RESUMO
The goal of high-throughput screening is to enable screening of compound libraries in an automated manner to identify quality starting points for optimization. This often involves screening a large diversity of compounds in an assay that preserves a connection to the disease pathology. Phenotypic screening is a powerful tool for drug identification, in that assays can be run without prior understanding of the target and with primary cells that closely mimic the therapeutic setting. Advanced automation and high-content imaging have enabled many complex assays, but these are still relatively slow and low throughput. To address this limitation, we have developed an automated workflow that is dedicated to processing complex phenotypic assays for flow cytometry. The system can achieve a throughput of 50,000 wells per day, resulting in a fully automated platform that enables robust phenotypic drug discovery. Over the past 5 years, this screening system has been used for a variety of drug discovery programs, across many disease areas, with many molecules advancing quickly into preclinical development and into the clinic. This report will highlight a diversity of approaches that automated flow cytometry has enabled for phenotypic drug discovery.
Assuntos
Descoberta de Drogas , Citometria de Fluxo , Ensaios de Triagem em Larga Escala , Automação Laboratorial , Plaquetas/efeitos dos fármacos , Linhagem Celular , Biologia Computacional/métodos , Análise de Dados , Descoberta de Drogas/instrumentação , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Ensaios de Triagem em Larga Escala/instrumentação , Ensaios de Triagem em Larga Escala/métodos , Humanos , Hibridomas , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismoRESUMO
Tumor progenitor cells represent a population of drug-resistant cells that can survive conventional chemotherapy and lead to tumor relapse. However, little is known of the role of tumor progenitors in prostate cancer metastasis. The studies reported herein show that the CXCR4/CXCL12 axis, a key regulator of tumor dissemination, plays a role in the maintenance of prostate cancer stem-like cells. The CXCL4/CXCR12 pathway is activated in the CD44(+)/CD133(+) prostate progenitor population and affects differentiation potential, cell adhesion, clonal growth and tumorigenicity. Furthermore, prostate tumor xenograft studies in mice showed that a combination of the CXCR4 receptor antagonist AMD3100, which targets prostate cancer stem-like cells, and the conventional chemotherapeutic drug Taxotere, which targets the bulk tumor, is significantly more effective in eradicating tumors as compared to monotherapy.
Assuntos
Células-Tronco Neoplásicas/metabolismo , Neoplasias da Próstata/metabolismo , Receptores CXCR4/metabolismo , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Benzilaminas , Adesão Celular , Proliferação de Células , Quimiocina CXCL12/metabolismo , Ciclamos , Docetaxel , Compostos Heterocíclicos/farmacologia , Humanos , Masculino , Camundongos , Metástase Neoplásica , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Taxoides/farmacologiaRESUMO
Glioblastoma multiforme (GBM) is a highly aggressive form of brain cancer associated with a very poor prognosis. Recently, the initiation and growth of GBM has been linked to brain tumor-initiating cells (BTICs), which are poorly differentiated and share features with neural stem cells (NSCs). Here we describe a kinome-wide RNA interference screen to identify factors that control the tumorigenicity of BTICs. We identified several genes whose silencing induces differentiation of BTICs derived from multiple GBM patients. In particular, knockdown of the adaptor protein TRRAP significantly increased differentiation of cultured BTICs, sensitized the cells to apoptotic stimuli, and negatively affected cell cycle progression. TRRAP knockdown also significantly suppressed tumor formation upon intracranial BTIC implantation into mice. Together, these findings support a critical role for TRRAP in maintaining a tumorigenic, stem cell-like state.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/análise , Neoplasias Encefálicas/química , Diferenciação Celular , Transformação Celular Neoplásica/química , Glioblastoma/química , Células-Tronco Neoplásicas/química , Proteínas Nucleares/análise , Interferência de RNA , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Apoptose , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Glioblastoma/patologia , Humanos , Camundongos , Camundongos SCID , Células-Tronco Neoplásicas/citologia , Proteínas Nucleares/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A series of peptidic fluorogenic substrates were synthesized to develop a flow cytometry assay (FACS) to monitor the proteolytic activity of cathepsin C in live cells. Of the 16 substrates tested, (NH(2)-aminobutyric-homophenylalanine)(2)-rhodamine demonstrated the best reactivity and selectivity profile in the FACS assay using the B721 human B-lymphoblastoid cell line. The resulting FACS assay was validated through correlation of the IC(50) values with a competitive radiolabeling assay against a series of small molecule inhibitors of cathepsin C.
Assuntos
Catepsina C/metabolismo , Corantes Fluorescentes/química , Rodaminas/química , Catepsina C/antagonistas & inibidores , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Concentração Inibidora 50 , Marcação por Isótopo , Radioisótopos/química , Rodaminas/síntese química , Especificidade por SubstratoRESUMO
Resistance of Bcr-Abl-positive leukemic stem cells (LSCs) to imatinib treatment in patients with chronic myeloid leukemia (CML) can cause relapse of disease and might be the origin for emerging drug-resistant clones. In this study, we identified Smo as a drug target in Bcr-Abl-positive LSCs. We show that Hedgehog signaling is activated in LSCs through upregulation of Smo. While Smo(-/-) does not impact long-term reconstitution of regular hematopoiesis, the development of retransplantable Bcr-Abl-positive leukemias was abolished in the absence of Smo expression. Pharmacological Smo inhibition reduced LSCs in vivo and enhanced time to relapse after end of treatment. Our results indicate that Smo inhibition might be an effective treatment strategy to reduce the LSC pool in CML.
