Name: focus formation: a cell-based assay to determine the oncogenic potential of a gene
Uploaded: 2014-12-31T15:00:00.000+00:00
Duration: 8 min 18 s
Description: Watch this Scientific Journal Video about Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene at JoVE.com

Este trabalho foi financiado por uma bolsa da New Innovator Award Program Director do NIH (ED). AA foi apoiado em parte por prêmios de graduação do Instituto Nacional do Câncer e da Fundação Nacional de Ciência.

1. Fazer os vetores virais As sequências de codificação para o gene de interesse, bem como os controlos positivos e negativos, são inseridos em pBABEpuro por métodos tradicionais de clonagem (amplificação por PCR, digestão de restrição e de ligação). Existem quatro locais de restrição no vector em que o DNA pode ser inserido: BamHI, SnaBI, EcoRI e Sall. <ol><li> Prepare plasmídeo DNA transfecção-grade a partir de células competentes, segundo o kit plasmídeo midi QIAGEN. </li><li> Medir a concentração de DNA usando um espectrofotômetro NanoDrop2000. </li></ol> 2. Retrovirus Produção A linha celular de empacotamento Plat-E vai ser utilizado para produzir retrovírus ecotrópico que permitirá obter o ADNc de interesse a células NIH 3T3. <ol><li> Estabelecer Plat-E culturas de células congeladas <ol><li> Rapidamente descongelar células Plat-E de um ° C banho de água 37 e transferir para um tubo de 15 ml. Lentamente, adicionar 9 ml de meio de Plat-E (DuMeio Eagle Modificado de lbecco (DMEM) + 10% de Soro Fetal Bovino (FBS) + penicilina e estreptomicina). </li><li> Centrifugar o tubo em 180 x g durante 5 min e desprezar o sobrenadante. </li><li> Re-suspender as células com 10 ml de Plat-E e meio de transferência para uma placa de cultura de 10 centímetros. </li><li> Incubar as células em uma temperatura de 37 ° C, 5% de CO2 até que eles são de 80-90% confluentes (cerca de 2 dias). </li></ol></li><li> Splitting celular <ol><li> Aspirar o meio e lave as células uma vez com PBS. </li><li> Adicionar 2 ml de 0,05% de tripsina-EDTA e incubar durante 1 minuto à temperatura ambiente. </li><li> Retire as células por finger tapping, adicione 10 ml de meio Plat-E, e transferir a suspensão de células para um tubo de 15 ml. </li><li> Centrifugar o tubo em 180 x g durante 5 min e desprezar o sobrenadante. </li><li> Ressuspender as células com 10 ml de meio Plat-E e semear-los em novas placas a 1: 4-1: 6 diluições. </li></ol></li><li> Plat-E Sementeira e Transfection <ol><li> Semente de 2 x 10 6células por 10 cm prato de cultura, utilizando meio de Plat-E sem quaisquer antibióticos. </li><li> Incubar as células S / N em uma temperatura de 37 ° C, 5% de CO2. </li><li> No dia seguinte, transferir 300 ul de Opti-MEM em 1,5 ml de tubo de microcentrífuga. </li><li> Adicionar 27 ul de polietilenimina (PEI), um reagente de transfecção de baixo custo 12, o tubo preparado com Opti-MEM. Misturar suavemente batendo por dedo e incubar durante 5 min à TA. </li><li> Adicionar 9 ug de ADN plasmídeo de transfecção de grau para o tubo de Opti-MEM / PEI, misturar suavemente em vórtex e incubar durante 15 min à temperatura ambiente. </li><li> Adicionar o ADN / PEI gota a gota complexo no prato Plat-E, e incubar O / N a 37 ° C, 5% de CO 2. </li><li> No dia seguinte, aspirar o meio contendo o reagente de transfecção e adicione 10 ml de meio Plat-E fresco (sem antibióticos). </li><li> Retorne as células para a incubadora. </li></ol></li></ol> 3. As células NIH 3T3 e Infecção <ol><li> Estabelecer NIHCultures 3T3 e propagação <ol><li> Rapidamente descongelar células NIH 3T3 numa ° C num banho de água a 37 e transferir para um tubo de 15 ml. Adiciona-se lentamente 9 ml de meio de NIH 3T3 (DMEM + 10% FBS). </li><li> Centrifugar o tubo em 180 x g durante 5 min e desprezar o sobrenadante. </li><li> Ressuspender as células com 10 ml de NIH 3T3 médio e transferir para um 10 centímetros prato de cultura. </li><li> Incubar as células em uma temperatura de 37 ° C, 5% de CO2. É muito importante que as células NIH 3T3 são sempre mantidas underconfluent (50-60%), tal como a frequência de transformação espontânea (e, por conseguinte, níveis basais de formação de focos) aumenta uma vez que uma cultura atinge a confluência. </li><li> Semente de 3 x 10 5 células por placa de 10 cm e incubar O / N para a infecção, no dia seguinte. </li></ol></li><li> Infecção <ol><li> Colher o sobrenadante retroviral do prato Plat-E usando uma seringa descartável de 10 ml, filtração através de um filtro de membrana de nylon de 0,45 um de tamanho de poro, e transferindo-o para um 15 mltubo. </li><li> Adicionar 10 ml de meio Plat-E fresco às células (sem antibióticos), e devolvê-las para a incubadora. </li><li> Aspirar o meio do prato de células NIH 3T3 de estarem infectados, e adicione 5 ml de meio de NIH 3T3 regular e 5 ml de sobrenadante filtrado que contém o vírus. </li><li> Adicionar ao prato polibreno a uma concentração de 6 ug / ml. </li><li> Incubar as células S / N a 37 ° C, 5% de CO 2. </li><li> No dia seguinte, repita os passos 3.2.1 - 3.2.5 para uma segunda rodada de infecção. </li><li> Substituir o meio contendo vírus NIH 3T3 com meio regular. </li><li> Permitir que as células NIH-3T3 para crescer durante 2-3 semanas, substituindo o meio, conforme necessário. Verifique a expressão da proteína de interesse por electroforese e imunotransferência de proteína convencional em lisados ​​de células inteiras a partir de placas réplica. </li></ol></li></ol> 4. Coloração e Quantificação <ol><li> Violeta Cristal Coloração <ol><li> Aspirar o meio de NIH 3T3 e colocar os pratosem gelo. </li><li> Lavam-se as placas duas vezes com PBS gelado. </li><li> Fixar as células com metanol gelado durante 10 min. </li><li> Remover os pratos de gelo, aspirar o metanol, e adicionar 3 ml de solução de violeta cristal 0,5% feitos em 25% de metanol (RT). </li><li> Incubar as placas durante 5 min à TA. </li><li> Aspirar a solução de cristal violeta e lavar cuidadosamente o prato com Milli-Q H2O até sem cor sai na lavagem. </li><li> Permitir que os pratos para secar O / N em uma bancada (descoberto). </li></ol></li><li> Focos Quantificação <ol><li> Uma vez que os pratos estão secos, use uma régua para medir as coleções de cor escura de células na placa. Contar apenas aqueles que são maiores do que 5 mm de diâmetro, como &quot;focos&quot;. Anote o número de focos e calcular médias e a importância em comparação com os controles. </li></ol></li></ol>

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A FFA fornece um método rápido e fácil de avaliar a transformação maligna in vitro. É passível de exibição de um número relativamente grande de genes candidatos e seus requisitos técnicos modestos torná-lo rentável. Além disso, dois ou mais genes pode ser co-expressa (por vezes referido como um ensaio de &quot;cooperação&quot;) para avaliar o potencial tumorigénico de combinação. As vantagens deste ensaio contar com a sua técnica simples, a sua facilidade de quantificação e a sua relativam...

As células tumorais podem ser distinguidos dos seus homólogos normais por uma ampla gama de alterações, a partir de padrões de expressão de genes para epigenomics a alterações morfológicas e proliferativas. Entre estes últimos, redução da dependência do soro, a perda de contacto (densidade) de inibição, a aquisição de proliferação independente de ancoragem e, em última análise, a capacidade de formar tumores quando injectadas em animais são indicadores úteis, mensuráveis ​​de transformação maligna 3. Vários in vitro e em ensaios in vivo têm sido desenvolvidos para a transformação celular. Os ensaios in vitro por objectivo a identificação e medindo alterações na morfologia da cultura (ensaio de formação de foco), dinâmica de cultura (taxa de crescimento, a densidade de saturação) e o factor de crescimento (crescimento em soro reduzido) ou ancoradouro (crescimento em agar mole) requisitos. O padrão de ouro para a determinação do carácter maligno de um tipo de célula permanece a formação de tumores (xenoenxertos) em animais experimentais. No entanto, tele custa e comprimento de estudos in vivo nem sempre fazê-los justificável como uma primeira etapa de validação ou screening de oncogenes candidatos. Embora nenhum ensaio in vitro fornece uma avaliação definitiva do potencial oncogénico de um gene, que não fornecem informações sobre potencial oncogénico que pode afinar futuro em estudos in vivo. Um dos sistemas mais utilizados para a avaliação do potencial oncogênico in vitro é o foco Formação Ensaio 2. Esta abordagem baseia-se na utilização de NIH 3T3 de fibroblastos de rato, uma linha de células não transformadas que mostram forte inibição de contacto. A sobre-expressão de um oncogene resulta em perda do crescimento dependente de densidade; As células transformadas podem então crescer em múltiplas camadas, formando &quot;focos&quot;, facilmente visualizado contra a monocamada de células não transformadas fundo. O Ensaio de formação de focos, em seguida, mede a capacidade de um oncogene candidato para induzir a transformação maligna, como evidenciado pela perda de contacto Inibiç como um fenótipo mensurável. O FFA tem sido utilizado para avaliar a sobre-expressão por transformação de proteína-quinases (por exemplo, Src 4, 5 BRAF), factores de transcrição (por exemplo, N-myc 6) receptores, L-acoplados à proteína (por exemplo, P2RY8 7) e GTPases (p.ex. , Ras 1), entre outros. A relativa facilidade deste ensaio faz-se uma boa escolha que vai proporcionar uma resposta rápida e visualmente claro para se sobre-expressão do gene é suficiente para transformar células de fibroblastos NIH 3T3 de rato in vitro. O FFA descrito neste protocolo utiliza a linha celular de empacotamento Plat-E 8, o qual fornece as proteínas virais de empacotamento, e o vector retroviral pBABEpuro 9 (Addgene plasmídeo 1764) para a produção de retrovírus. Após transfecção com a construção pBABEpuro contendo o gene de interesse, a linha celular Plat-E vai produzir retrovírus ecotrópico que pode ser utilizado para infectar as células NIH 3T3. Tseu método de entrega gene viral é mais eficiente do que os métodos de transfecção química tradicionais e oferece uma maneira de expressar de forma sustentável o gene 10. Uma vez incorporado no genoma das células NIH 3T3, a sobre-expressão do gene de interesse é impulsionado pelo repetições terminais longas virais (LTR) 11. Esta expressão constante pode ser usado para determinar se o gene de interesse tem actividade oncogénica, tal como medido pela formação de focos, sobre as células NIH 3T3.

<table><tbody><tr><td>pBABE-puro vector</td><td>Addgene</td><td>Plasmid 1764</td><td>cloning vector</td></tr><tr><td>Platinum-E Retroviral Packaging Cell Line, Ecotropic</td><td>Cell Biolabs, Inc.</td><td>RV-101</td><td>cell line for viral production</td></tr><tr><td>NIH 3T3 Cell Line murine</td><td>Sigma-Aldrich</td><td>93061524</td><td>cell line for focus formation assay</td></tr><tr><td>10 ml BD Luer-Lok tip syringe</td><td>BD Biosciences</td><td>309604</td><td>viral production reagent</td></tr><tr><td>0.45 &amp;mu;m Puradisc Syringe Filter</td><td>Whatman</td><td>6750-2504</td><td>viral production reagent</td></tr><tr><td>Polyethylenimine (PEI)</td><td>Polysciences, Inc.</td><td>23966-2</td><td>cell transfection reagent</td></tr><tr><td>Polybrene Infection / Transfection Reagent</td><td>EMD Millipore</td><td>TR-1003-G</td><td>cell transfection reagent</td></tr><tr><td>Crystal Violet</td><td>Fisher Scientific</td><td>C581-25</td><td>cell stain reagent</td></tr><tr><td>Plasmid Plus Midi Kit</td><td>QIAGEN</td><td>12945</td><td>plasmid purification</td></tr><tr><td>BD Falcon Tissue Culture Dishes</td><td>BD Biosciences</td><td>353003</td><td>cell culture supplies</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>11995-065</td><td>cell culture media</td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Gibco</td><td>25300-054</td><td>cell culture supplies</td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Gibco</td><td>31985-062</td><td>cell culture media</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>16000-044</td><td>cell culture media</td></tr></tbody></table>

focus formation: a cell-based assay to determine the oncogenic potential of a gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

MXD3 é um fator fundamental zipper hélice-alça-hélice leucina (bHLHZ) de transcrição que é um membro da MYC / MAX / network MAD. Ele é um membro da família atípica MAD 13-15, e tem sido referida como estando envolvida na carcinogénese 16,17. Quando comparado com pBABEpuro (controlo negativo) e MYC (controlo positivo), os pratos de NIH 3T3 que sobre-expressas foi MXD3 tinham significativamente menos focos (Figura 1A). Os dados na Figura 1B foram recolhido...

Watch this Scientific Journal Video about Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene at JoVE.com

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

O ensaio de formao de foco fornece um método simples para avaliar o potencial de transformação de um oncogene candidato.

Concentre Formação: Um Ensaio Celular para Determinar o Potencial oncogénica de um Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

focus-formation-cell-based-assay-to-determine-oncogenic-potential

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27.8K visualizações.  University of California, Davis.  O ensaio de formao de foco fornece um método simples para avaliar o potencial de transformação de um oncogene candidato. 

Vídeo: Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

Utilizando Genómica Funcional de triagem para identificar alvos de drogas potencialmente romance em Spheroid culturas de células de câncer

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Pesquisa do câncer

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

Formadoras de Colônias celular Assay (CFC) para células hematopoiéticas humanas

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

Imunologia e Infecção

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

Métodos para avaliar o papel de c-Fos e Dusp1 em dependência do Oncogene

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples from cancer patients often directly influences diagnosis, prognosis, and/or therapy selection. As a result, the accurate detection of gene fusions has become a critical component of clinical management for many disease types. Until recently, clinical gene fusion detection was predominantly accomplished through the use of single-gene assays. However, the ever-growing list of gene fusions with clinical significance has created a need for assessing fusion status of multiple genes simultaneously. Next generation sequencing (NGS)-based testing has met this demand through the ability to sequence nucleic acid in massively parallel fashion. Multiple NGS-based approaches that employ different strategies for gene target enrichment are now available for use in clinical molecular diagnostics, each with its own strengths and weaknesses. This article describes the use of anchored multiplex PCR (AMP)-based target enrichment and library preparation followed by NGS to assess for gene fusions in clinical solid tumor specimens. AMP is unique among amplicon-based enrichment approaches in that it identifies gene fusions regardless of the identity of the fusion partner. Detailed here are both the wet-bench and data analysis steps that ensure accurate gene fusion detection from clinical samples.

This article details the use of an anchored multiplex polymerase chain reaction-based library preparation kit followed by next-generation sequencing to assess for oncogenic gene fusions in clinical solid tumor samples. Both wet-bench and data analysis steps are described.

Detecção de fusão gênica oncogênica usando a reação em cadeia de polimerase multiplex ancorada seguida por sequenciamento da próxima geração

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

O uso de células tumorais do rato mamário em um ensaio de invasão in vitro como medida de comportamento de células oncogênicas

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

Genética

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common mutations associated with RMS and allowed discrimination of different RMS subtypes, major challenges still exist for the development of novel treatments to further improve prognosis. Although identified by the expression of myogenic markers, there is still significant controversy over whether RMS has myogenic or non-myogenic origins, as the cell of origin is still poorly understood. In the present study, a reliable method is provided for the tumorsphere assay for mouse RMS. The assay is based on functional properties of tumor cells and allows the identification of rare populations in the tumor with tumorigenic functions. Also described are procedures for testing recombinant proteins, integrating transfection protocols with the tumorsphere assay, and evaluating candidate genes involved in tumor development and growth. Described further is a procedure for allograft transplantation of tumorspheres into recipient mice to validate tumorigenic function in vivo. Overall, the described method allows reliable identification and testing of rare RMS tumorigenic populations that can be applied to RMS arising in different contexts. Finally, the protocol can be utilized as a platform for drug screening and future development of therapeutics.

This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.

Derivação e tratamento de tumorsphere de células tumorais primárias isoladas de rabdomiossarcoma de camundongo

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

Mapeando as Relações Estrutura-Função de Fatores de Transcrição Oncogênica Desordenados usando análise transcriômica

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

A plataforma de triagem baseada luciferase diversificadas para interrogar associados ao câncer transdução de sinal em células cultivadas

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Medicina

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

A Formação Ensaio macio Agar Colony

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...

Biologia

Methyl Cellulose-Based CFU Assay: A Method to Determine the Effect of Anti-cancer Compounds on Colony Formation in Leukemic Cells

Source: Kesarwani, M., et al. <a href="https://www.jove.com/t/58194">Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence.</a> J. Vis. Exp. (2019).
This video describes the protocol for methyl cellulose-based colony formation unit or CFU assay, which helps identify and count the number of colonies formed by leukemic cells in response to the treatment with anticancer compounds.

Ensaio de UFC à base de metilcelulose: um método para determinar o efeito de compostos anticancerígenos na formação de colônias em células leucêmicas

Source: Kesarwani, M., et al. Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence. J. Vis. Exp. (2019).
This video describes the ...