RESUMEN
Polycystic kidney disease (PKD) is an inherited disorder that results in large kidneys, numerous fluid-filled cysts, and ultimately end-stage kidney disease. PKD is either autosomal dominant caused by mutations in PKD1 or PKD2 genes or autosomal recessive caused by mutations in the PKHD1 or DZIP1L genes. While the genetic basis of PKD is known, the downstream molecular mechanisms and signaling pathways that lead to deregulation of proliferation, apoptosis, and differentiation are not completely understood. The Notch pathway plays critical roles during kidney development including directing differentiation of various progenitor cells, and aberrant Notch signaling results in gross alternations in cell fate. In the present study, we generated and studied transgenic mice that have overexpression of an intracellular fragment of mouse Notch1 ('NotchIC') in renin-expressing cells. Mice with overexpression of NotchIC in renin-expressing cells developed numerous fluid-filled cysts, enlarged kidneys, anemia, renal insufficiency, and early death. Cysts developed in both glomeruli and proximal tubules, had increased proliferation marks, and had increased levels of Myc. The present work implicates the Notch signaling pathway as a central player in PKD pathogenesis and suggests that the Notch-Myc axis may be an important target for therapeutic intervention.
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Riñón Poliquístico Autosómico Dominante , Riñón Poliquístico Autosómico Recesivo , Ratones , Animales , Renina/genética , Transducción de Señal , Fenotipo , Ratones Transgénicos , Riñón Poliquístico Autosómico Dominante/genética , Riñón/patología , Canales Catiónicos TRPP/genética , Receptores de Superficie Celular/genéticaRESUMEN
[Figure: see text].
Asunto(s)
Coartación Aórtica/metabolismo , Presión Arterial , Núcleo Celular/metabolismo , Células Endocrinas/metabolismo , Riñón/metabolismo , Mecanotransducción Celular , Presorreceptores/metabolismo , Renina/metabolismo , Animales , Coartación Aórtica/genética , Coartación Aórtica/patología , Coartación Aórtica/fisiopatología , Línea Celular , Núcleo Celular/genética , Núcleo Celular/patología , Ensamble y Desensamble de Cromatina , Modelos Animales de Enfermedad , Células Endocrinas/patología , Femenino , Homeostasis , Integrina beta1/genética , Integrina beta1/metabolismo , Riñón/patología , Riñón/fisiopatología , Lamina Tipo A/genética , Lamina Tipo A/metabolismo , Masculino , Ratones Noqueados , Presorreceptores/fisiopatología , Renina/genética , Estrés MecánicoRESUMEN
Secondary hemophagocytic lymphohistiocytosis (sHLH) is a rare hyperinflammatory disorder caused by an aberrant immune response to a number of infectious or inflammatory conditions. Successful treatment of this potentially fatal condition requires early recognition and prompt therapy directed at the underlying trigger. In this report, we describe the clinical presentation, diagnostic findings, management, and outcome of a child with Lemierre's syndrome-associated sHLH. This is the first reported association of these 2 rare conditions and expands the number of known triggers for sHLH.
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Síndrome de Lemierre/complicaciones , Linfohistiocitosis Hemofagocítica/etiología , Adolescente , Diagnóstico Diferencial , Manejo de la Enfermedad , Femenino , Humanos , Linfohistiocitosis Hemofagocítica/diagnóstico , Linfohistiocitosis Hemofagocítica/terapia , Resultado del TratamientoRESUMEN
Renin-expressing cells appear early in the embryo and are distributed broadly throughout the body as organogenesis ensues. Their appearance in the metanephric kidney is a relatively late event in comparison with other organs such as the fetal adrenal gland. The functions of renin cells in extra renal tissues remain to be investigated. In the kidney, they participate locally in the assembly and branching of the renal arterial tree and later in the endocrine control of blood pressure and fluid-electrolyte homeostasis. Interestingly, this endocrine function is accomplished by the remarkable plasticity of renin cell descendants along the kidney arterioles and glomeruli which are capable of reacquiring the renin phenotype in response to physiological demands, increasing circulating renin and maintaining homeostasis. Given that renin cells are sensors of the status of the extracellular fluid and perfusion pressure, several signaling mechanisms (ß-adrenergic receptors, Notch pathway, gap junctions and the renal baroreceptor) must be coordinated to ensure the maintenance of renin phenotype--and ultimately the availability of renin--during basal conditions and in response to homeostatic threats. Notably, key transcriptional (Creb/CBP/p300, RBP-J) and posttranscriptional (miR-330, miR125b-5p) effectors of those signaling pathways are prominent in the regulation of renin cell identity. The next challenge, it seems, would be to understand how those factors coordinate their efforts to control the endocrine and contractile phenotypes of the myoepithelioid granulated renin-expressing cell.
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Riñón/embriología , Riñón/metabolismo , Organogénesis/fisiología , Renina/metabolismo , Células Madre/metabolismo , Animales , HumanosRESUMEN
AIM: Ureteral obstruction leads to significant changes in kidney renin expression. It is unclear whether those changes are responsible for the progression of kidney damage, repair, or regeneration. In the current study, we aimed to elucidate the contribution of renin-producing cells (RPCs) and the cells of the renin lineage (CoRL) towards kidney damage and regeneration using a model of partial and reversible unilateral ureteral obstruction (pUUO) in neonatal mice. METHODS: Renin cells are progenitors for other renal cell types collectively called CoRL. We labeled the CoRL with green fluorescent protein (GFP) using genetic approaches. We performed lineage tracing to analyze the changes in the distribution of CoRL during and after the release of obstruction. We also ablated the RPCs and CoRL by cell-specific expression of Diphtheria Toxin Sub-unit A (DTA). Finally, we evaluated the kidney damage and regeneration during and after the release of obstruction in the absence of CoRL. RESULTS: In the obstructed kidneys, there was a 163% increase in the renin-positive area and a remarkable increase in the distribution of GFP+ CoRL. Relief of obstruction abrogated these changes. In addition, DTA-expressing animals did not respond to pUUO with increased RPCs and CoRL. Moreover, reduction in CoRL significantly compromised the kidney's ability to recover from the damage after the release of obstruction. CONCLUSIONS: CoRL play a role in the regeneration of the kidneys post-relief of obstruction.
Asunto(s)
Riñón , Obstrucción Ureteral , Ratones , Animales , Riñón/metabolismo , Renina/metabolismo , Animales Recién Nacidos , Obstrucción Ureteral/metabolismo , Ratones Transgénicos , RegeneraciónRESUMEN
Pediatric patients with relapsed or refractory rhabdomyosarcoma (RMS) have dismal cure rates, and effective therapy is urgently needed. The oncogenic receptor tyrosine kinase fibroblast growth factor receptor 4 (FGFR4) is highly expressed in RMS and lowly expressed in healthy tissues. Here, we describe a second-generation FGFR4-targeting chimeric antigen receptor (CAR), based on an anti-human FGFR4-specific murine monoclonal antibody 3A11, as an adoptive T cell treatment for RMS. The 3A11 CAR T cells induced robust cytokine production and cytotoxicity against RMS cell lines in vitro. In contrast, a panel of healthy human primary cells failed to activate 3A11 CAR T cells, confirming the selectivity of 3A11 CAR T cells against tumors with high FGFR4 expression. Finally, we demonstrate that 3A11 CAR T cells are persistent in vivo and can effectively eliminate RMS tumors in two metastatic and two orthotopic models. Therefore, our study credentials CAR T cell therapy targeting FGFR4 to treat patients with RMS.
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Receptores Quiméricos de Antígenos , Rabdomiosarcoma , Animales , Niño , Humanos , Ratones , Línea Celular Tumoral , Inmunoterapia Adoptiva , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 4 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptores Quiméricos de Antígenos/genética , Rabdomiosarcoma/tratamiento farmacológicoRESUMEN
Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood and adolescence, accounting for approximately 7% of childhood cancers. Current therapies include nonspecific cytotoxic chemotherapy regimens, radiation therapy, and surgery; however, these multimodality strategies are unsuccessful in the majority of patients with high-risk disease. It is generally believed that these tumors represent arrested or aberrant skeletal muscle development, and, accordingly, developmental signaling pathways critical to myogenesis such as Notch, WNT, and Hedgehog may represent new therapeutic targets. In this paper, we summarize the current preclinical studies linking these embryonic pathways to rhabdomyosarcoma tumorigenesis and provide support for the investigation of targeted therapies in this embryonic cancer.
RESUMEN
The hormone renin plays a crucial role in the regulation of blood pressure and fluid-electrolyte homeostasis. Normally, renin is synthesized by juxtaglomerular (JG) cells, a specialized group of myoepithelial cells located near the entrance to the kidney glomeruli. In response to low blood pressure and/or a decrease in extracellular fluid volume (as it occurs during dehydration, hypotension, or septic shock) JG cells respond by releasing renin to the circulation to reestablish homeostasis. Interestingly, renin-expressing cells also exist outside of the kidney, where their function has remained a mystery. We discovered a unique type of renin-expressing B-1 lymphocyte that may have unrecognized roles in defending the organism against infections. These cells synthesize renin, entrap and phagocyte bacteria and control bacterial growth. The ability of renin-bearing lymphocytes to control infections-which is enhanced by the presence of renin-adds a novel, previously unsuspected dimension to the defense role of renin-expressing cells, linking the endocrine control of circulatory homeostasis with the immune control of infections to ensure survival.
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Bacterias/inmunología , Infecciones Bacterianas/inmunología , Diferenciación Celular/inmunología , Regulación Enzimológica de la Expresión Génica/inmunología , Linfocitos/inmunología , Renina/inmunología , Animales , Ratones , Ratones Transgénicos , Renina/genéticaRESUMEN
Secondary hemophagocytic lymphohistiocytosis (sHLH) is a reactive, proliferative disorder of the immune system resulting in lymphohistiocytic proliferation, hemophagocytosis, and cytokine dysregulation. The most common infectious trigger in sHLH is Epstein-Barr virus (EBV-HLH). Current treatment protocols for EBV-HLH have a cure rate of approximately 75%; however, there are significant toxicities associated with these therapies. We present two patients with EBV-HLH who experienced spontaneous resolution of their disease prior to the initiation of therapy, suggesting there may be a subgroup of patients with EBV-HLH who do well with conservative management and can avoid potentially toxic therapies.
Asunto(s)
Infecciones por Virus de Epstein-Barr/complicaciones , Linfohistiocitosis Hemofagocítica/diagnóstico , Adolescente , Adulto , Femenino , Humanos , MasculinoRESUMEN
Juxtaglomerular cells are crucial for blood pressure and fluid-electrolyte homeostasis. The factors that maintain the life of renin cells are unknown. In vivo, renin cells receive constant cell-to-cell, mechanical, and neurohumoral stimulation that maintain their identity and function. Whether the presence of this niche is crucial for the vitality of the juxtaglomerular cells is unknown. Integrins are the largest family of cell adhesion molecules that mediate cell-to-cell and cell-to-matrix interactions. Of those, ß1-integrin is the most abundant in juxtaglomerular cells. However, its role in renin cell identity and function has not been ascertained. To test the hypothesis that cell-matrix interactions are fundamental not only to maintain the identity and function of juxtaglomerular cells but also to keep them alive, we deleted ß1-integrin in vivo in cells of the renin lineage. In mutant mice, renin cells died by apoptosis, resulting in decreased circulating renin, hypotension, severe renal-vascular abnormalities, and renal failure. Results indicate that cell-to-cell and cell-to-matrix interactions via ß1-integrin is essential for juxtaglomerular cells survival, suggesting that the juxtaglomerular niche is crucial not only for the tight regulation of renin release but also for juxtaglomerular cell survival-a sine qua non condition to maintain homeostasis.
Asunto(s)
Integrina beta1/metabolismo , Aparato Yuxtaglomerular/metabolismo , Enfermedades Renales/metabolismo , Arteria Renal/metabolismo , Renina/metabolismo , Animales , Apoptosis/fisiología , Supervivencia Celular/fisiología , Homeostasis/fisiología , Integrina beta1/genética , Aparato Yuxtaglomerular/citología , Enfermedades Renales/genética , Ratones , Ratones NoqueadosRESUMEN
A 16-year-old girl presented to the emergency department with intermittent fevers and worsening abdominal pain of 5 weeks duration. She had a history of travel to a less developed country and exposure to possible infectious diseases. Abdominal imaging and blood tests revealed diffuse mesenteric lymphadenopathy, elevated transaminases, and elevation of inflammatory markers. Gastroesophageal and colon endoscopies revealed gastric ulcers, and the patient was discharged with a presumptive diagnosis of systemic juvenile idiopathic arthritis given the lymphadenopathy seen on imaging, serositis, sacroiliac joint stiffness noted on physical examination, and pain relief with celecoxib. She presented again 4 days later with worsening abdominal tenderness, elevated transaminases, and new-onset abdominal distention. Tissue biopsy yielded the diagnosis and directed appropriate treatment.
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Dolor Abdominal/etiología , Fiebre/etiología , Linfoma Anaplásico de Células Grandes/diagnóstico , Adolescente , Animales , Antiinflamatorios no Esteroideos/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Artritis Juvenil/diagnóstico , Biomarcadores/sangre , Biopsia , Diagnóstico Diferencial , Servicio de Urgencia en Hospital , Endoscopía Gastrointestinal , Enterobiasis/diagnóstico , Femenino , Humanos , Inflamación/diagnóstico , Linfadenopatía/diagnóstico , Linfoma Anaplásico de Células Grandes/diagnóstico por imagen , Linfoma Anaplásico de Células Grandes/tratamiento farmacológico , Linfoma Anaplásico de Células Grandes/patología , Tomografía Computarizada por Tomografía de Emisión de Positrones , Úlcera Gástrica/inducido químicamente , Úlcera Gástrica/diagnóstico , Tomografía Computarizada por Rayos X , Transaminasas/sangreRESUMEN
Conditional deletion of RBP-J, the major transcriptional effector of Notch signaling, specifically within renin-expressing cells leads to the development of B-cell leukemia. However, the influence of contributing factors such as mouse strain, cell of origin and Cre recombinase copy number are unknown. In this study, we compared RBP-J deletion efficiency using one versus two copies of Cre recombinase. Further, we compared the incidence and timing of leukemia development in two unique strains of mice, C57BL/6 and 129/SV, as well as at different B-cell developmental stages. We found that animals expressing two copies of Cre recombinase developed B-cell leukemia at an earlier age and with more fulminant disease, compared with control animals and animals expressing one copy of Cre recombinase. In addition, we found a difference in leukemia incidence between C57BL/6 and 129/SV mouse strains. Whereas deletion of RBP-J in renin-expressing cells of C57BL/6 mice leads to the development of B-cell leukemia, 129/SV mice develop dermatitis with a reactive, myeloproliferative phenotype. The difference in phenotypes is explained, in part, by the differential expression of extra-renal renin; C57BL/6 mice have more renin-expressing cells within hematopoietic tissues. Finally, we found that deletion of RBP-J in Mb1- or CD19-expressing B lymphocytes does not result in leukemia development. Together, these studies establish that renin progenitors are vulnerable cells for neoplastic transformation and emphasize the importance of genetic background on the development of inflammatory and malignant conditions.This article has an associated First Person interview with the first author of the paper.
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Carcinogénesis/genética , Eliminación de Gen , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Leucemia/genética , Animales , Linfocitos B/patología , Linaje de la Célula , Transformación Celular Neoplásica/patología , Dermatitis/patología , Dosificación de Gen , Hematopoyesis , Integrasas/metabolismo , Leucemia/patología , Ratones Endogámicos C57BL , Fenotipo , Renina/metabolismo , Piel/patología , Células Madre/metabolismoRESUMEN
Renin cells are crucial for survival - they control fluid-electrolyte and blood pressure homeostasis, vascular development, regeneration, and oxygen delivery to tissues. During embryonic development, renin cells are progenitors for multiple cell types that retain the memory of the renin phenotype. When there is a threat to survival, those descendants are transformed and reenact the renin phenotype to restore homeostasis. We tested the hypothesis that the molecular memory of the renin phenotype resides in unique regions and states of these cells' chromatin. Using renin cells at various stages of stimulation, we identified regions in the genome where the chromatin is open for transcription, mapped histone modifications characteristic of active enhancers such as H3K27ac, and tracked deposition of transcriptional activators such as Med1, whose deletion results in ablation of renin expression and low blood pressure. Using the rank ordering of super-enhancers, epigenetic rewriting, and enhancer deletion analysis, we found that renin cells harbor a unique set of super-enhancers that determine their identity. The most prominent renin super-enhancer may act as a chromatin sensor of signals that convey the physiologic status of the organism, and is responsible for the transformation of renin cell descendants to the renin phenotype, a fundamental process to ensure homeostasis.
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Epigénesis Genética , Código de Histonas , Histonas/metabolismo , Homeostasis , Subunidad 1 del Complejo Mediador/metabolismo , Renina/biosíntesis , Células Madre/metabolismo , Animales , Histonas/genética , Subunidad 1 del Complejo Mediador/genética , Ratones , Ratones Transgénicos , Renina/genética , Células Madre/citologíaRESUMEN
Rhabdomyosarcoma (RMS), a cancer characterized by skeletal muscle features, is the most common soft-tissue sarcoma of childhood. While low- and intermediate-risk groups have seen improved outcomes, high-risk patients still face a 5-year survival rate of <30%, a statistic that has not changed in over 40 years. Understanding the biologic underpinnings of RMS is critical. The developmental pathways of Notch and YAP have been identified as potent but independent oncogenic signals that support the embryonal variant of RMS (eRMS). Here, the cross-talk between these pathways and the impact on eRMS tumorigenesis is reported. Using human eRMS cells grown as three-dimensional (3D) rhabdospheres, which enriches in stem cells, it was found that Notch signaling transcriptionally upregulates YAP1 gene expression and YAP activity. Reciprocally, YAP transcriptionally upregulates the Notch ligand genes JAG1 and DLL1 and the core Notch transcription factor RBPJ This bidirectional circuit boosts expression of key stem cell genes, including SOX2, which is functionally required for eRMS spheres. Silencing this circuit for therapeutic purposes may be challenging, because the inhibition of one node (e.g., pharmacologic Notch blockade) can be rescued by upregulation of another (constitutive YAP expression). Instead, dual inhibition of Notch and YAP is necessary. Finally, supporting the existence of this circuit beyond a model system, nuclear Notch and YAP protein expression are correlated in human eRMS tumors, and YAP suppression in vivo decreases Notch signaling and SOX2 expression.Implications: This study identifies a novel oncogenic signaling circuit driving eRMS stemness and tumorigenesis, and provides evidence and rationale for combination therapies co-targeting Notch and YAP. Mol Cancer Res; 15(12); 1777-91. ©2017 AACR.
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Carcinogénesis/genética , Proteínas Nucleares/genética , Receptores Notch/genética , Rabdomiosarcoma Embrionario/genética , Factores de Transcripción/genética , Proteínas de Unión al Calcio , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proliferación Celular/genética , Regulación Neoplásica de la Expresión Génica , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Proteína Jagged-1/genética , Proteínas de la Membrana/genética , Células Madre Neoplásicas/patología , Rabdomiosarcoma Embrionario/tratamiento farmacológico , Rabdomiosarcoma Embrionario/patología , Factores de Transcripción SOXB1/genética , Transducción de Señal/genéticaRESUMEN
The cardiac endothelium plays a crucial role in the development of a functional heart. However, the precise identification of the endocardial precursors and the mechanisms they require for their role in heart morphogenesis are not well understood. Using in vivo and in vitro cell fate tracing concomitant with specific cell ablation and embryonic heart transplantation studies, we identified a unique set of precursors which possess hemogenic functions and express the stem cell leukemia (SCL) gene driven by its 5' enhancer. These hemo-vascular precursors give rise to the endocardium, atrioventricular cushions and coronary vascular endothelium. Furthermore, deletion of the sphingosine-1-phosphate receptor 1 (S1P1) in these precursors leads to ventricular non-compaction cardiomyopathy, a poorly understood condition leading to heart failure and early mortality. Thus, we identified a distinctive population of hemo-vascular precursors which require S1P1 to exert their functions and are essential for cardiac morphogenesis.
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Cardiomiopatía Dilatada/genética , Células Madre Embrionarias/metabolismo , Endotelio Vascular/citología , Corazón/embriología , No Compactación Aislada del Miocardio Ventricular/genética , Miocardio/citología , Receptores de Lisoesfingolípidos/metabolismo , Animales , Células Madre Embrionarias/citología , Endotelio Vascular/embriología , Endotelio Vascular/metabolismo , Elementos de Facilitación Genéticos , Ratones , Ratones Endogámicos C57BL , Morfogénesis , Miocardio/metabolismo , Receptores de Lisoesfingolípidos/genética , Proteína 1 de la Leucemia Linfocítica T Aguda/genética , Proteína 1 de la Leucemia Linfocítica T Aguda/metabolismoRESUMEN
The Notch signaling pathway is required to maintain renin expression within juxtaglomerular (JG) cells. However, the specific ligand which activates Notch signaling in renin-expressing cells remains undefined. In this study, we found that among all Notch ligands, Jagged1 is differentially expressed in renin cells with higher expression during neonatal life. We therefore hypothesized that Jagged1 was involved in renin expression and/or vascular integrity. We used a conditional knockout approach to delete Jagged1 in cells of the renin lineage. Deletion of Jagged1 specifically within renin cells did not result in decreased renin production within the kidney. However, animals with conditional deletion of Jagged1 did develop focal kidney fibrosis and elevated blood urea nitrogen. Our data demonstrate that Jagged1-mediated Notch signaling is dispensable in renin cells of the kidney in regard to renin expression. However, deletion of Jagged1 in renin cells descendants affects perivascular-interstitial integrity leading to focal fibrosis and diminished renal function.
RESUMEN
The cell of origin and triggering events for leukaemia are mostly unknown. Here we show that the bone marrow contains a progenitor that expresses renin throughout development and possesses a B-lymphocyte pedigree. This cell requires RBP-J to differentiate. Deletion of RBP-J in these renin-expressing progenitors enriches the precursor B-cell gene programme and constrains lymphocyte differentiation, facilitated by H3K4me3 activating marks in genes that control the pre-B stage. Mutant cells undergo neoplastic transformation, and mice develop a highly penetrant B-cell leukaemia with multi-organ infiltration and early death. These renin-expressing cells appear uniquely vulnerable as other conditional models of RBP-J deletion do not result in leukaemia. The discovery of these unique renin progenitors in the bone marrow and the model of leukaemia described herein may enhance our understanding of normal and neoplastic haematopoiesis.
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Células de la Médula Ósea/metabolismo , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Leucemia de Células B/etiología , Leucemia Experimental/etiología , Renina/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Médula Ósea/patología , Células de la Médula Ósea/patología , Epigénesis Genética , Femenino , Hematopoyesis , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Leucemia de Células B/metabolismo , Leucemia de Células B/patología , Leucemia Experimental/metabolismo , Leucemia Experimental/patología , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Bazo/patología , Adulto JovenRESUMEN
Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials conducted in Europe and North America, outcomes for high risk patients with this disease have not significantly improved in the last several decades, and survival of metastatic or relapsed disease remains extremely poor. Accrual into new clinical trials is slow and difficult, so in vitro cell-line research and in vivo xenograft models present an attractive alternative for preclinical research for this cancer type. Currently, 30 commonly used human RMS cell lines exist, with differing origins, karyotypes, histologies, and methods of validation. Selecting an appropriate cell line for RMS research has important implications for outcomes. There are also potential pitfalls in using certain cell lines including contamination with murine stromal cells, cross-contamination between cell lines, discordance between the cell line and its associated original tumor, imposter cell lines, and nomenclature errors that result in the circulation of two or more presumed unique cell lines that are actually from the same origin. These pitfalls can be avoided by testing for species-specific isoenzymes, microarray analysis, assays for subtype-specific fusion products, and short tandem repeat analysis.