RESUMEN
Muscle invasive bladder cancers (BCs) can be divided into 2 major subgroups-basal/squamous (BASQ) tumors and luminal tumors. Since Pparg has low or undetectable expression in BASQ tumors, we tested the effects of rosiglitazone, Pparg agonist, in a mouse model of BASQ BC. We find that rosiglitazone reduces proliferation while treatment with rosiglitazone plus trametinib, a MEK inhibitor, induces apoptosis and reduces tumor volume by 91% after 1 month. Rosiglitazone and trametinib also induce a shift from BASQ to luminal differentiation in tumors, which our analysis suggests is mediated by retinoid signaling, a pathway known to drive the luminal differentiation program. Our data suggest that rosiglitazone, trametinib, and retinoids, which are all FDA approved, may be clinically active in BASQ tumors in patients.
Asunto(s)
Apoptosis , Proliferación Celular , Modelos Animales de Enfermedad , Piridonas , Pirimidinonas , Rosiglitazona , Neoplasias de la Vejiga Urinaria , Animales , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/genética , Piridonas/farmacología , Piridonas/uso terapéutico , Pirimidinonas/farmacología , Pirimidinonas/uso terapéutico , Rosiglitazona/farmacología , Rosiglitazona/uso terapéutico , Ratones , Apoptosis/efectos de los fármacos , Humanos , Proliferación Celular/efectos de los fármacos , Línea Celular Tumoral , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Invasividad Neoplásica , Femenino , PPAR gamma/metabolismo , PPAR gamma/agonistas , Tiazolidinedionas/farmacología , Tiazolidinedionas/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Retinoides/farmacología , Retinoides/uso terapéuticoRESUMEN
Bladder cancers (BCs) can be divided into 2 major subgroups displaying distinct clinical behaviors and mutational profiles: basal/squamous (BASQ) tumors that tend to be muscle invasive, and luminal/papillary (LP) tumors that are exophytic and tend to be non-invasive. Pparg is a likely driver of LP BC and has been suggested to act as a tumor suppressor in BASQ tumors, where it is likely suppressed by MEK-dependent phosphorylation. Here we tested the effects of rosiglitazone, a Pparg agonist, in a mouse model of BBN-induced muscle invasive BC. Rosiglitazone activated Pparg signaling in suprabasal epithelial layers of tumors but not in basal-most layers containing highly proliferative invasive cells, reducing proliferation but not affecting tumor survival. Addition of trametinib, a MEK inhibitor, induced Pparg signaling throughout all tumor layers, and eradicated 91% of tumors within 7-days of treatment. The 2-drug combination also activated a luminal differentiation program, reversing squamous metaplasia in the urothelium of tumor-bearing mice. Paired ATAC-RNA-seq analysis revealed that tumor apoptosis was most likely linked to down-regulation of Bcl-2 and other pro-survival genes, while the shift from BASQ to luminal differentiation was associated with activation of the retinoic acid pathway and upregulation of Kdm6a, a lysine demethylase that facilitates retinoid-signaling. Our data suggest that rosiglitazone, trametinib, and retinoids, which are all FDA approved, may be clinically active in BASQ tumors in patients. That muscle invasive tumors are populated by basal and suprabasal cell types with different responsiveness to PPARG agonists will be an important consideration when designing new treatments.
RESUMEN
How cancer-associated chromatin abnormalities shape tumor-immune interaction remains incompletely understood. Recent studies have linked DNA hypomethylation and de-repression of retrotransposons to anti-tumor immunity through the induction of interferon response. Here, we report that inactivation of the histone H3K36 methyltransferase NSD1, which is frequently found in squamous cell carcinomas (SCCs) and induces DNA hypomethylation, unexpectedly results in diminished tumor immune infiltration. In syngeneic and genetically engineered mouse models of head and neck SCCs, NSD1-deficient tumors exhibit immune exclusion and reduced interferon response despite high retrotransposon expression. Mechanistically, NSD1 loss results in silencing of innate immunity genes, including the type III interferon receptor IFNLR1, through depletion of H3K36 di-methylation (H3K36me2) and gain of H3K27 tri-methylation (H3K27me3). Inhibition of EZH2 restores immune infiltration and impairs the growth of Nsd1-mutant tumors. Thus, our work uncovers a druggable chromatin cross talk that regulates the viral mimicry response and enables immune evasion of DNA hypomethylated tumors.
Asunto(s)
Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Histona Metiltransferasas , Escape del Tumor , Animales , Ratones , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Cromatina , Metilación de ADN , Neoplasias de Cabeza y Cuello/genética , Histona Metiltransferasas/genética , Histona Metiltransferasas/metabolismo , Histonas/genética , Histonas/metabolismo , Interferones/genética , Proteínas Nucleares/metabolismo , Receptores de Interferón/genética , Retroelementos , Escape del Tumor/genéticaRESUMEN
Pparg, a nuclear receptor, is downregulated in basal subtype bladder cancers that tend to be muscle invasive and amplified in luminal subtype bladder cancers that tend to be non-muscle invasive. Bladder cancers derive from the urothelium, one of the most quiescent epithelia in the body, which is composed of basal, intermediate, and superficial cells. We find that expression of an activated form of Pparg (VP16;Pparg) in basal progenitors induces formation of superficial cells in situ, that exit the cell cycle, and do not form tumors. Expression in basal progenitors that have been activated by mild injury however, results in luminal tumor formation. We find that these tumors are immune deserted, which may be linked to down-regulation of Nf-kb, a Pparg target. Interestingly, some luminal tumors begin to shift to basal subtype tumors with time, down-regulating Pparg and other luminal markers. Our findings have important implications for treatment and diagnosis of bladder cancer.
Asunto(s)
PPAR gamma/metabolismo , Transducción de Señal , Neoplasias de la Vejiga Urinaria/inmunología , Neoplasias de la Vejiga Urinaria/patología , Animales , Biomarcadores de Tumor/metabolismo , Carcinogénesis , Carcinógenos/toxicidad , Diferenciación Celular , Proliferación Celular , Proteína Vmw65 de Virus del Herpes Simple/genética , Proteína Vmw65 de Virus del Herpes Simple/metabolismo , Humanos , Ratones , Ratones Transgénicos , PPAR gamma/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Neoplasias de la Vejiga Urinaria/inducido químicamente , Neoplasias de la Vejiga Urinaria/metabolismo , Urotelio/efectos de los fármacos , Urotelio/inmunología , Urotelio/patologíaRESUMEN
The urothelium is an epithelial barrier lining the bladder that protects against infection, fluid exchange and damage from toxins. The nuclear receptor Pparg promotes urothelial differentiation in vitro, and Pparg mutations are associated with bladder cancer. However, the function of Pparg in the healthy urothelium is unknown. Here we show that Pparg is critical in urothelial cells for mitochondrial biogenesis, cellular differentiation and regulation of inflammation in response to urinary tract infection (UTI). Superficial cells, which are critical for maintaining the urothelial barrier, fail to mature in Pparg mutants and basal cells undergo squamous-like differentiation. Pparg mutants display persistent inflammation after UTI, and Nf-KB, which is transiently activated in response to infection in the wild type urothelium, persists for months. Our observations suggest that in addition to its known roles in adipogegnesis and macrophage differentiation, that Pparg-dependent transcription plays a role in the urothelium controlling mitochondrial function development and regeneration.
Asunto(s)
Diferenciación Celular , Células Epiteliales/metabolismo , Expresión Génica , Genes Mitocondriales/genética , PPAR gamma/metabolismo , Urotelio/metabolismo , Animales , Humanos , Inflamación/complicaciones , Inflamación/genética , Ratones Noqueados , Ratones Transgénicos , Mutación , PPAR gamma/genética , Vejiga Urinaria/citología , Neoplasias de la Vejiga Urinaria/genética , Infecciones Urinarias/complicaciones , Urotelio/citologíaRESUMEN
Hematopoietic stem cell transplantation (HSCT) is a curative therapy for blood and immune diseases with potential for many settings beyond current standard-of-care. Broad HSCT application is currently precluded largely due to morbidity and mortality associated with genotoxic irradiation or chemotherapy conditioning. Here we show that a single dose of a CD117-antibody-drug-conjugate (CD117-ADC) to saporin leads to > 99% depletion of host HSCs, enabling rapid and efficient donor hematopoietic cell engraftment. Importantly, CD117-ADC selectively targets hematopoietic stem cells yet does not cause clinically significant side-effects. Blood counts and immune cell function are preserved following CD117-ADC treatment, with effective responses by recipients to both viral and fungal challenges. These results suggest that CD117-ADC-mediated HSCT pre-treatment could serve as a non-myeloablative conditioning strategy for the treatment of a wide range of non-malignant and malignant diseases, and might be especially suited to gene therapy and gene editing settings in which preservation of immunity is desired.
Asunto(s)
Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/efectos de los fármacos , Inmunoconjugados/farmacología , Proteínas Proto-Oncogénicas c-kit/inmunología , Animales , Médula Ósea/efectos de los fármacos , Trasplante de Médula Ósea , Candida albicans/patogenicidad , Muerte Celular , Línea Celular , Femenino , Terapia Genética , Humanos , Inmunoconjugados/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Neoplasias , Donantes de TejidosRESUMEN
The urothelium is an epithelia barrier lined by a luminal layer of binucleated, octoploid, superficial cells. Superficial cells are critical for production and transport of uroplakins, a family of proteins that assemble into a waterproof crystalline plaque that helps protect against infection and toxic substances. Adult urothelium is nearly quiescent, but rapidly regenerates in response to injury. Yet the mechanism by which binucleated, polyploid, superficial cells are produced remains unclear. Here, we show that superficial cells are likely to be derived from a population of binucleated intermediate cells, which are produced from mononucleated intermediate cells via incomplete cytokinesis. We show that binucleated intermediate and superficial cells increase DNA content via endoreplication, passing through S phase without entering mitosis. The urothelium can be permanently damaged by repetitive or chronic injury or disease. Identification of the mechanism by which superficial cells are produced may be important for developing strategies for urothelial repair.
Asunto(s)
Citocinesis , Endorreduplicación , Mitosis , Poliploidía , Urotelio/fisiopatología , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Femenino , Masculino , Ratones , Urotelio/lesionesRESUMEN
Hematopoietic stem cell transplantation is a potential curative therapy for malignant and nonmalignant diseases. Improving the efficiency of stem cell collection and the quality of the cells acquired can broaden the donor pool and improve patient outcomes. We developed a rapid stem cell mobilization regimen utilizing a unique CXCR2 agonist, GROß, and the CXCR4 antagonist AMD3100. A single injection of both agents resulted in stem cell mobilization peaking within 15 min that was equivalent in magnitude to a standard multi-day regimen of granulocyte colony-stimulating factor (G-CSF). Mechanistic studies determined that rapid mobilization results from synergistic signaling on neutrophils, resulting in enhanced MMP-9 release, and unexpectedly revealed genetic polymorphisms in MMP-9 that alter activity. This mobilization regimen results in preferential trafficking of stem cells that demonstrate a higher engraftment efficiency than those mobilized by G-CSF. Our studies suggest a potential new strategy for the rapid collection of an improved hematopoietic graft.
Asunto(s)
Movilización de Célula Madre Hematopoyética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/inmunología , Adulto , Animales , Bencilaminas , Quimiocina CXCL2/farmacología , Ciclamas , Femenino , Células Madre Hematopoyéticas/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Humanos , Masculino , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Endogámicos ICR , Polimorfismo GenéticoRESUMEN
Physiological stem cell function is regulated by secreted factors produced by niche cells. In this study, we describe an unbiased approach based on the differential single-cell gene expression analysis of mesenchymal osteolineage cells close to, and further removed from, hematopoietic stem/progenitor cells (HSPCs) to identify candidate niche factors. Mesenchymal cells displayed distinct molecular profiles based on their relative location. We functionally examined, among the genes that were preferentially expressed in proximal cells, three secreted or cell-surface molecules not previously connected to HSPC biology-the secreted RNase angiogenin, the cytokine IL18, and the adhesion molecule Embigin-and discovered that all of these factors are HSPC quiescence regulators. Therefore, our proximity-based differential single-cell approach reveals molecular heterogeneity within niche cells and can be used to identify novel extrinsic stem/progenitor cell regulators. Similar approaches could also be applied to other stem cell/niche pairs to advance the understanding of microenvironmental regulation of stem cell function.
Asunto(s)
Células Madre Hematopoyéticas/citología , Análisis de la Célula Individual/métodos , Nicho de Células Madre , Animales , Células de la Médula Ósea/citología , Huesos/citología , Linaje de la Célula/genética , Autorrenovación de las Células/genética , Separación Celular , Eliminación de Gen , Perfilación de la Expresión Génica , Células Madre Hematopoyéticas/metabolismo , Interleucina-18/metabolismo , Glicoproteínas de Membrana/metabolismo , Ribonucleasa Pancreática/metabolismo , Factores de Tiempo , Transcripción Genética , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Hematopoietic stem cell transplantation (HSCT) offers curative therapy for patients with hemoglobinopathies, congenital immunodeficiencies, and other conditions, possibly including AIDS. Autologous HSCT using genetically corrected cells would avoid the risk of graft-versus-host disease (GVHD), but the genotoxicity of conditioning remains a substantial barrier to the development of this approach. Here we report an internalizing immunotoxin targeting the hematopoietic-cell-restricted CD45 receptor that effectively conditions immunocompetent mice. A single dose of the immunotoxin, CD45-saporin (SAP), enabled efficient (>90%) engraftment of donor cells and full correction of a sickle-cell anemia model. In contrast to irradiation, CD45-SAP completely avoided neutropenia and anemia, spared bone marrow and thymic niches, enabling rapid recovery of T and B cells, preserved anti-fungal immunity, and had minimal overall toxicity. This non-genotoxic conditioning method may provide an attractive alternative to current conditioning regimens for HSCT in the treatment of non-malignant blood diseases.
Asunto(s)
Daño del ADN/inmunología , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas/inmunología , Antígenos Comunes de Leucocito/inmunología , Proteínas Inactivadoras de Ribosomas Tipo 1/genética , Proteínas Inactivadoras de Ribosomas Tipo 1/inmunología , Animales , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Células Cultivadas , Daño del ADN/genética , Femenino , Mejoramiento Genético/métodos , Fenómenos Inmunogenéticos/genética , Inmunotoxinas , Ratones , Ratones Endogámicos C57BL , SaporinasRESUMEN
The laboratory mouse is the model most frequently used in hematologic studies and assessment of blood parameters across a broad range of disciplines. Often, analysis of blood occurs in a nonterminal manner. However, the small body size of the mouse limits collection based on volume, frequency, and accessible sites. Commonly used sites in the mouse include the retro-orbital sinus, facial vein, tail vein, saphenous vein, and heart. The method of blood acquisition varies considerably across laboratories and is often not reported in detail. In this study, we report significant alterations in blood parameters, particularly of total white blood cells, specific populations of dendritic cells and myeloid-derived suppressor cells, and hematopoietic progenitor cells, as a result of site and manner of sampling. Intriguingly, warming of mice prior to tail bleeding was found to significantly alter blood values. Our findings suggest that the same method should be used across an entire study, that mice should be warmed prior to tail bleeds to make levels uniform, and that accurate description of bleeding methods in publications should be provided to allow for interpretation of comparative reports and inter- and intralaboratory experimental variability.
Asunto(s)
Animales de Laboratorio , Sangre , Manejo de Especímenes , Animales , RatonesRESUMEN
Chemotherapy, irradiation, and other agents are widely used to target the process of cell division in neoplastic cells. However, while these therapies are effective against most cancers, the high proliferative rate of the cells of the hematopoietic system that produce billions of blood cells needed daily throughout life is extremely sensitive to these agents, resulting in loss of blood cell populations, which can be life threatening. Neutropenia is the most serious hematologic toxicity of chemotherapy, which can result in patient morbidity and mortality due to opportunistic infection and often is the limiting factor in dose escalation or duration of chemotherapeutic administration. Neutropenic patients often require hospitalization and incur substantial medical costs associated with anti-infective therapy. Treatment of iatrogenic and congenic neutropenia was changed in the early 1990s with the introduction of filgrastim (Neupogen(®)) and pegfilgrastim (Neulasta(®)). With the expiration of patent lives of both of these drugs, biosimilars have begun to emerge. In this review, we will summarize the chemical characteristics, pharmacokinetics, safety and efficacy of lipegfilgrastim (Lonquex(®)), the first long-acting biosimilar filgrastim to receive regulatory approval and enter the marketplace.
Asunto(s)
Neutropenia Febril Inducida por Quimioterapia/tratamiento farmacológico , Factor Estimulante de Colonias de Granulocitos , Filgrastim , Factor Estimulante de Colonias de Granulocitos/efectos adversos , Factor Estimulante de Colonias de Granulocitos/química , Factor Estimulante de Colonias de Granulocitos/farmacocinética , Factor Estimulante de Colonias de Granulocitos/uso terapéutico , Humanos , Polietilenglicoles , Proteínas Recombinantes/efectos adversos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/uso terapéuticoRESUMEN
Hematopoietic stem cell transplantation (HSCT) can be performed with hematopoietic stem and progenitor cells (HSPC) acquired directly from bone marrow, from umbilical cord blood or placental tissue, or from the peripheral blood after treatment of the donor with agents that enhance egress of HSPC into the circulation, a process known as "mobilization." Mobilized peripheral blood stem cells (PBSC) have become the predominate hematopoietic graft for HSCT, particularly for autologous transplants. Despite the success of PBSC transplant, many patients and donors do not achieve optimal levels of mobilization. Thus, accurate animal models and basic laboratory investigations are needed to further investigate the mechanisms that lead to PBSC mobilization and define improved or new mobilizing agents and/or strategies to enhance PBSC mobilization and transplant. This chapter outlines assays and techniques for exploration of hematopoietic mobilization using mice as a model organism.
Asunto(s)
Movilización de Célula Madre Hematopoyética/métodos , Células Madre Hematopoyéticas/citología , Animales , Recuento de Células Sanguíneas , Vías de Administración de Medicamentos , Eritrocitos/citología , Femenino , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Factor Estimulante de Colonias de Granulocitos/farmacología , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/efectos de los fármacos , Cinética , Leucocitos Mononucleares/citología , Masculino , RatonesRESUMEN
More than 36,000 Americans die and 200,000 more are hospitalized because of influenza every\year. Despite the wide availability of a vaccine to prevent influenza, the vast majority of Americans go unimmunized. The Maryland Department of Health and Mental Hygiene (DHMH) and the Maryland Partnership for Prevention (MPP) collect data about the state's local health departments' influenza season practices and experiences and compile them into the annual Maryland Influenza Season Final Report. The report becomes a tool for DHMH, MPP, and the state's 24 local health departments to plan improvements in influenza vaccination services. This article chronicles four influenza seasons. Influenza season challenges experienced in three of the last four influenza seasons underscore the importance of coordination and communication efforts to ensure that vaccine is efficiently delivered to the most vulnerable populations. The partnership between DHMH and MPP has facilitated access to information on ordering and administration practices, communication systems, community partnerships, and lessons learned, thus enabling the state of Maryland to continually optimize its influenza vaccination promotion efforts.
Asunto(s)
Comunicación , Servicios de Salud Comunitaria/organización & administración , Programas de Gobierno/organización & administración , Programas de Inmunización/organización & administración , Vacunas contra la Influenza , Gripe Humana/prevención & control , Programas de Gobierno/estadística & datos numéricos , Humanos , Programas de Inmunización/estadística & datos numéricos , Gripe Humana/epidemiología , Gripe Humana/mortalidad , Maryland , Estaciones del AñoRESUMEN
In February 1999, the Maryland Department of Health and Mental Hygiene initiated pandemic influenza planning for the state of Maryland. This process involved several major steps, including the development of the Maryland Pandemic Influenza Preparedness Plan, and culminated in a high-level tabletop exercise to test the plan in April 2004. During the tabletop exercise, participants were presented with nine different fictitious scripts encompassing a single scenario. They were asked to respond to the information presented in each script, discuss organization-specific questions posed by the exercise facilitator, and make decisions regarding action steps that their organization would take in response to the various issues raised. The exercise identified a number of important gaps that need to be addressed, including (1) additional surge capacity specific to a pandemic, (2) greater understanding of the realities and implications of pandemic influenza among elected officials and decision-makers, (3) coordination of pandemic influenza planning with the existing emergency response infrastructure coupled with additional training in incident command, (4) further steps to operationalize several aspects of the Maryland Pandemic Influenza Preparedness Plan, and (5) additional federal guidance.