We thank Dr. Jordi Ochando from the Flow Cytometry Shared Resources at the Mount Sinai Medical Center for their assistance in flow cytometry analysis. We thank Dr. Rumana Huq from the Microscopy Shared Resource Facility at the Mount Sinai Medical Center for their imaging assistance. The authors thank the TJ Martell Foundation for its support in this project.

Este protocolo foi realizado em nossa instituição com a aprovação do conselho de ética em pesquisa da instituição e está em conformidade com todas as diretrizes institucionais, nacionais e internacionais para o bem-estar humano. 1. Recolha de Sangue Periférico de pacientes com câncer de próstata avançado Nota: Selecione os pacientes com câncer de próstata metastático. Obter o consentimento escrito do paciente e registrar características clínicas dos pacientes, incluindo idade em isolamento e quimioterapia anterior e tratamentos hormonais. Os doentes com doença metastática potencialmente terão maior concentração de CTC no sangue periférico. <ol><li> Recolha de amostras de sangue, após consentimento informado e no âmbito de um Conselho de Revisão Institucional apropriado protocolo aprovado. Usar luvas de látex e uma bata de laboratório durante todo o procedimento. </li><li> Conecte-25 G borboleta agulha e tubos para suporte de agulha. Use um algodão embebido em álcool para limpar a área deveia antecubital entre bíceps e antebraço, em seguida, aplicar torniquete a parte superior do braço do paciente. </li><li> Insira a agulha na veia antecubital borboleta e empurre tubo de coleta de sangue para suporte de agulha para começar a coleção. Recolha cerca de 10 ml de sangue em tubos de recolha que contêm ácido etilenodiaminotetracético (EDTA) para impedir a coagulação. </li><li> Remova a agulha borboleta do paciente e aplicar pressão com gaze esterilizada sobre o local da punção da veia por 1 min. Local Cubra com uma gaze esterilizada. </li></ol> 2. Isolamento de Células Mononucleares Nota: O sangue recolhido a partir do passo 1 conterá células mononucleares do sangue periférico (PBMC) (por exemplo, linfócitos e monócitos), para além dos CTC mononucleares. <ol><li> Pipeta todo o sangue em tubo de poliestireno de 50 mL cónico, com uma pipeta de 5 mL e adicionar serológica Solução Salina Equilibrada de Hanks (HBSS) em uma proporção de 1: 1. Suavemente mistura pipeta para homogeneizar. </li><li> Adicionar 15 ml de uma solução preparada comercialmente (Ficoll-Paque) para separar componentes de sangue por gradiente de densidade baixa velocidade de centrifugação para um tubo cónico de 50 ml de poliestireno vazio. </li><li> Pipeta suavemente sangue total e mistura HBSS, a partir do passo 2.1, para dentro do tubo de poliestireno a partir do passo 2.2, para formar a camada superior distinta. Set pipeta a configuração mais baixa para minimizar a mistura de soluções, o que irá garantir a separação eficiente. </li><li> Centrifuga-se a 400 xg durante 30 min à temperatura ambiente (25 ° C). Definir desaceleração a configuração mais baixa para evitar a mistura de soluções após a separação. A aceleração pode ser ajustado para qualquer velocidade. </li><li> Após centrifugação, a banda branca identificar-cinza fina camada de PBMC e CTC ensanduichada entre o plasma em solução de topo e de separação no fundo do tubo. </li><li> Recolher as células a partir da faixa branca-acinzentada no passo 2.5 para um tubo de 50 mL de poliestireno, usando uma pipeta de transferência de plástico. </li><li> Adicionar HBSS a 50 ml de poliestireno tubo cónico com PBMCs e CTC e Centrifuge novamente a 400 xg durante 10 min temperatura ambiente para lavar. </li><li> Decantar sedimento líquido e ressuspender em 50 ml de HBSS e centrifugar novamente a 400 xg durante 3 minutos à temperatura ambiente para lavar. </li><li> Descartar o sobrenadante, o sedimento restante ressuspender com 5 ml de tampão de lise de glóbulos vermelhos e incubar a solução durante 5 min à TA. </li><li> Remover o tampão de lise de glóbulos vermelhos por centrifugação a 400 xg durante 3 minutos à temperatura ambiente. </li><li> Descartar o sobrenadante e ressuspender o sedimento em 1 ml de PBS 1x suplementado com soro fetal bovino a 10% (FBS) para bloquear antes da coloração do anticorpo. </li></ol> 3. As células mononucleares com coloração CD45-FITC de anticorpos para fluorescência celular ativado (FACS) <ol><li> Quantificar o número de células viáveis ​​(Trypan Blue-negativa) (do passo 2.11), utilizando um hemocitómetro ou contador de células automatizado. Prepara-se uma 6 x 10 células / suspensão de 1 ml (em PBS 1x com 10% de FBS) e colocá-lo em gelo durante 1 hr. </li><li> Distribua o quantificadosuspensão de células a partir do passo anterior em dois tubos. Tubos de etiquetas de controle e coloração CD45. </li><li> No tubo de controlo, adicionar IgG1K-FITC (2,5 ug / ml), a uma diluição de 1: 250. No tubo de coloração CD45, CD45 adicionar FITC (2,5 ug / mL) conjugada com o anticorpo primário a uma diluição de 1: 250 e incuba-se as suspensões de células sobre gelo durante 30 min. Rotulagem antigénio CD45 é utilizado para excluir células hematopoiéticas. </li><li> Centrifugar as células a 400 xg durante 3 min a 4 ° C, e desprezar o sobrenadante. </li><li> Lavar as células duas vezes, suspendendo cada pellet estéril com 1 x PBS suplementado com FBS a 10%, seguido por centrifugação a 400 xg durante 3 min a 4 ° C. </li><li> Ressuspender as células numa solução de PBS contendo 1 x 4 &#39;, 6-diamidino-2-fenilindole (DAPI) a uma concentração de 10 ug / ml 1 ml. </li><li> Filtrar a solução final através de tampas de filtro 35 um em 12 milímetros tubos de poliestireno x 75 mm. </li></ol> 4. Isolamento de próstata por CTCFACS Nota: Utilize um citômetro de fluxo para coletar ao vivo CTCs negativos CD45. <ol><li> Defina controles de compensação usando células do tubo rotulado, &quot;Control&quot;. </li><li> Criar portões que excluem detritos e aglomerados de células usando parâmetros para a frente-de dispersão e side-de dispersão apropriados. </li><li> Estabelecer as portas FITC utilizando as suspensões de células a partir do tubo rotulado, &quot;CD45-FITC.&quot; </li><li> Portão células viáveis ​​(DAPI-negativos) usando o Pacífico azul-A do canal. </li><li> Recolhe população negativa para CD45 num tubo cónico de 15 ml de poliestireno estéril, contendo 2 ml de Roswell Park Memorial Institute (RPMI) 1640, suplementado com FBS a 10%. </li><li> Centrifugar a suspensão de células de tumor da próstata classificados a 400 xg durante 3 min, em seguida, voltar a suspender as pelotas em 200 - 500 ul de meio RPMI suplementado com FBS a 10%. </li></ol> 5. Injeção de CTCs em ratos e Monitoramento de Crescimento Xenoenxerto Nota:Realizar todos os procedimentos de animais em conformidade com os protocolos aprovados pelo comitê de cuidados com os animais institucional. Este protocolo foi realizado em nossa instituição no âmbito de um protocolo específico ao Uso de Animais aprovado pelo nosso Comitê Animal Care em conformidade e cumprimento de todas as agências regulatórias e institucionais, regulamentos e orientações relevantes. <ol><li> Misture a suspensão de células de tumor da próstata classificados com matriz extracelular em uma proporção de 1: 1 e o local em gelo. </li><li> Anestesiar rato antes da implantação subcutânea em uma câmara de fornecimento de 5% (v / v) de isoflurano inalado em 1 L / min de oxigénio. Certifique-se anestesia adequada verificando a perda do reflexo corneal e dedo do pé no mouse. </li><li> Usando uma agulha G 25 e uma seringa de 1 ml, injectar 250 ul de células de tumor da próstata e na matriz extracelular de células subcutaneamente suspensão em ambos os flancos superiores de um 8-10 semanas de idade do sexo masculino murganho imunodeficiente. Injectar 250 ul, independentemente da concentração de tetracloreto de carbono como o volume de injecção é o ivalor mportant. Administração máxima SQ no rato não é mais do que 1 ml. &#160;</li><li> Monitorar ratos através da realização de palpação semanal de locais de injecção do mouse para o crescimento de densidades nodulares subcutâneas e medindo os ratos de peso duas vezes por semana. </li><li> Euthanize ratos por asfixia dióxido de carbono, seguido por deslocamento cervical e colheita subcutâneos xenoenxertos de cancro da próstata humano, quando o tamanho do tumor é mais do que 0,5 cm e menos de 1 cm de maior diâmetro para assegurar tecido tumoral suficiente para análises moleculares e passagem serial. Euthanize camundongos com sinais de sofrimento ou perda de peso, de 15%, apesar de nenhum tumor é palpável. </li></ol>

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The authors declare that they have no competing financial interests.

Este manuscrito descreve um método para a geração de modelos de cancro da próstata a partir de PDX CTC. O uso do CTC para a geração de PDX modelos tem várias vantagens potenciais importantes quando comparado com métodos existentes. Em primeiro lugar, de recolha acessível CTC partir de sangue periférico permite a geração de modelos experimentais a partir do mesmo paciente em diferentes estádios da doença. Em segundo lugar, a colheita de sangue representa um método barato e mais seguro pa...

A correction was made to <a href="http://www.jove.com/video/53182">Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells</a>. There was a spelling error in one of the authors' surname. The author's name was corrected from:
Veronica Rodriquez-Bravo
to:
Veronica Rodriguez-Bravo

A correction was made to Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells.

Erratum: Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Paciente xenotransplantes derivados são cada vez mais populares modelos experimentais utilizadas para a investigação do cancro. Eles podem ser usados ​​para a caracterização de biomarcadores e vias biológicas, avaliação pré-clínica da eficácia do medicamento, e criação de avatares para terapias de câncer personalizado 1,2. Previamente, outros grupos de pesquisa têm desenvolvido modelos PDX quer por implantação ou injecção de suspensões de células tumorais únicas ou explantes de tumores em ratinhos inteiro 1 imunocomprometidos. Estes PDX modelos exige a recolha cirúrgica do tumor sólido fresco, ascite maligna ou derrame pleural de um paciente submetido a um procedimento cirúrgico que é caro e expõe o paciente a um risco aumentado de morbidade iatrogênica. Um desenvolvimento recente importante na investigação do cancro tem sido a detecção, isolamento e caracterização de células tumorais circulantes. Estas células tumorais escapar da massa do tumor primário e entra na circulaçãoonde eles desempenham um papel crítico na metástase e recidiva, a causa mais comum de cancro relacionados com a mortalidade 3. A avaliação e caracterização dos CTC a partir de vários tipos de tumores sólidos forneceram informação clínica para o diagnóstico, prognóstico e monitorização da doença residual 3. Uma variedade de abordagens actualmente usados ​​dependem tanto nas propriedades físicas, à expressão dos biomarcadores ou as características funcionais do CTC pode ser utilizado para isolar CTC 4 eficientemente. Métodos existentes macroescala isolamento CTC incluem centrifugação em gradiente de densidade, de filtração com poros de filtro física e separação contra moléculas da superfície. As metodologias de isolamento mais amplamente utilizados baseiam-se no CTC captura à base de anticorpo da CTC. Tanto a selecção positiva e negativa de marcadores de superfície celular podem ser empregues para isolar CTC partir de sangue periférico. A selecção positiva para a CTC na circulação periférica geralmente usa marcadores epiteliais (por exemplo, que um EpCAM)está expressa em células CTC mas não hematopoiéticas. A desvantagem deste método é que CTC com potencial metastático têm muitas vezes sido submetidos epitelial-mesenquimal-a transição (EMT), que regula negativamente a marcadores de superfície epitelial 3. A fim de isolar CTC com potencial metastático, uma metodologia de selecção negativa que utiliza o marcador de superfície hematopoiéticas, CD45, para esgotar a população de células normais de leucócitos pode ser utilizado 5. O câncer de próstata é o câncer mais comumente diagnosticado e uma das principais causas de mortes relacionadas ao câncer em homens 6. Os mecanismos de progressão tumoral e agressividade não são completamente compreendidos e, por conseguinte, a geração e caracterização de modelos experimentais que recapitulam a heterogeneidade molecular de cancro da próstata são de interesse significativo. PDX modelos de câncer de próstata têm sido gerado anteriormente pelo enxerto de células cancerosas humanas da próstata em immunocomcamundongos prometidos 7,8. No entanto, a geração de tais modelos tem sido dificultada pela baixa taxa de enxerto de cancro da próstata em ratinhos imunocomprometidos, a qual é atribuída principalmente à natureza indolente da doença. Recentemente, CTC foram usadas para gerar o cancro da mama 9, cancro do pulmão e cancro da próstata 10 11 modelos PDX. Estes estudos de prova de conceito introduzido a possibilidade de gerar modelos PDX independente da necessidade de recolha de amostra cirúrgica. Neste artigo vamos descrever em detalhes um método para a geração deste modelo experimental romance.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Roswell Park Memorial Institute (RPMI) 1640</td>
			<td>Gibco Life Technologies</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco Life Technologies</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Gibco Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Corning Cell Gro</td>
			<td>21-031-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>35 &#181;m Cell Strainer</td>
			<td>BD Falcon</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml polystyrene conical tube</td>
			<td>Crystalgen</td>
			<td>23-2263</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood cell lysing buffer</td>
			<td>Sigma</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>d3571</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus</td>
			<td>GE Healthcare</td>
			<td>17-1440</td>
			<td></td>
		</tr>
		<tr>
			<td>12 mm x 75 mm Polystyrene tubes with cell strainer cap</td>
			<td>BD Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer Lavender Blood Collection Tubes with EDTA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Winged Blood Collection Set with Push Button Retract Needle 23 gauge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer One Use Needle Holder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable Latex Tourniquet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Latex or non-latex gloves</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>alcohol swabs</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>2x2 cotton gauze pads</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive bandage</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>25 gauge needle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml syringe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of prostate cancer patient derived xenograft models from circulating tumor cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

Este protocolo conduz à geração de modelos de PDX isolados CD45 CTC cancro da próstata negativo. Com base no método de selecção negativa utilizada no nosso protocolo é necessário para excluir as células mortas utilizando DAPI mancha. A percentagem de células CD45 negativas detectada por citometria de fluxo é variável e depende do peso do tumor do paciente (Figura 1A). Coloração de imunofluorescência de células não triados usando CD45 e DAPI (para identificar os núcleos celulares) reve...

Watch this Scientific Journal Video about Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells at JoVE.com

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Geração de paciente com câncer de próstata Derivado modelos de xenotransplante de células tumorais circulantes

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

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14.0K visualizações.  Icahn School of Medicine at Mount Sinai. This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Vídeo: Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Lapso de tempo de imagem de pré-neoplásicas primárias células epiteliais mamárias derivados de modelos de rato geneticamente modificado do cancro da mama

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Medicina

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Cultura de Células e Isolamento paciente Derivado de CD133<sup&gt; +</sup&gt; Células-tronco de câncer putativos Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Adaptação do Semi-Automática de circulação Tumor de Células (CTC) Ensaios para aplicações clínicas e pré-clínica de Pesquisa

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolamento de células-tronco cancerosas em amostras de câncer de próstata humano

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to extravasate and form a secondary niche.&#160;The extravasation of tumor cells from the blood vascular system can be studied using endothelial cells (ECs) and tumor cells obtained from different cell lines.&#160;Initial studies were conducted using static conditions but it has been well documented that ECs behave differently under physiological flow conditions.&#160;Therefore, different flow chamber assemblies are currently being used to studying cancer cell interactions with ECs.&#160;Current flow chamber assemblies offer reproducible results using either different cell lines or fluid at different shear stress conditions.&#160;However, to observe and study interactions with rare cells such as circulating tumor cells (CTCs), certain changes are required to be made to the conventional flow chamber assembly.&#160;CTCs are a rare cell population among millions of blood cells. Consequently, it is difficult to obtain a pure population of CTCs.&#160;Contamination of CTCs with different types of cells normally found in the circulation is inevitable using present enrichment or depletion techniques.&#160;In the present report, we describe a unique method to fluorescently label circulating prostate cancer cells and study their interactions with ECs in a self-assembled flow chamber system.&#160;This technique can be further applied to observe interactions between prostate CTCs and any protein of interest.

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

Our report describes a unique method to visualize and analyze CTC/EC interactions in prostate cancer under physiological flow conditions.

Método in vitro para observar interações E-selectina mediadas entre próstata células tumorais circulantes derivadas de pacientes e células endoteliais humanas

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to ...

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in&#160;vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy1. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2&#8211;3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

Geração de pluripotentes induzidas células-tronco de distrofia muscular pacientes: Reprogramação livre de integração eficiente de urina células derivadas

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

Isolamento e Imortalidade de linhas celulares derivadas do paciente a partir de biópsia muscular para a doença Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

Em Enriquecimento vitro de câncer de ovário de iniciação Células Tumorais

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Criação de um Modelo de Xenoenxerto Humano mieloma múltiplo na galinha para estudar o crescimento tumoral, invasão e angiogênese

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.