Este trabajo es apoyado por el BRC Imperial, el Instituto Nacional de Investigación en Salud y Acción Contra el Cáncer con mención especial a Hilary Artesanía y Sir Douglas Myers.

Los procedimientos a continuación han sido éticamente aprobado por el Imperial College de Londres. 1. Generación de mammospheres primarias de Mama Humano líneas celulares de cáncer NOTA: Realice los siguientes pasos debajo de una campana de cultivo estéril. <ol><li> Prepare mammosphere los medios de comunicación que contiene DMEM / F12 suplementado con 2 mM L-glutamina, 100 U / ml de penicilina, 100 U / ml de estreptomicina. Preparar medio completo inmediatamente antes del uso mediante la adición de 20 factor de ng / ml recombinante de crecimiento epidérmico humano (EGF; Sigma), 10 ng / ml de factor recombinante humano de crecimiento básico de fibroblastos (bFGF; R &amp; D Systems) y suplemento 1x B27. </li><li> Aspirar los medios de frasco que contiene las células MCF-7 o MDA-MB-231 células adherentes (o una línea celular de cáncer de mama de su elección; 70-80% de confluencia), se lavan dos veces en PBS 1x y trypsinize células. </li><li> Centrifugar las células a 200 xg a temperatura ambiente durante 5 min. Sobrenadante y volver a suspender las células de decantación en 1-5 ml de medio mammosphere. Pipetaarriba y abajo y si es necesario, utilice un filtro de tapa filtro de células de 40 micras para obtener la suspensión de una sola célula. Utilice un hemocitómetro para asegurar una suspensión de células se ha formado (si no, utilizar una aguja de 25 G a la jeringa de la suspensión celular hasta 3 veces). </li><li> Si CD44 + CD24- aislado necesario subconjunto celular a través de FACS usando anti-CD24-ficoeritrina (PE) e isotiocianato (FITC) anticuerpos monoclonales anti-CD44 9-fluoresceína. Alternativamente, aislar dicha población usando una célula magnético activadas por la clasificación del sistema (MACS) con anti-CD44 y anti-combinados microperlas CD24-biotina según las instrucciones del fabricante. Realice la selección positiva utilizando LS columnas, y la selección negativa utilizando columnas de LD y confirmar los fenotipos de todas las células aisladas por citometría de flujo 17. </li><li> Calcular el número de células viables por ml utilizando azul de tripano. NOTA: La viabilidad celular se calcula como el número de células viables dividido por el número total de células dentro de las rejillas en el hemocyfotómetro. Las células que ocupan azul tripán se consideran no viables. El siguiente procedimiento se utiliza para determinar con precisión la proporción de células viables. <ol><li> Preparar una solución de 0,4% de azul de tripano en PBS. Añadir 0,1 ml de solución madre de azul de tripano a 1 ml de las células. Cargar un hemocitómetro y examinar inmediatamente bajo un microscopio a bajo aumento. Contar el número de células totales y el número de células de tinción con azul. Las células viables = [1,00 - (Número de células azules ÷ Número de células totales)] × 100. </li><li> Para calcular el número de células viables por ml de cultivo, utilizar la siguiente fórmula. Recuerde que debe corregir el factor de dilución. Número de células viables × 10 × 1,1 = 4 células / ml de cultivo </li></ol></li><li> Resuspender una cantidad predeterminada de células en 2 ml de medio mammosphere completos en cada pocillo de una placa de 6 pocillos adherente ultra-bajo. La densidad de siembra es normalmente 500-4.000 células / cm 2 células por pocillo. Uso Opcionalmente low de fijación placas de 24 pocillos, mientras que la siembra de células a la misma densidad en 0,5 ml de medios de comunicación. Nota: Se recomienda la optimización de la densidad y la hora de la cultura de siembra para cada líneas celulares de interés. </li><li> Incubar bajo fijación 6 y placas de ultra a 37 ° C y 5% de CO 2 durante 5-10 días (dependiendo de la línea celular y el tamaño de mammospheres) sin molestar a las placas, en particular durante la fase de crecimiento de los primeros 5 días . </li></ol> 2. Generación de mammospheres primarias de muestras clínicas de cáncer de mama humano NOTA: el tejido de cáncer de mama humano puede obtenerse de pacientes sometidos a cirugía para la eliminación de tumores de mama. Tejido de la tienda en hielo durante hasta 24 horas en tubos de 50 ml estériles en medios de cultivo que contiene 100 U / ml de penicilina y 100 U / ml de estreptomicina. Lleve a cabo los siguientes pasos bajo una campana de cultivo estéril. <ol><li> Transferir la muestra en un plato de 100 mm de tejido de Petri con un pequeño volumen de medios de comunicación. Remove el tejido adiposo con unas tijeras estériles, bisturí y pinzas. </li><li> Añadir 2-3 ml de DMEM / F12 y picar la muestra en 1 mm 3 piezas con un escalpelo estéril u hoja de afeitar hasta que no haya piezas grandes se mantienen. </li><li> Resuspender las piezas de tejido en pre-calentado 10 ml de DMEM que contiene enzimas proteolíticas (3000 U / ml de colagenasa y 1.000 U / ml de hialuronidasa) y se incuba a 37 ° C en un agitador rotatorio hasta que se digieren todos los fragmentos de tejido. Por lo general, la digestión completa tarda 1-3 horas. Ajuste el tiempo de digestión de acuerdo con caracteres patológicos de los tumores. (Por ejemplo adenocarcinoma de mama es generalmente más difícil de digerir en comparación con carcinoma mucinoso, que es más frágil y necesita más corto tiempo de digestión). Evaluar el grado de digestión cada media hora observando 20 l suspensión bajo hemocitómetro. </li><li> Permitir que los fragmentos sedimentar durante 5 min, luego transferir el sobrenadante en un tubo de polipropileno cónico de 15 ml y centrifugar a 200 xg durante 10 min a tem habitaciónambiente. Decantar con cuidado las células sobrenadante y resuspender en 1 - 5 ml de medios mammosphere. </li><li> Siga los pasos 1.1 a 1.4 descritas anteriormente para las líneas celulares para la obtención y la placa de suspensiones de células individuales. Pasan células a través de la jeringuilla 25 G un máximo de 2 veces si es necesario para limitar las células perjudiciales. </li></ol> 3. La formación de mammosphere Eficiencia (%) Cálculo <ol><li> Después de que el período de cultivo, contar los mammospheres (mayor que 40 m) de diámetro bajo un microscopio a 40 aumentos. Imagen digitalmente 5 campos aleatorios utilizando una cámara digital en un microscopio de luz y determinan el tamaño mammospheres utilizando el software de adquisición. Utilice cualquiera de Análisis de Imágenes Software. </li><li> Calcular mammosphere eficacia de formación (MFE%) usando la siguiente ecuación: MFE (%) = (# de mammospheres por pocillo) / (# de células sembradas por pocillo) x 100 </li></ol> 4. pasada en serie de mammospheres para la Evaluación de la auto-renovación <ol><li&gt; Pipeta los medios de comunicación que contiene las mammospheres de cada pocillo a un tubo de 15 ml. Lavar los pocillos con PBS y añadir a los medios de comunicación recogidos. Centrifugar a 115 xg durante 10 min a temperatura ambiente. </li><li> Desechar el sobrenadante y resuspender el sedimento en 500 l de 0,5% de tripsina / 0.2% EDTA pre-calentado. Incubar durante 2-3 minutos. </li><li> Añadir 500 l de FCS para neutralizar la tripsina. Centrifugar a 500 xg durante 5 min. Desechar el sobrenadante y pellet se resuspende en 100 l de mammospheres medios de comunicación, la pipeta hacia arriba y hacia abajo para desagregar las esferas. </li><li> Contar las células con un hemocitómetro y determinar si las células están en suspensión de células individuales. Si no pasa a través de una jeringa 25 G hasta 3 veces para obtener células individuales. Sembrar las células en un nuevo mínimo apego placa de 6 pocillos de ultra a la misma densidad utilizado en la generación primaria. </li><li> Después de 5-10 días, contar esferas&gt; 40 micras y calcular la eficiencia de formación de esferas mediante la fórmula informado antes. </li></ol>

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The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this article.

Evaluación exitosa de mammospheres primaria y secundaria se basa en las células se sembraron a densidades suficientemente bajas que mammospheres forma de clones individuales, con un mínimo de agregación esfera. Sin embargo a densidades que son demasiado bajo, demasiado pocos mammospheres pueden formar para distinguir los efectos de los tratamientos estadísticamente. Densidad de siembra debe ser optimizado para cada línea celular utilizada, ya que pueden variar considerablemente en sus formando esfera eficiencia (l...

La existencia de linajes de células tumorales encabezadas por las células madre del cáncer de tallo como ha contribuido en gran medida a nuestra comprensión de la heterogeneidad del tumor. Mientras que algunos diversidad fenotípica en los tumores no surgen de la excrecencia clonal de clones genéticamente distintas, un componente sustancial parece resultar de diferencias epigenéticas: las células cancerosas pueden transición (a veces de forma reversible) entre el vástago, progenitor, y los estados diferenciados través de la activación o represión de genes específicos programas de expresión 1-3. Esto puede reflejar celulares factores intrínsecos o extrínsecos, que refleja la expresión génica programa que actualmente se expresa en una célula con su señalización autocrina resultante en conjunción con la señalización paracrina de la vecina cáncer, del estroma o células inmunes entrega de factores moduladores, y las condiciones microenviromental tales como el grado de hipoxia 2,4,5. Aunque innovadores enfoques linaje de rastreoavanzan nuestra capacidad para estudiar las células madre del cáncer putativos en su in vivo nicho 6-8, los ensayos de formación de esferas siguen siendo un método popular y conveniente para estimar el potencial de las células de cáncer de mama de comportarse como células madre, al menos en las condiciones de ensayo utilizados. A menudo se utiliza junto con los métodos retrospectivos de las células madre del cáncer de purificación, por su expresión de marcadores de membrana CD44 y CD24 9, y los niveles de actividad de la enzima ALDH (aldehído deshidrogenasa) 10, los marcadores que se han propuesto para corresponder a más mesenchymal- y epitelial Las células madre del cáncer -como respectivamente 11. El enfoque de formación de esferas fue desarrollado primero como el ensayo neurosphere, lo que permite el crecimiento de las células madre putativas de clones individuales en condiciones libres no adherentes, de suero con la adición de factor de crecimiento epitelial (EGF) 12, después de ser útil aplicar a normal y canceroso tejidos de la mama. <p class = &quot;jove_content&quot;&gt; La identidad de la célula fundadora de formación de esferas, y los tipos de células mixtas que integran la masa esfera, son relevantes para las inferencias que se pueden hacer desde mamográfica, o ensayos de formación de otra esfera. Células óseas quiescente fide madre a largo plazo, que se cree que descansan en fase G0, no experimentará la combinación precisa de factores que favorecerían la activación in vivo. El ensayo mammosphere vez permite el crecimiento de células o bien preparada para la división mitótica o ya divisorias 13. Estos progenitores, aunque no realmente una célula quiescente, pueden ser la etapa de célula que prolifera con los mitógenos de EGF y el factor de crecimiento de fibroblastos básico (bFGF) utilizados en el ensayo. Sin embargo, contienen una serie de señalización asociada a las células madre activado vías 14. Además, la tasa de su formación se refiere a la tumorigenicidad del tejido que se tomaron de, cuando se mide por su potencia en ensayos de dilución limitada en xenoinjertos de ratón 2,15,16 &lt;/ Sup&gt;. Aquí nos proporcionan protocolos detallados para aislar células individuales y generar mammospheres primarias de ambas líneas celulares de cáncer de mama humano y muestras clínicas de los tumores de mama. También describe cómo realizar pases seriados de mammospheres primarios para evaluar la auto-renovación, y la forma de calcular la eficiencia esfera que permite la comparación entre diferentes densidades de siembra (véase el esquema de la figura 1) que forma.

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			<td>G8540</td>
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			<td>P4083</td>
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			<td height="25" style="height:25px;width:625px;">20ng/ml recombinant human epidermal growth factor (EGF)</td>
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			<td>E9644</td>
			<td></td>
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			<td height="22" style="height:22px;width:625px;">20ng/ml recombinant human basic fibroblast growth factor (bFGF)</td>
			<td style="width:139px;">R&#38;D systems</td>
			<td>233-FB-025</td>
			<td></td>
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			<td height="22" style="height:22px;width:625px;">1x B27 supplement&#160;</td>
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			<td>17504-044</td>
			<td></td>
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			<td height="27" style="height:27px;">Phosphate buffered saline (PBS);</td>
			<td style="width:139px;">Thermo Scientific</td>
			<td>12399902</td>
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			<td height="21" style="height:21px;">&#160;0.5% trypsin-0.2%EDTA;</td>
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			<td>59418C</td>
			<td></td>
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			<td height="21" style="height:21px;">Fetal Calf Serum</td>
			<td style="width:139px;">First Link UK</td>
			<td>02-00-850</td>
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			<td height="21" style="height:21px;">&#160;Trypan Blue</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>93595</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">&#160;Low attachment 6 well plates</td>
			<td style="width:139px;">Corning</td>
			<td>CLS3814</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Collagenase type 1A</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>C9891</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyaluronidase</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>H3506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile razor blades</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td>12443170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:625px;">Sterile scalpel</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:119px;">11758353</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:625px;">Sterile micro-dissecting scissors</td>
			<td style="width:139px;">Sigma Aldrich</td>
			<td>S3146</td>
			<td></td>
		</tr>
	</tbody>
</table>

mammosphere formation assay from human breast cancer tissues and cell lines

De manera similar a los tejidos sanos, muchos de sangre y tumores malignos sólidos están ahora cree que están organizados jerárquicamente, con un subconjunto de células de cáncer de tallo como la auto-renovarse mientras que da lugar a la progenie diferenciada. La comprensión y la orientación de estas células madre del cáncer en el cáncer de mama, que pueden poseer mejorado quimio y radio-resistencia en comparación con el volumen del tumor no tallo, se ha convertido en un área de investigación importante. Marcadores incluyendo CD44, CD24, y la actividad de ALDH se pueden evaluar usando células activadas por fluorescencia (FACS) para aislar de forma prospectiva células que muestran una mayor tumorigenicidad cuando se implantan en ratones inmunodeficientes: el ensayo mammosphere también se ha convertido en ampliamente utilizado por su capacidad para identificar de forma retrospectiva esfera- formación de células que se desarrollan a partir de clones de células madre similares sola. Aquí resumimos los enfoques para el cultivo apropiado de mammospheres partir de líneas celulares o muestras de pacientes primarios, su pases, y los cálculos para estimar esfera feficiencia orming (SFE). En primer lugar se discuten consideraciones clave y las trampas en la planificación y la interpretación de los experimentos mammosphere apropiado.

Diferentes muestras o los sometidos a diferentes tratamientos pueden variar en el número de mammospheres&gt; 40 micras que se forman después de la normalización para las células iniciales sembradas. Calcular formando mammosphere eficiencia (MFE) para cada tratamiento crecido por triplicado. Esto permite experimentos con diferentes densidades de siembra que deben compararse. Los datos se muestra mejor en un gráfico de barras, idealmente con controles positivos y negativos, y la visualización de la desviación está...

Watch this Scientific Journal Video about Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines at JoVE.com

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Mammosphere Ensayo Formación de cáncer de mama humano tejidos y líneas celulares

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

mammosphere-formation-assay-from-human-breast-cancer-tissues-cell

Research

JoVE Journal

Medicine

33.0K vistas.  Imperial College London. Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Video: Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Humanos neuroendocrinos líneas de células tumorales como un modelo tridimensional para el estudio de la terapia humana tumor neuroendocrino

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Investigación

Medicina

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

Procesamiento de mamoplastia de reducción y tejidos humanos Mastectomía para el cultivo celular

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

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.

Time-lapse de imágenes de Primaria preneoplásicas células epiteliales mamarias derivadas de los modelos de ratones genéticamente modificados del cáncer de mama

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

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3.
In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6 as well as other solid tumors 7. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.
Protein based analysis of RCC8 is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

El uso de matrices inversas de proteínas de fase (RPPA) para explorar la variación de expresión de proteínas individuales dentro de cánceres de células renales

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

La formación del epitelio prostático humano utilizando tejido recombinación de roedores Urogenital mesénquima seno y células madre humanas

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Perfiles de microARN regulados por estrógeno en las células del cáncer de mama

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

La cuantificación de la invasividad de las células del cáncer de mama El uso de un modelo (3D) Tres Dimensiones

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

El enriquecimiento de las células madre quimiorresistente cáncer ovárico de líneas celulares humanas

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

Una<em&gt; Ex vivo</em&gt; Modelo para estudiar la acción hormonal en el pecho humano

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

Métodos de explantes de tejidos El cultivo fémur humano para el Estudio de la colonización de la célula del cáncer de pecho de la Niche metastásico

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...