RESUMO
The rapid development of single-cell transcriptomic technologies has helped uncover the cellular heterogeneity within cell populations. However, bulk RNA-seq continues to be the main workhorse for quantifying gene expression levels due to technical simplicity and low cost. To most effectively extract information from bulk data given the new knowledge gained from single-cell methods, we have developed a novel algorithm to estimate the cell-type composition of bulk data from a single-cell RNA-seq-derived cell-type signature. Comparison with existing methods using various real RNA-seq data sets indicates that our new approach is more accurate and comprehensive than previous methods, especially for the estimation of rare cell types. More importantly, our method can detect cell-type composition changes in response to external perturbations, thereby providing a valuable, cost-effective method for dissecting the cell-type-specific effects of drug treatments or condition changes. As such, our method is applicable to a wide range of biological and clinical investigations.
Assuntos
Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Algoritmos , Conjuntos de Dados como Assunto , Estudos de Viabilidade , Feminino , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Análise dos Mínimos Quadrados , Melanoma/genética , Neoplasias Ovarianas/genética , Neoplasias Cutâneas/genética , Transcriptoma/genéticaRESUMO
MICA and MICB are expressed by many human cancers as a result of cellular stress, and can tag cells for elimination by cytotoxic lymphocytes through natural killer group 2D (NKG2D) receptor activation. However, tumors evade this immune recognition pathway through proteolytic shedding of MICA and MICB proteins. We rationally designed antibodies targeting the MICA α3 domain, the site of proteolytic shedding, and found that these antibodies prevented loss of cell surface MICA and MICB by human cancer cells. These antibodies inhibited tumor growth in multiple fully immunocompetent mouse models and reduced human melanoma metastases in a humanized mouse model. Antitumor immunity was mediated mainly by natural killer (NK) cells through activation of NKG2D and CD16 Fc receptors. This approach prevents the loss of important immunostimulatory ligands by human cancers and reactivates antitumor immunity.
Assuntos
Anticorpos Bloqueadores/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Antígenos de Histocompatibilidade Classe I/imunologia , Células Matadoras Naturais/imunologia , Melanoma/terapia , Animais , Anticorpos Bloqueadores/imunologia , Anticorpos Monoclonais/imunologia , Antígenos de Histocompatibilidade Classe I/química , Humanos , Imunocompetência , Ligantes , Melanoma/imunologia , Melanoma/patologia , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Metástase Neoplásica , Domínios Proteicos/imunologia , Receptores de IgG/imunologiaRESUMO
Many human cancers are resistant to immunotherapy, for reasons that are poorly understood. We used a genome-scale CRISPR-Cas9 screen to identify mechanisms of tumor cell resistance to killing by cytotoxic T cells, the central effectors of antitumor immunity. Inactivation of >100 genes-including Pbrm1, Arid2, and Brd7, which encode components of the PBAF form of the SWI/SNF chromatin remodeling complex-sensitized mouse B16F10 melanoma cells to killing by T cells. Loss of PBAF function increased tumor cell sensitivity to interferon-γ, resulting in enhanced secretion of chemokines that recruit effector T cells. Treatment-resistant tumors became responsive to immunotherapy when Pbrm1 was inactivated. In many human cancers, expression of PBRM1 and ARID2 inversely correlated with expression of T cell cytotoxicity genes, and Pbrm1-deficient murine melanomas were more strongly infiltrated by cytotoxic T cells.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Citotoxicidade Imunológica/genética , Melanoma Experimental/imunologia , Melanoma Experimental/terapia , Neoplasias Cutâneas/imunologia , Neoplasias Cutâneas/terapia , Linfócitos T Citotóxicos/imunologia , Fatores de Transcrição/metabolismo , Animais , Proteínas de Bactérias , Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA , Endonucleases , Testes Genéticos , Proteínas HMGB/genética , Proteínas HMGB/metabolismo , Humanos , Imunoterapia , Interferon gama/uso terapêutico , Melanoma Experimental/genética , Camundongos , Neoplasias Cutâneas/genética , Fatores de Transcrição/genéticaRESUMO
Importance: The persistent shortage of dermatologists in the United States affects access to care and patient outcomes. Objective: To characterize the effect of geographic variations in dermatologist density on the provision of dermatology procedures within Medicare. Design, Setting, and Participants: This was a cross-sectional study using the 2013 Medicare Provider Utilization and Payment Database. Dermatology-related procedures were defined by the top 50 billing codes accounting for more than 95% of procedures billed by dermatologists. Billing codes corresponding to evaluation and monitoring visits and dermatopathology were excluded. Total costs were estimated from the Centers for Medicare & Medicaid Services physician fee schedule, based on the nonfacility national payment amount with no modifiers. Nationally representative administrative database that includes 100% of charges billed by noninstitutional clinicians covered under Medicare Part B. A total of 10â¯391 dermatologists practicing within the 50 states and Washington, DC, were included. The Medicare-eligible population was defined as all persons 65 years or older. Exposures: Density of dermatologists, categorized into first (5.3 per 100â¯000 persons ≥65 years) through fifth (54.8 per 100â¯000 persons ≥65 years) quintiles. Main Outcomes and Measures: Utilization of dermatology procedures (mean volume per 100â¯000 persons ≥65 years) and total cost (mean amount billed per person ≥65 years) by clinician type across quintiles of dermatologist density. Results: In 2013, dermatologists billed Medicare for 28 million procedures costing $2.21 billion. Mean billed amount by dermatologists per person 65 years or older was $15.87 in the lowest-density quintile vs $92.02 in the highest-density quintile. This trend suggests that each interval increase of 10 dermatologists per 100â¯000 persons 65 years or older is correlated with a $14.81 increase in Medicare spending on dermatology procedures (95% CI, 8.28-21.34; P = .005). Utilization of these procedures differed among clinician types, with dermatologists largely performing destruction of premalignant lesions and PCPs primarily doing injections. Conclusions and Relevance: There is evidence of supply-sensitive variation in the provision of dermatology procedures for the Medicare-eligible population; higher dermatologist density is correlated with increased utilization of dermatology procedures and subsequent billed charges to Medicare. Further research is needed to determine the effect of such variations on outcomes and whether incentives can better align dermatologists with areas of clinical need.
Assuntos
Dermatologistas/provisão & distribuição , Dermatologia/organização & administração , Planos de Pagamento por Serviço Prestado/economia , Custos de Cuidados de Saúde , Medicare/economia , Idoso , Idoso de 80 Anos ou mais , Estudos Transversais , Bases de Dados Factuais , Feminino , Humanos , Masculino , Avaliação das Necessidades , Avaliação de Resultados em Cuidados de Saúde , Estados UnidosRESUMO
The advent of single-cell sequencing has been revolutionary to the field of cancer genomics. Perfectly suited to capture cancer's heterogeneous nature, single-cell analyses provide information bulk sequencing could never hope to uncover. Many mechanisms of cancer have yet to be fully understood, and single-cell approaches are showing promise in their abilities to uncover these mysteries. Here we focus on the most recent single-cell methods for cancer genomics, and how they are not only providing insights into the inner workings of cancer, but are also transforming individualized therapy and non-invasive monitoring and diagnosis.
Assuntos
Genômica , Sequenciamento de Nucleotídeos em Larga Escala/tendências , Neoplasias/genética , Análise de Célula Única/tendências , Humanos , Neoplasias/diagnósticoRESUMO
The incidence of eosinophilic esophagitis (EoE) has increased in the past several years, yet our understanding of its pathogenesis remains limited. To test the hypothesis that microRNAs (miRNAs) are altered in children with EoE, miRNAs were profiled in esophageal mucosa biopsies obtained from patients with active disease (n = 5) and healthy control subjects (n = 6). Fourteen miRNAs were significantly altered between groups; four of these miRNAs were decreased in EoE patients. A panel of five miRNAs (miR-203, miR-375, miR-21, miR-223, and miR-142-3p) were selected for validation in an independent set of samples from control (n = 22), active disease (n = 22), inactive disease (n = 22), and gastroesophageal reflux disease (n = 6) patients. Each panel miRNA was significantly altered among groups. miRNA changes in esophageal biopsies were not reflected in the circulating RNA pool, as no differences in panel miRNA levels were observed in sera collected from the four patient groups. In addition, in contrast to previous studies, no change in esophageal miRNA levels was detected following treatment that resolved esophageal eosinophilia. In an effort to identify the ramifications of reduced esophageal miR-203, miR-203 activity was inhibited in cultured epithelial cells via expression of a tough decoy miRNA inhibitor. Luciferase reporter assays demonstrated that miR-203 does not directly regulate human IL-15 through targeting of the IL-15 3'-untranslated region. From these experiments, it is concluded that miRNAs are perturbed in the esophageal mucosa, but not the serum, of pediatric EoE patients. Further investigation is required to decipher pathologically relevant consequences of miRNA perturbation in this context.
Assuntos
Esofagite Eosinofílica/metabolismo , Esôfago/metabolismo , MicroRNAs/metabolismo , Adolescente , Estudos de Casos e Controles , Células Cultivadas , Criança , Pré-Escolar , Esofagite Eosinofílica/sangue , Células Epiteliais/metabolismo , Esôfago/patologia , Feminino , Refluxo Gastroesofágico/sangue , Refluxo Gastroesofágico/metabolismo , Humanos , Masculino , MicroRNAs/sangue , MicroRNAs/genéticaRESUMO
BACKGROUND AND AIMS: Changes in intestinal microRNAs have been reported in adult patients with ulcerative colitis or Crohn's disease. The goal of this study was to identify changes in microRNA expression associated with colitis in children with inflammatory bowel disease. METHODS: Rectal mucosal biopsies (n = 50) and blood samples (n = 47) were collected from patients with known or suspected inflammatory bowel disease undergoing endoscopy. Rectal and serum microRNA levels were profiled using the nCounter platform and the TaqMan low-density array platform, respectively. Significantly altered microRNAs were validated in independent sample sets via quantitative RT-PCR. In vitro luciferase reporter assays were performed in the human colorectal Caco-2 cell line to determine the effect of miR-192 on NOD2 expression. RESULTS: Profiling of rectal RNA identified 21 microRNAs significantly altered between control, UC, and colonic CD sample groups. Nine of the ten microRNAs selected for validation were confirmed as significantly changed. Rectal miR-24 was increased 1.47-fold in UC compared to CD samples (p = 0.0052) and was the only microRNA altered between IBD subtypes. Three colitis-associated microRNAs were significantly altered in sera of disease patients and displayed diagnostic utility. However, no serum microRNAs were found to distinguish ulcerative colitis from Crohn's colitis. Finally, miR-192 inhibition did not affect luciferase reporter activity, suggesting that miR-192 does not regulate human NOD2. CONCLUSION: This study has demonstrated that rectal and serum microRNAs are perturbed in pediatric inflammatory bowel disease. Future studies identifying targets of inflammatory bowel disease-associated microRNAs may lead to novel therapies.