuals with narcolepsy suffer from abnormal sleep patterns due to loss of neurons that uniquely supply hypocretin (HCRT). Previous studies found associations of narcolepsy with the human leukocyte antigen (HLA)-DQ6 allele and T-cell receptor α (TRA) J24 gene segment and also suggested that in vitro-stimulated T cells can target HCRT. Here, we present evidence of in vivo expansion of DQ6-HCRT tetramer+/TRAJ24+/CD4+ T cells in DQ6+ individuals with and without narcolepsy. We identify related TRAJ24+ TCRαß clonotypes encoded by identical α/ß gene regions from two patients and two controls. TRAJ24-G allele+ clonotypes only expand in the two patients, whereas a TRAJ24-C allele+ clonotype expands in a control. A representative tetramer+/G-allele+ TCR shows signaling reactivity to the epitope HCRT87-97. Clonally expanded G-allele+ T cells exhibit an unconventional effector phenotype. Our analysis of in vivo expansion of HCRT-reactive TRAJ24+ cells opens an avenue for further investigation of the autoimmune contribution to narcolepsy development.
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Autoinmunidad/inmunología , Linfocitos T CD4-Positivos/inmunología , Narcolepsia/inmunología , Orexinas/inmunología , Animales , Autoinmunidad/genética , Estudios de Casos y Controles , Proliferación Celular , Cristalografía por Rayos X , Drosophila , Antígenos HLA-DQ/genética , Antígenos HLA-DQ/inmunología , Humanos , Región de Unión de la Inmunoglobulina/genética , Narcolepsia/genética , Tolerancia Periférica , Fenotipo , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Receptores de Antígenos de Linfocitos T alfa-beta/inmunologíaRESUMEN
Expression of the chromatin-associated protein HMGA2 correlates with progression, metastasis and therapy resistance in pancreatic ductal adenocarcinoma (PDAC). Hmga2 has also been identified as a marker of a transient subpopulation of PDAC cells that has increased metastatic ability. Here, we characterize the requirement for Hmga2 during growth, dissemination, and metastasis of PDAC in vivo using conditional inactivation of Hmga2 in well-established autochthonous mouse models of PDAC. Overall survival, primary tumour burden, presence of disseminated tumour cells in the peritoneal cavity or circulating tumour cells in the blood, and presence and number of metastases were not significantly different between mice with Hmga2-wildtype or Hmga2-deficient tumours. Treatment of mice with Hmga2-wildtype and Hmga2-deficient tumours with gemcitabine did not uncover a significant impact of Hmga2-deficiency on gemcitabine sensitivity. Hmga1 and Hmga2 overlap in their expression in both human and murine PDAC, however knockdown of Hmga1 in Hmga2-deficient cancer cells also did not decrease metastatic ability. Thus, Hmga2 remains a prognostic marker which identifies a metastatic cancer cell state in primary PDAC, however Hmga2 has limited if any direct functional impact on PDAC progression and therapy resistance.
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Adenocarcinoma/secundario , Carcinoma Ductal Pancreático/secundario , Proliferación Celular , Desoxicitidina/análogos & derivados , Resistencia a Antineoplásicos , Proteína HMGA2/metabolismo , Neoplasias Pancreáticas/patología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Animales , Antimetabolitos Antineoplásicos/farmacología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Desoxicitidina/farmacología , Proteína HMGA2/genética , Humanos , Ratones , Ratones Noqueados , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Pronóstico , Tasa de Supervivencia , Análisis de Matrices Tisulares , Células Tumorales Cultivadas , GemcitabinaRESUMEN
Inhibitory signals that govern the cytolytic functions of CD8(+) T lymphocytes have been linked to the expression of natural killer cell receptors (NKRs) on CTLs. There is limited knowledge about the induction of inhibitory NKR (iNKR) expression in vivo. Up-regulation of iNKRs has been linked to the modulation of the virus- and/or tumor-specific immune responses in animal models. In the present study, we directly examined the expression of various NKRs on tumor-infiltrating lymphocytes (TILs) derived from human cervical cancer. We found that in human cervical cancer, the percentage expression of immunoglobulin-like NKR(+)CD8(+) T lymphocytes were similar in gated CD8(+)-autologous TILs and peripheral blood mononuclear cells. On the contrary, cervical cancer-infiltrating CD8(+) T lymphocytes expressed up-regulated C-type lectin NKRs CD94/NKG2A compared with either peripheral blood CD8(+) T cells or normal cervix-infiltrating CD8(+) T lymphocytes. Dual NKR coexpression analyses showed that CD94 and NKG2A were mainly expressed on CD56(-)CD161(-)CD8(+) TILs within the cancer milieu. Immunohistochemical study showed that cervical cancer cells expressed abundant interleukin 15 (IL-15) and transforming growth factor-beta (TGF-beta). In kinetic coculture assay, cervical cancer cells can promote the expression of CD94/NKG2A on CD8(+) T lymphocytes. The cancer-derived effects can be reversed by addition of rIL-15Ralpha/Fc and anti-TGF-beta antibody. Functional analyses illustrated that intracellular perforin expression of CD8(+) T cells was minimal upon up-regulation of CD94/NKG2A. Kinetic cytotoxicity assays showed that up-regulated expressions of CD94/NKG2A restrain CD8(+) T lymphocyte cytotoxicity. Our study strongly indicated that cervical cancer cells could promote the expression of iNKRs via an IL-15- and possibly TGF-beta-mediated mechanism and abrogate the antitumor cytotoxicity of TILs.
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Antígenos CD/biosíntesis , Linfocitos T CD8-positivos/inmunología , Lectinas Tipo C/biosíntesis , Linfocitos Infiltrantes de Tumor/inmunología , Glicoproteínas de Membrana/biosíntesis , Receptores Inmunológicos/biosíntesis , Neoplasias del Cuello Uterino/inmunología , Antígenos CD/inmunología , Linfocitos T CD8-positivos/metabolismo , Citotoxicidad Inmunológica , Femenino , Humanos , Interleucina-15/farmacología , Lectinas Tipo C/inmunología , Prueba de Cultivo Mixto de Linfocitos , Linfocitos Infiltrantes de Tumor/metabolismo , Glicoproteínas de Membrana/inmunología , Subfamília C de Receptores Similares a Lectina de Células NK , Subfamília D de Receptores Similares a Lectina de las Células NK , Perforina , Proteínas Citotóxicas Formadoras de Poros , Receptores Inmunológicos/inmunología , Receptores de Células Asesinas Naturales , Proteínas Recombinantes/farmacología , Linfocitos T Citotóxicos/inmunología , Factor de Crecimiento Transformador beta/farmacología , Regulación hacia ArribaRESUMEN
Large-scale genomic analyses of human cancers have cataloged somatic point mutations thought to initiate tumor development and sustain cancer growth. However, determining the functional significance of specific alterations remains a major bottleneck in our understanding of the genetic determinants of cancer. Here, we present a platform that integrates multiplexed AAV/Cas9-mediated homology-directed repair (HDR) with DNA barcoding and high-throughput sequencing to simultaneously investigate multiple genomic alterations in de novo cancers in mice. Using this approach, we introduce a barcoded library of non-synonymous mutations into hotspot codons 12 and 13 of Kras in adult somatic cells to initiate tumors in the lung, pancreas, and muscle. High-throughput sequencing of barcoded Kras HDR alleles from bulk lung and pancreas reveals surprising diversity in Kras variant oncogenicity. Rapid, cost-effective, and quantitative approaches to simultaneously investigate the function of precise genomic alterations in vivo will help uncover novel biological and clinically actionable insights into carcinogenesis.
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Carcinogénesis/genética , Análisis Mutacional de ADN/métodos , Neoplasias/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Reparación del ADN por Recombinación/genética , Animales , Sistemas CRISPR-Cas/genética , Análisis Costo-Beneficio , Análisis Mutacional de ADN/economía , Estudios de Factibilidad , Femenino , Genómica/economía , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Masculino , Ratones , Mutación , Neoplasias/patología , Reproducibilidad de los ResultadosRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is one of the most metastatic and deadly cancers. Despite the clinical significance of metastatic spread, our understanding of molecular mechanisms that drive PDAC metastatic ability remains limited. By generating a genetically engineered mouse model of human PDAC, we uncover a transient subpopulation of cancer cells with exceptionally high metastatic ability. Global gene expression profiling and functional analyses uncovered the transcription factor BLIMP1 as a driver of PDAC metastasis. The highly metastatic PDAC subpopulation is enriched for hypoxia-induced genes, and hypoxia-mediated induction of BLIMP1 contributes to the regulation of a subset of hypoxia-associated gene expression programs. These findings support a model in which upregulation of BLIMP1 links microenvironmental cues to a metastatic stem cell character.Significance: PDAC is an almost uniformly lethal cancer, largely due to its tendency for metastasis. We define a highly metastatic subpopulation of cancer cells, uncover a key transcriptional regulator of metastatic ability, and define hypoxia as an important factor within the tumor microenvironment that increases metastatic proclivity. Cancer Discov; 7(10); 1184-99. ©2017 AACR.See related commentary by Vakoc and Tuveson, p. 1067This article is highlighted in the In This Issue feature, p. 1047.
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Carcinoma Ductal Pancreático/patología , Perfilación de la Expresión Génica/métodos , Neoplasias Pancreáticas/patología , Factor 1 de Unión al Dominio 1 de Regulación Positiva/genética , Análisis de Secuencia de ARN/métodos , Regulación hacia Arriba , Animales , Carcinoma Ductal Pancreático/genética , Hipoxia de la Célula , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Ingeniería Genética , Humanos , Ratones , Metástasis de la Neoplasia , Trasplante de Neoplasias , Neoplasias Pancreáticas/genética , Microambiente TumoralRESUMEN
Lung cancer is the leading cause of cancer deaths worldwide, with the majority of mortality resulting from metastatic spread. However, the molecular mechanism by which cancer cells acquire the ability to disseminate from primary tumors, seed distant organs, and grow into tissue-destructive metastases remains incompletely understood. We combined tumor barcoding in a mouse model of human lung adenocarcinoma with unbiased genomic approaches to identify a transcriptional program that confers metastatic ability and predicts patient survival. Small-scale in vivo screening identified several genes, including Cd109, that encode novel pro-metastatic factors. We uncovered signaling mediated by Janus kinases (Jaks) and the transcription factor Stat3 as a critical, pharmacologically targetable effector of CD109-driven lung cancer metastasis. In summary, by coupling the systematic genomic analysis of purified cancer cells in distinct malignant states from mouse models with extensive human validation, we uncovered several key regulators of metastatic ability, including an actionable pro-metastatic CD109-Jak-Stat3 axis.
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Adenocarcinoma/genética , Antígenos CD/genética , Regulación Neoplásica de la Expresión Génica/genética , Quinasas Janus/genética , Neoplasias Pulmonares/genética , Proteínas de Neoplasias/genética , Factor de Transcripción STAT3/genética , Adenocarcinoma/metabolismo , Animales , Western Blotting , Línea Celular Tumoral , Modelos Animales de Enfermedad , Técnicas de Silenciamiento del Gen , Janus Quinasa 1/genética , Janus Quinasa 3/genética , Neoplasias Pulmonares/metabolismo , Ratones , Terapia Molecular Dirigida , Metástasis de la Neoplasia/genética , Reacción en Cadena de la Polimerasa , Inhibidores de Proteínas Quinasas , Proteínas Proto-Oncogénicas p21(ras)/genética , Transducción de Señal , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Although blood-brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt-ß-catenin signaling. Constitutive activation of Wnt-ß-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption.
Asunto(s)
Barrera Hematoencefálica/metabolismo , Células Endoteliales/metabolismo , Glioblastoma/genética , Infarto de la Arteria Cerebral Media/genética , Hemorragias Intracraneales/genética , Receptores Acoplados a Proteínas G/genética , Uniones Estrechas/metabolismo , Animales , Barrera Hematoencefálica/ultraestructura , Modelos Animales de Enfermedad , Células Endoteliales/ultraestructura , Matriz Extracelular/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Glioblastoma/metabolismo , Infarto de la Arteria Cerebral Media/metabolismo , Hemorragias Intracraneales/metabolismo , Ratones , Ratones Noqueados , Microscopía Electrónica , Microvasos , Pericitos/ultraestructura , Reacción en Cadena en Tiempo Real de la Polimerasa , Uniones Estrechas/ultraestructura , Vía de Señalización WntRESUMEN
Tumor-infiltrating lymphocytes (TILs) develop as manifestations of the recognition and defense against malignant cells by the host immune system. TILs were literally defined as "tumor-infiltrating lymphocytes", which a posteriori locate within the tumor tissues. Although such cells can be found, they fail to control the growth of tumor. Many have proposed diverse mechanisms for dysfunction of TILs with regard to the roles of immunosurveillance against cancer. However, only a few cancer types, e.g. melanoma, have seen the benefits brought by activating these cells for immunotherapy. Functional defects of TILs have been linked to abnormalities of signaling molecules; however, there is conflicting data. The death of TILs was attributed to expression of cancer-derived FasL, PD-1 and RCAS1, and cancer-induced activation-induced cell death (AICD). Confirmed by studies using TILs and animal models, the compromise of tumor-specific immune responses was thought to result from not only mechanisms of clonal anergy but also exhaustion and/or deletion. Furthermore, functional cytotoxic CD8(+) TILs might be rendered incompetent by cancer-induced up-regulation of inhibitory NK receptors or proximal signaling abnormalities. Additionally, immune privilege was partly attributed to recruitment of regulatory T cells to the tumor sites. The failure of IL-2 signaling, which stands at the center of T cell functionalities, had been linked to the enzymatic activity of cancer-derived matrix metalloproteinases (MMPs). Finally, the exploitation of IDO expression, an important enzyme in pregnancy-related immunosuppression, by cancer cells might play a role in tumor immunity. The disparity of cancer types, origin, developmental stages and individual genetic backgrounds likely account for differences, or even contradictions, which might be the reason why immunotherapy works only on a few cancer types. Delineating the mechanisms behind functional defects of TILs can help not only boost chances of the development of a successful cure but understand the not fully identified roles played by immune system in the face of malignancies.