RESUMEN

Glioblastoma (GBM) is the most common and aggressive form of malignant glioma. The GBM tumor microenvironment (TME) is a complex ecosystem of heterogeneous cells and signaling factors. Glioma associated macrophages and microglia (GAMs) constitute a significant portion of the TME, suggesting that their functional attributes play a crucial role in cancer homeostasis. In GBM, an elevated GAM population is associated with poor prognosis and therapeutic resistance. Neoplastic cells recruit these myeloid populations through release of chemoattractant factors and dysregulate their induction of inflammatory programs. GAMs become protumoral advocates through production a variety of cytokines, inflammatory mediators, and growth factors that can drive cancer proliferation, invasion, immune evasion, and angiogenesis. Among these inflammatory factors, cyclooxygenase-2 (COX-2) and its downstream product, prostaglandin E2 (PGE2), are highly enriched in GBM and their overexpression is positively correlated with poor prognosis in patients. Both tumor cells and GAMs have the ability to signal through the COX-2 PGE2 axis and respond in an autocrine/paracrine manner. In the GBM TME, enhanced signaling through the COX-2/PGE2 axis leads to pleotropic effects that impact GAM dynamics and drive tumor progression.

RESUMEN

Chronic activation of microglia is a driving factor in the progression of neuroinflammatory diseases, and mechanisms that regulate microglial inflammatory signaling are potential targets for novel therapeutics. Regulator of G protein Signaling 10 is the most abundant RGS protein in microglia, where it suppresses inflammatory gene expression and reduces microglia-mediated neurotoxicity. In particular, microglial RGS10 downregulates the expression of pro-inflammatory mediators including cyclooxygenase 2 (COX-2) following stimulation with lipopolysaccharide (LPS). However, the mechanism by which RGS10 affects inflammatory signaling is unknown and is independent of its canonical G protein targeted mechanism. Here, we sought to identify non-canonical RGS10 interacting partners that mediate its anti-inflammatory mechanism. Through RGS10 co-immunoprecipitation coupled with mass spectrometry, we identified STIM2, an endoplasmic reticulum (ER) localized calcium sensor and a component of the store-operated calcium entry (SOCE) machinery, as a novel RGS10 interacting protein in microglia. Direct immunoprecipitation experiments confirmed RGS10-STIM2 interaction in multiple microglia and macrophage cell lines, as well as in primary cells, with no interaction observed with the homologue STIM1. We further determined that STIM2, Orai channels, and the calcium-dependent phosphatase calcineurin are essential for LPS-induced COX-2 production in microglia, and this pathway is required for the inhibitory effect of RGS10 on COX-2. Additionally, our data demonstrated that RGS10 suppresses SOCE triggered by ER calcium depletion and that ER calcium depletion, which induces SOCE, amplifies pro-inflammatory genes. In addition to COX-2, we also show that RGS10 suppresses the expression of pro-inflammatory cytokines in microglia in response to thrombin and LPS stimulation, and all of these effects require SOCE. Collectively, the physical and functional links between RGS10 and STIM2 suggest a complex regulatory network connecting RGS10, SOCE, and pro-inflammatory gene expression in microglia, with broad implications in the pathogenesis and treatment of chronic neuroinflammation.

Asunto(s)

Canales de Calcio/metabolismo , Señalización del Calcio , Calcio/metabolismo , Regulación de la Expresión Génica , Microglía/metabolismo , Proteínas RGS/metabolismo , Molécula de Interacción Estromal 2/metabolismo , Animales , Canales de Calcio/genética , Inflamación/genética , Inflamación/metabolismo , Ratones , Células RAW 264.7 , Proteínas RGS/genética , Molécula de Interacción Estromal 2/genética

RESUMEN

Triple-negative breast cancers (TNBCs) are heterogeneous and aggressive, with high mortality rates. TNBCs frequently respond to chemotherapy, yet many patients develop chemoresistance. The molecular basis and roles for tumor cell-stromal crosstalk in establishing chemoresistance are complex and largely unclear. Here we report molecular studies of paired TNBC patient-derived xenografts (PDXs) established before and after the development of chemoresistance. Interestingly, the chemoresistant model acquired a distinct KRASQ61R mutation that activates K-Ras. The chemoresistant KRAS-mutant model showed gene expression and proteomic changes indicative of altered tumor cell metabolism. Specifically, KRAS-mutant PDXs exhibited increased redox ratios and decreased activation of AMPK, a protein involved in responding to metabolic homeostasis. Additionally, the chemoresistant model exhibited increased immunosuppression, including expression of CXCL1 and CXCL2, cytokines responsible for recruiting immunosuppressive leukocytes to tumors. Notably, chemoresistant KRAS-mutant tumors harbored increased numbers of granulocytic myeloid-derived suppressor cells (gMDSCs). Interestingly, previously established Ras/MAPK-associated gene expression signatures correlated with myeloid/neutrophil-recruiting CXCL1/2 expression and negatively with T cell-recruiting chemokines (CXCL9/10/11) across patients with TNBC, even in the absence of KRAS mutations. MEK inhibition induced tumor suppression in mice while reversing metabolic and immunosuppressive phenotypes, including chemokine production and gMDSC tumor recruitment in the chemoresistant KRAS-mutant tumors. These results suggest that Ras/MAPK pathway inhibitors may be effective in some breast cancer patients to reverse Ras/MAPK-driven tumor metabolism and immunosuppression, particularly in the setting of chemoresistance.

Asunto(s)

Antineoplásicos/farmacología , Glucólisis , MAP Quinasa Quinasa 1/metabolismo , Células Supresoras de Origen Mieloide/patología , Neoplasias de la Mama Triple Negativas/patología , Proteínas ras/metabolismo , Animales , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Proliferación Celular , Resistencia a Antineoplásicos , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/genética , Ratones , Ratones Desnudos , Células Supresoras de Origen Mieloide/efectos de los fármacos , Células Supresoras de Origen Mieloide/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas ras/antagonistas & inhibidores , Proteínas ras/genética

RESUMEN

Immunotherapies targeting the PD-1 pathway produce durable responses in many cancers, but the tumor-intrinsic factors governing response and resistance are largely unknown. MHC-II expression on tumor cells can predict response to anti-PD-1 therapy. We therefore sought to determine how MHC-II expression by tumor cells promotes PD-1 dependency. Using transcriptional profiling of anti-PD-1-treated patients, we identified unique patterns of immune activation in MHC-II+ tumors. In patients and preclinical models, MHC-II+ tumors recruited CD4+ T cells and developed dependency on PD-1 as well as Lag-3 (an MHC-II inhibitory receptor), which was upregulated in MHC-II+ tumors at acquired resistance to anti-PD-1. Finally, we identify enhanced expression of FCRL6, another MHC-II receptor expressed on NK and T cells, in the microenvironment of MHC-II+ tumors. We ascribe this to what we believe to be a novel inhibitory function of FCRL6 engagement, identifying it as an immunotherapy target. These data suggest a MHC-II-mediated context-dependent mechanism of adaptive resistance to PD-1-targeting immunotherapy.

Asunto(s)

Antígenos CD/metabolismo , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase II/metabolismo , Inmunoterapia , Receptores de Superficie Celular/metabolismo , Inmunidad Adaptativa , Animales , Anticuerpos Neutralizantes , Neoplasias de la Mama/metabolismo , Linfocitos T CD4-Positivos , Línea Celular Tumoral , Antígenos HLA-DR/metabolismo , Antígenos de Histocompatibilidad Clase II/genética , Humanos , Células Asesinas Naturales/inmunología , Ligandos , Ratones , Receptor de Muerte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfocitos T , Linfocitos T/inmunología , Microambiente Tumoral , Proteína del Gen 3 de Activación de Linfocitos

RESUMEN

Immunotherapies targeting programmed cell death protein 1 (PD-1) or its ligand, programmed cell death ligand 1 (PD-L1), dramatically improve the survival of melanoma patients. However, only ∼40% of treated patients demonstrate a clinical response to single-agent anti-PD-1 therapy. An intact tumor response to type-II interferon (i.e. IFN-γ) correlates with response to anti-PD-1, and patients with de novo or acquired resistance may harbor loss-of-function alterations in the JAK/STAT pathway, which lies downstream of the interferon gamma receptor (IFNGR1/2). In this study, we dissected the specific roles of individual JAK/STAT pathway members on the IFN-γ response, and identified JAK1 as the primary mediator of JAK/STAT signaling associated with IFN-γ-induced expression of antigen-presenting molecules MHC-I and MHC-II, as well as PD-L1 and the cytostatic response to IFN-γ. In contrast to the crucial role of JAK1, JAK2 was largely dispensable in mediating most IFN-γ effects. In a mouse melanoma model, inhibition of JAK1/2 in combination with anti-PD-L1 therapy partially blocked anti-tumor immunologic responses, while selective JAK2 inhibition appeared to augment therapy. Amplification of JAK/STAT signaling in tumor cells through genetic inhibition of the negative regulator PTPN2 potentiated IFN-γ response in vitro and in vivo, and may be a target to enhance immunotherapy efficacy.

RESUMEN

Amplifications at 9p24 have been identified in breast cancer and other malignancies, but the genes within this locus causally associated with oncogenicity or tumor progression remain unclear. Targeted next-generation sequencing of postchemotherapy triple-negative breast cancers (TNBCs) identified a group of 9p24-amplified tumors, which contained focal amplification of the Janus kinase 2 (JAK2) gene. These patients had markedly inferior recurrence-free and overall survival compared to patients with TNBC without JAK2 amplification. Detection of JAK2/9p24 amplifications was more common in chemotherapy-treated TNBCs than in untreated TNBCs or basal-like cancers, or in other breast cancer subtypes. Similar rates of JAK2 amplification were confirmed in patient-derived TNBC xenografts. In patients for whom longitudinal specimens were available, JAK2 amplification was selected for during neoadjuvant chemotherapy and eventual metastatic spread, suggesting a role in tumorigenicity and chemoresistance, phenotypes often attributed to a cancer stem cell-like cell population. In TNBC cell lines with JAK2 copy gains or amplification, specific inhibition of JAK2 signaling reduced mammosphere formation and cooperated with chemotherapy in reducing tumor growth in vivo. In these cells, inhibition of JAK1-signal transducer and activator of transcription 3 (STAT3) signaling had little effect or, in some cases, counteracted JAK2-specific inhibition. Collectively, these results suggest that JAK2-specific inhibitors are more efficacious than dual JAK1/2 inhibitors against JAK2-amplified TNBCs. Furthermore, JAK2 amplification is a potential biomarker for JAK2 dependence, which, in turn, can be used to select patients for clinical trials with JAK2 inhibitors.

Asunto(s)

Cromosomas Humanos Par 9/genética , Amplificación de Genes , Sitios Genéticos , Janus Quinasa 2/genética , Neoplasias de la Mama Triple Negativas/enzimología , Neoplasias de la Mama Triple Negativas/genética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Estudios de Cohortes , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Persona de Mediana Edad , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT6/metabolismo , Transducción de Señal/efectos de los fármacos , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/patología , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología

RESUMEN

Cardiomyopathy refers to a set of diseases that are characterized by myocardial dysfunction. Classically, two-dimensional echocardiography has been used in the diagnosis of these disorders and to help guide their management. Three-dimensional transthoracic echocardiography is now increasingly being used in the diagnosis, management, and prognostication of intrinsic cardiomyopathies. In this article, we summarize the available data on the use of three-dimensional transthoracic echocardiography in various forms of intrinsic cardiomyopathy as well as some of its advantages over traditional two-dimensional transthoracic echocardiography.

Asunto(s)

Cardiomiopatías/complicaciones , Cardiomiopatías/diagnóstico por imagen , Ecocardiografía Tridimensional/métodos , Disfunción Ventricular Izquierda/diagnóstico por imagen , Disfunción Ventricular Izquierda/etiología , Sistemas de Computación , Humanos

RESUMEN

OBJECTIVES: The aim of this study was to test the hypothesis that patients with normal regadenoson myocardial perfusion imaging (MPI) have a low rate of cardiac events, similar to patients with normal adenosine MPI. BACKGROUND: Regadenoson, a new selective adenosine A(2A) receptor agonist, is now a widely used stress agent for MPI. The low rate of cardiac events in patients with normal adenosine MPI is well-documented, but the prognostic implications of a normal regadenoson MPI have not been examined and compared with those with adenosine. METHODS: Data on primary composite endpoint (cardiac death, myocardial infarction, and coronary revascularization) were collected for 2,000 patients (1,000 regadenoson, and 1,000 adenosine stress) with normal myocardial perfusion and left ventricular ejection fraction referred for vasodilator MPI. In addition, propensity scores were used to assemble a balanced cohort of 505 pairs of patients who were balanced on 36 baseline characteristics. RESULTS: The primary endpoint occurred in 21 (2.1%; 1.1%/year) patients in the regadenoson group and 33 (3.3%; 1.7%/year) patients in the adenosine group (hazard ratio [HR] for regadenoson vs. adenosine: 0.62; 95% confidence interval [CI]: 0.36 to 1.08; p = 0.090). In the propensity-matched pairs, the primary endpoint occurred in 7 (1.4%; 0.7%/year) patients in the regadenoson group and 13 (2.6%; 1.3%/year) patients in the adenosine group (matched HR: 0.58; 95% CI: 0.23 to 1.48; p = 0.257). Cardiac deaths were infrequent in the entire sample and in the propensity-matched groups; the cardiac death rate was 0.9%/year and 1.15%/year in the regadenoson and adenosine groups (HR: 0.77; 95% CI: 0.42 to 1.43; p = 0.404) in the pre-match sample and 0.5%/year and 0.7%/year in the matched groups, respectively (HR: 0.83; 95% CI: 0.25 to 2.73; p = 0.763). CONCLUSIONS: Major cardiac events are infrequent in patients with normal regadenoson MPI. These findings provide assurance that normal MPI using a simpler stress protocol with regadenoson provides prognostic data similar to normal adenosine MPI.

Asunto(s)

Agonistas del Receptor de Adenosina A2 , Adenosina , Tomografía Computarizada por Emisión de Fotón Único Sincronizada Cardíaca , Cardiopatías/diagnóstico por imagen , Imagen de Perfusión Miocárdica/métodos , Purinas , Pirazoles , Vasodilatadores , Anciano , Causas de Muerte , Distribución de Chi-Cuadrado , Femenino , Cardiopatías/mortalidad , Cardiopatías/fisiopatología , Cardiopatías/terapia , Humanos , Incidencia , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Análisis Multivariante , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/fisiopatología , Revascularización Miocárdica , Valor Predictivo de las Pruebas , Pronóstico , Puntaje de Propensión , Modelos de Riesgos Proporcionales , Valores de Referencia , Factores de Riesgo , Volumen Sistólico , Factores de Tiempo , Función Ventricular Izquierda

RESUMEN

Diagnosing congenital heart disease (CHD) depends heavily on imaging. Traditionally, two-dimensional (2D) echocardiography has been the most widely used but since this imaging modality utilizes a 2D technique to evaluate three-dimensional (3D) structures it suffers from inherent limitations. The more recently developed 3D echocardiography is poised to be superior in providing comprehensive evaluation prior to intervention on such complex conditions. In this review, we summarize the applications of 3D echocardiography in evaluating patients with CHD.

Asunto(s)

Ecocardiografía Tridimensional/métodos , Cardiopatías Congénitas/diagnóstico por imagen , Humanos

RESUMEN

BACKGROUND: Myocardial perfusion imaging (MPI) is a useful method for risk assessment in patients with diabetes mellitus (DM) and chronic kidney disease (CKD), but these patients have a residual risk that is not accounted for by MPI. The objective of this study is to determine whether the heart rate response (HRR) to adenosine has an incremental prognostic value to MPI in high-risk patients. METHODS: The study group included 879 (age 61 ± 13 years, 48% women, 58% white, 40% DM, 49% CKD) consecutive patients who underwent adenosine MPI. Chronic kidney disease was defined as an estimated glomerular filtration rate <60 mL/min per 1.73 m(2) or dialysis replacement therapy. An HRR <10% (change from baseline) was considered blunted. The outcome of interest was overall mortality. RESULTS: During a follow-up period of 40 ± 14 months, 212 patients (24%) died. Patients with DM (23.4% ± 16.3% vs 29.4% ± 21.4%, P < .0001) and CKD (22.7% ± 17.6% vs 30.5% ± 20.4%, P < .0001) had lower HRR as compared with patients without DM and CKD, respectively. A blunted HRR was associated with increased mortality in the overall population and in those with DM and CKD and helped in risk stratification when added to traditional MPI findings. In a Cox regression model, a blunted HRR was the strongest predictor of mortality (hazard ratio 2.8, P < .0001) and provided additional prognostic data to MPI (hazard ratio 1.9, P < .0001) after controlling for age, gender, race, history of myocardial infarction, DM, CKD, ß-blocker use, and presence of chest pain. CONCLUSIONS: A blunted HRR to adenosine is an independent predictor of poor outcome, adds incremental value to MPI, and helps in better risk stratification in high-risk patient groups.

Asunto(s)

Adenosina , Enfermedad Coronaria/diagnóstico , Diabetes Mellitus/fisiopatología , Frecuencia Cardíaca/efectos de los fármacos , Enfermedades Renales/complicaciones , Imagen de Perfusión Miocárdica/métodos , Medición de Riesgo/métodos , Adenosina/administración & dosificación , Alaska/epidemiología , Antiarrítmicos/administración & dosificación , Enfermedad Crónica , Enfermedad Coronaria/epidemiología , Enfermedad Coronaria/fisiopatología , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Incidencia , Enfermedades Renales/fisiopatología , Masculino , Persona de Mediana Edad , Pronóstico , Estudios Retrospectivos , Factores de Riesgo

RESUMEN

BACKGROUND: Regadenoson myocardial perfusion imaging (MPI) is a useful method for risk assessment. We hypothesized that the heart rate response (HRR) to regadenoson carries incremental prognostic information to that derived from perfusion pattern and left ventricular (LV) ejection fraction (EF). METHODS AND RESULTS: The study population included 1,156 (60 ± 13 years, 46% women, 40% diabetes mellitus, 53% chronic kidney disease) patients. During a follow-up period of 22 ± 5 months, 103 patients died (9%). Independent determinants of the HRR included age, gender, race, diabetes mellitus, coronary revascularization, LVEF, use of insulin and aldosterone antagonists. Decreasing HRR was associated with stepwise increase in mortality (log-rank P < .0001). In a Cox proportional model for mortality that adjusted for age, gender, diabetes mellitus, renal disease, and MPI findings, HRR in the lowest quartile was independently associated with fivefold increase in mortality compared to the highest quartile [HR 5.2, 95% CI 2.3-12.0, P < .0001]. Patients with a normal HRR had a relatively low annualized total mortality despite the presence of risk factors. The addition of HRR to traditional MPI findings had a net reclassification improvement of 15%, P = .02. CONCLUSION: A blunted HRR to regadenoson is an independent predictor of poor outcome, adds incremental value to MPI, and helps in better risk stratification.

Asunto(s)

Frecuencia Cardíaca/efectos de los fármacos , Imagen de Perfusión Miocárdica/estadística & datos numéricos , Purinas , Pirazoles , Volumen Sistólico/efectos de los fármacos , Disfunción Ventricular Izquierda/diagnóstico , Disfunción Ventricular Izquierda/mortalidad , Antagonistas del Receptor de Adenosina A2/uso terapéutico , Alabama/epidemiología , Prueba de Esfuerzo/estadística & datos numéricos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Prevalencia , Pronóstico , Reproducibilidad de los Resultados , Medición de Riesgo , Factores de Riesgo , Sensibilidad y Especificidad , Análisis de Supervivencia , Tasa de Supervivencia , Resultado del Tratamiento

RESUMEN

This unit describes the technology in general, to give a feel for the interplay between the various parts of a flow cytometer. Topics include cell preparation, detectors, analysis, and sorting. A brief chronology of flow instrumentation development illustrates the long history of the field.

Asunto(s)

Células/citología , Citometría de Flujo/instrumentación , Animales , Separación Celular/instrumentación , Citogenética , Citometría de Flujo/tendencias , Colorantes Fluorescentes

Asunto(s)

Citometría de Flujo/historia , Citometría de Imagen/historia , Cooperación Internacional , Sociedades Científicas/historia , Historia del Siglo XX , Humanos , Sociedades Científicas/organización & administración