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Methods Mol Biol ; 1869: 69-77, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30324514


As a useful biotechnology, flow cytometry has revolutionized the field of cell analysis through its dynamic system that employs fluidics, optics, and electronics. It was first used to analyze DNA, but is often used to determine biomarker expression, as well as to characterize and sort cells, in accordance with various parameters. A common application of flow cytometry is the identification and isolation of a distinct cancer cell population, known as cancer stem cells (CSCs). Various biomarkers have been used to elucidate this proportion of cells within the brain, termed brain tumor initiating cells (BTICs). Here, we discuss methodology to prepare BTICs for flow cytometric analysis that includes the expression of markers.

Neoplasias Encefálicas/patología , Citometría de Flujo/métodos , Células Madre Neoplásicas/patología , Adhesión Celular , Línea Celular Tumoral , Supervivencia Celular , Colorantes Fluorescentes/metabolismo , Humanos , Coloración y Etiquetado
Methods Mol Biol ; 1869: 85-91, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30324516


Differentiation is a central key capability of stem cells. Their ability to be multipotent and undergo self-renewal are key identifying features of stem cells. A differentiation assay allows for study of one of the essential features of stem cells, the ability to differentiate into all of the cell types of its lineage, in order to ensure that the cells cultured and utilized in key experiments indeed have stem cell properties. Neural stem cells when plated in differentiation media, differentiate into all three neural lineages: Neurons, Astrocytes, and Oligodendrocytes. Brain tumor initiating cells (BTICs) are cells present in brain tumors that possess stem cell properties and are able to self-renew and differentiate into neural lineages. In the current chapter, we discuss protocols involved in immunofluorescence staining and identification of differentiated cells from BTIC populations.

Neoplasias Encefálicas/patología , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular , Células Madre Neoplásicas/patología , Permeabilidad de la Membrana Celular , Citometría de Flujo , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Células-Madre Neurales/metabolismo
Cancer Res ; 78(17): 5124-5134, 2018 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-29986997


Brain metastases (BM) result from the spread of primary tumors to the brain and are a leading cause of cancer mortality in adults. Secondary tissue colonization remains the main bottleneck in metastatic development, yet this "premetastatic" stage of the metastatic cascade, when primary tumor cells cross the blood-brain barrier and seed the brain before initiating a secondary tumor, remains poorly characterized. Current studies rely on specimens from fully developed macrometastases to identify therapeutic options in cancer treatment, overlooking the potentially more treatable "premetastatic" phase when colonizing cancer cells could be targeted before they initiate the secondary brain tumor. Here we use our established brain metastasis initiating cell (BMIC) models and gene expression analyses to characterize premetastasis in human lung-to-BM. Premetastatic BMIC engaged invasive and epithelial developmental mechanisms while simultaneously impeding proliferation and apoptosis. We identified the dopamine agonist apomorphine to be a potential premetastasis-targeting drug. In vivo treatment with apomorphine prevented BM formation, potentially by targeting premetastasis-associated genes KIF16B, SEPW1, and TESK2 Low expression of these genes was associated with poor survival of patients with lung adenocarcinoma. These results illuminate the cellular and molecular dynamics of premetastasis, which is subclinical and currently impossible to identify or interrogate in human patients with BM. These data present several novel therapeutic targets and associated pathways to prevent BM initiation.Significance: These findings unveil molecular features of the premetastatic stage of lung-to-brain metastases and offer a potential therapeutic strategy to prevent brain metastases. Cancer Res; 78(17); 5124-34. ©2018 AACR.

Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Terapia Molecular Dirigida , Metástasis de la Neoplasia/tratamiento farmacológico , Apomorfina/farmacología , Apoptosis/efectos de los fármacos , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/secundario , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dopamina/metabolismo , Agonistas de Dopamina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Cinesina/genética , Pulmón/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Proteínas Serina-Treonina Quinasas/genética , Selenoproteína W/genética
Cancer Res ; 78(17): 5023-5037, 2018 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-29945963


Glioblastoma (GBM) carries a dismal prognosis and inevitably relapses despite aggressive therapy. Many members of the Eph receptor tyrosine kinase (EphR) family are expressed by GBM stem cells (GSC), which have been implicated in resistance to GBM therapy. In this study, we identify several EphRs that mark a therapeutically targetable GSC population in treatment-refractory, recurrent GBM (rGBM). Using a highly specific EphR antibody panel and CyTOF (cytometry by time-of-flight), we characterized the expression of all 14 EphR in primary and recurrent patient-derived GSCs to identify putative rGBM-specific EphR. EPHA2 and EPHA3 coexpression marked a highly tumorigenic cell population in rGBM that was enriched in GSC marker expression. Knockdown of EPHA2 and EPHA3 together led to increased expression of differentiation marker GFAP and blocked clonogenic and tumorigenic potential, promoting significantly higher survival in vivo Treatment of rGBM with a bispecific antibody against EPHA2/A3 reduced clonogenicity in vitro and tumorigenic potential of xenografted recurrent GBM in vivo via downregulation of AKT and ERK and increased cellular differentiation. In conclusion, we show that EPHA2 and EPHA3 together mark a GSC population in rGBM and that strategic cotargeting of EPHA2 and EPHA3 presents a novel and rational therapeutic approach for rGBM.Significance: Treatment of rGBM with a novel bispecific antibody against EPHA2 and EPHA3 reduces tumor burden, paving the way for the development of therapeutic approaches against biologically relevant targets in rGBM. Cancer Res; 78(17); 5023-37. ©2018 AACR.

Efrina-A2/genética , Glioblastoma/genética , Recurrencia Local de Neoplasia/genética , Proteínas Tirosina Quinasas Receptoras/genética , Animales , Biomarcadores de Tumor/genética , Carcinogénesis/genética , Diferenciación Celular/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos/genética , Efrina-A2/antagonistas & inhibidores , Regulación Neoplásica de la Expresión Génica/genética , Técnicas de Silenciamiento del Gen , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Glioblastoma/radioterapia , Humanos , Ratones , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/radioterapia , Células Madre Neoplásicas/patología , Pronóstico , Radiación , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptores de la Familia Eph/antagonistas & inhibidores , Receptores de la Familia Eph/genética , Temozolomida/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto
J Neurooncol ; 126(1): 57-67, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26498281


Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults with average disease relapse at 9 months and median survival rarely extending beyond 15 months. Brain tumor stem cells (BTSCs) have been implicated in not only initiating GBM but also conferring resistance to therapy. However, it is not clear whether the BTSC population that initiates tumor growth is also responsible for GBM recurrence. In this study, we have developed a novel in vitro treatment model to profile the evolution of primary treatment-naïve GBM BTSCs through chemoradiotherapy. We report that our in vitro model enriched for a CD15+/CD133- BTSC population, mirroring the phenotype of BTSCs in recurrent GBM. We also show that in vitro treatment increased stem cell gene expression as well as self-renewal capacity of primary GBMs. In addition, the chemoradiotherapy-refractory gene signature obtained from gene expression profiling identified a hyper-aggressive subtype of glioma. The delivery of in vitro chemoradiotherapy to primary GBM BTSCs models several aspects of recurrent GBM biology, and could be used as a discovery and drug-screening platform to uncover new biological drivers and therapeutic targets in GBM.

Neoplasias Encefálicas/patología , Regulación Neoplásica de la Expresión Génica/fisiología , Glioblastoma/patología , Células Madre Neoplásicas/patología , Anciano , Anciano de 80 o más Años , Análisis de Varianza , Antígenos CD/metabolismo , Antinematodos/farmacología , Antineoplásicos/farmacología , Autorrenovación de las Células/fisiología , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , Masculino , Persona de Mediana Edad , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/efectos de la radiación , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Células Tumorales Cultivadas