Damos las gracias a J. Vaughen por la lectura crítica del manuscrito. Este trabajo fue apoyado por subvenciones de JSPS KAKENHI Grant Números 26891025, 15H01500 y la Fundación de Ciencias de Takeda Subvención de investigación a YT

1. Cruces de la mosca y inducción del clon <ol><li> Eliminar todas las moscas en el vial 12 h antes de recoger virgen moscas. </li><li> Anestesiar las moscas en el vial inyectando gas CO 2 y colocar moscas en una almohadilla de mosca de CO 2 . </li><li> Transferir de 10 a 20 hembras virgenes y 10 machos de la almohadilla de mosca de CO 2 a un vial nuevo e incubar durante 1 día a 25 ° C. </li><li> Transferir estas moscas en un vial nuevo e incubar durante 12 h a 25 ° C. NOTA: Deseche el primer vial ya que las hembras virgen no ponen suficientes huevos en el primer día. </li><li> Para líneas enhancer-GAL4, retire las moscas adultas e incube el vial a 25 ° C hasta disección. </li><li> Para la inducción de los clones del mosaico mediante el flip-out-GAL4, retire las moscas adultas e incube el vial durante 2-4 días a 25 ° C. El tiempo de incubación antes del choque térmico depende del propósito experimental. Un choque térmico 2 días después de la deposición del huevo (AED) genera clones de mosaico en segundos inmadurosInstar epitelios imaginal. Un shock térmico después de 3 ó 4 días de DEA genera clones de mosaico en epitelios imaginarios diferenciados de instar temprano a mediados del tercer instar. NOTA: Es importante examinar el efecto del tiempo de choque térmico alterado en los fenotipos tumorigénicos. </li><li> Para la inducción de los clones de mosaico mediante flip-out-GAL4, calentar el vial por inmersión en un baño de agua a 37 ° C durante 10 - 45 min seguido de incubación durante 2 - 3 días a 25 ° C. NOTA: La información del tiempo de choque térmico, el tiempo y el tiempo de incubación en cada experimento se muestran en las leyendas de la figura. </li></ol> 2. Disección de las larvas <ol><li> Recoja las larvas de tercer estadio errantes con una pinza de madera o fórceps romos, genotipo por marcadores fluorescentes apropiados ( por ejemplo , EGFP) bajo un microscopio estereoscópico de fluorescencia y colóquelas en un plato de disección con PBS (solución salina tamponada con fosfato). </li><li> Lavar las larvas en PBS pipeteando con pipetas de transferencia de plástico de 2 ml. </li><li>Pellizque el centro de la larva con una pinza y rompa el cuerpo por la mitad con la otra pinza. </li><li> Pellizque el gancho de la boca de la mitad anterior con una pinza y empuje la boca hacia el cuerpo con la otra pinza para girar el cuerpo de adentro hacia afuera. </li><li> Eliminar los materiales innecesarios, como las glándulas salivales o los cuerpos de grasa con fórceps. </li></ol> 3. Fijación y tinción de anticuerpos <ol><li> Transferir la mitad anterior disecada del cuerpo larval (incluyendo los discos imaginarios del ala) a un tubo plástico de 1,5 ml y fijar en 1 mL de solución Fix (formaldehído 4% en PBS) durante 10 min a temperatura ambiente en la oscuridad con rotación suave. PRECAUCIÓN: El formaldehído es tóxico y tiene un potencial carcinógeno. Use guantes y ropa protectora para evitar el contacto con la piel. NOTA: En esta parte, todas las etapas tienen lugar en un nutator a temperatura ambiente en la oscuridad a menos que se indique lo contrario. </li><li> Quite la solución Fix y deséchela. Lave los tejidos con 1 mL de PBT (0.3% TritEn X-100 en PBS) tres veces durante 15 min cada uno. </li><li> Eliminar el PBT y añadir 1 ml de PBTG (0,2% de suero de bóvidos bovinos y 5% de suero normal de cabra en PBT) para bloquear y nutate 1 h a temperatura ambiente o durante la noche a 4ºC. </li><li> Quitar PBTG y añadir la solución de anticuerpo primario diluido apropiadamente con PBTG (véase Tabla de Materiales ) y nutate durante la noche a 4 ° C. </li><li> Eliminar la solución de anticuerpo primario y lavar los tejidos con 1 mL de PBT tres veces durante 15 minutos cada uno. </li><li> Eliminar PBT y añadir una solución de anticuerpo secundaria debidamente diluida con PBTG (1: 400). Nutate durante 2 h a temperatura ambiente o durante una noche a 4 ° C. </li><li> Eliminar la solución de anticuerpos secundarios y lavar los tejidos con 1 mL de PBT dos veces durante 15 minutos cada uno. </li><li> Para teñir F-actina, eliminar PBT y añadir Phalloidin solución diluida adecuadamente en PBS (1:40). A continuación, nutate durante 20 min. Retire la solución de Phalloidin y lave los tejidos con 1 mL de PBT dos veces durante 15 min cada uno. </li><li> Para contra-tinción de ADN nuclear, eliminar PBT y añadir DAPI (4 &#39;, 6-diamidino-2-fenilindole) solución (0,5 μ g / mL de DAPI en PBS). A continuación, nutate 10 min. PRECAUCIÓN: DAPI tiene potencial carcinógeno. Use guantes y ropa protectora para evitar el contacto con la piel. </li><li> Eliminar la solución DAPI y lavar los tejidos con 1 mL de PBT dos veces durante 15 minutos cada uno. </li><li> Enjuagar una vez en 1 mL de PBS durante 10 min a temperatura ambiente. </li><li> Eliminar PBS y añadir 500 μ l de 100% de glicerol como el medio de pre-montaje. </li></ol> 4. Montaje en portaobjetos de microscopio <ol><li> Coloque los tejidos teñidos en una diapositiva de microscopio con una pipeta de transferencia de plástico de 2 ml. </li><li> Transferir los tejidos a gotas de medio de montaje en otra diapositiva de microscopio con fórceps. </li><li> Mantenga presionado el extremo del tejido diseccionado con una pinza y retire los discos antenales del cerebro y los ojos con las otras pinzas. NOTA: Mantenga los cerebros disecados para colocarlos cerca de los discos imaginales del ala. Los cerebros actúan como una plataforma que impide que el cubreobjeto aplaste los discos imaginal del ala. </li><li> Para aislar el ala los discos imaginales mantienen el extremo del tejido disecado con una pinza y rascan suavemente la pared del cuerpo y arrancan los discos con las otras pinzas. NOTA: Si es difícil encontrar discos imaginal del ala, pelar la tráquea desde el lado posterior al lado anterior. Los discos imaginarios del ala se adhieren a la tráquea. </li><li> Cubra suavemente los discos imaginales con un cubreobjetos y selle con esmalte de uñas; Almacenar a 4 ° C. </li></ol> 5. Microscopía confocal <ol><li> Para adquirir imágenes confocales usando un microscopio confocal, fije los parámetros de adquisición de imágenes, incluyendo el rango de longitud de onda de emisión, intensidad láser, ganancia, offset, velocidad de exploración y tamaño de imagen 24 . NOTA: Para el procedimiento detallado de los ajustes de adquisición de imágenes, consulte el manual de instrucciones suministrado por cada fabricante de microscopio. </li><li> Para capturar solo coSecciones nfocal de un disco imaginal del ala entera, utilice un objetivo 20X. </li><li> Adquiera imágenes tridimensionales mediante escaneo de pilas en z con tamaño de paso de 0,5 a 1,0 μm usando lentes de 40X o 60X. NOTA: Para analizar los fenotipos celulares en alta resolución, un tamaño de imagen debe ser mayor de 512 x 512 píxeles. </li></ol> 6. Análisis de imágenes con ImageJ <ol><li> Utilice Fiji, un software de código abierto ImageJ enfocado en el análisis de imágenes biológicas (https://fiji.sc/) 25 , para adquirir y analizar las imágenes confocal de la z-pila. </li><li> Para obtener secciones verticales, abra las imágenes de pila z en ImageJ y seleccione el elemento de menú &quot;Imagen / pilas / Reslice&quot;. NOTA: Tanto el eje XZ como el eje YZ se pueden seleccionar en el menú Reslice. La sección vertical puede obtenerse también en una dirección arbitraria dibujando una línea recta o rectángulo sobre la imagen de la pila z. </li></ol> 7. Diagnóstico de los fenotipos neoplásicos <ol><li> Abrir la vertical(Eje XZ o YZ) obtenidos a partir de un conjunto de imágenes de pila z y analizar fenotipos morfológicos y tinción de anticuerpos. </li><li> Para el diagnóstico de los fenotipos tumorales, se centran principalmente en los siguientes tres puntos: (1) si una masa celular, incluyendo clones knockdown, se desvía de la capa epitelial principal, (2) si la localización subcelular de las proteínas de unión se altera en esta masa celular , Y (3) si el diámetro de esta masa celular es mayor que 4 células. </li><li> Clasificar los fenotipos tumorales de acuerdo con el diagrama de flujo descrito en la Figura 1 . </li></ol>

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Los autores no tienen nada que revelar.

El sistema GAL4-UAS es una de las herramientas genéticas más potentes para la expresión génica dirigida en Drosophila 26 y facilita en gran medida la inducción y el análisis de células tumorales in vivo 4 . Este sistema permite la generación de clones que tienen knockdown de genes supresores de tumores o sobreexpresión de oncogenes dentro de tejido epitelial de tipo salvaje, una situación muy similar a las etapas iniciales de cáncer humano en d...

Los tejidos epiteliales son sistemas altamente organizados que tienen la notable capacidad homeostática de mantener su organización a través del desarrollo y la renovación celular. Este robusto sistema de auto-organización, sin embargo, se interrumpe progresivamente durante el desarrollo del tumor. Al comienzo del desarrollo del tumor, las células mutantes individuales que surgen de la activación del oncogén o la inactivación del gen supresor del tumor emergen dentro de una capa epitelial. Cuando esta transformada &quot;célula pro-tumor&quot; evade un ambiente supresor, interrumpe la organización epitelial, y comienza la proliferación incontrolada, la tumorigénesis se produce 1 . Durante las últimas décadas, avances tecnológicos sobresalientes en genética y biología molecular han hecho progresos notables en la investigación del cáncer. En particular, los estudios recientes que utilizan los instrumentos de análisis genéticamente mosaico en Drosophila melanogaster , tales como la recombinación mitótica FLP-FRT (recombinante flippase / flase recombinase)ación 2 y flip-out-GAL4-UAS sistemas (secuencia de activación aguas arriba) 3, han contribuido enormemente a una mejor comprensión de los mecanismos genéticos implicados en la formación y metástasis de tumores 4, 5, 6. Los estudios de un grupo de genes supresores de tumores conservados de Drosophila , larvas gigantes letales ( lgl ), discos grandes ( dlg ) y garabatos ( scrib ), resaltaron la relación crítica entre la pérdida de organización epitelial y el desarrollo tumoral, ya que estos genes desempeñan papeles clave En la regulación de la polaridad celular apical-basal y la proliferación celular en los tejidos epiteliales [ 7] . Mientras que los discos imaginarios de Drosophila son normalmente epitelios monocapa, las mutaciones homocigóticas en cualquiera de estos tres genes hacen que las células pierdan estructura y polaridad, no difierenRentizar, sobreproliferar y, finalmente, formar masas amorfas multicapa que se funden con tejidos adyacentes 7 . Del mismo modo, la interrupción de estos genes en los mamíferos está involucrado en el desarrollo de tumores malignos [ 8 , 9] . Los fenotipos neoplásicos exhibidos por los tejidos mutantes han llevado a la clasificación de estos tres genes conservados, genes neoplásicos supresores de tumores (nTSGs) [ 7 , 8] . Sin embargo, cuando homozygous nTSG células mutantes son esporádicos generados en el desarrollo de tipo salvaje discursos imaginales utilizando FLP-FRT mediada por la recombinación mitótica, las células mutantes son eliminados del tejido a través de c-Jun N-terminal quinasa (JNK) -dependiente apoptosis 10 , 11 , 12 , 13 , 14 , extrusión 15 </suP&gt; , 16 , o el engolfamiento y la fagocitosis por los vecinos [ 17] . En este epitelio genéticamente mosaico, la apoptosis se detecta principalmente en las células mutantes nTSG localizadas en el límite del clon, lo que sugiere que las células normales adyacentes desencadenar la apoptosis de nTSG células mutantes [ 10 , 11 , 12 , 18] . Estudios recientes en células de mamíferos han confirmado que esta eliminación celular dependiente de la competencia de las células pro-tumor es un mecanismo evolutivo conservado epiteliales de autodefensa contra el cáncer 19 , 20 , 21 , 22 , 23 . Un reciente estudio en Drosophila imaginal discos, sin embargo, mostró que mosaico nTSG-knockdown clones induce tumores neoplásicos en especificaIc de los discos imaginal de ala 16 . La formación inicial de tumores se encontró siempre en la región periférica de la &quot;bisagra&quot; y nunca se observó en la región central de la &quot;bolsa&quot; del epitelio del disco de ala, lo que sugiere que el potencial tumorigénico de las células knockdown nTSG depende del entorno local. La región de la bolsa central funciona como una &quot;mancha fría del tumor&quot; donde las células pro-tumor no muestran sobrecrecimiento displásico, mientras que la región bisagra periférica se comporta como un &quot;hotspot tumoral&quot; 16 . En las regiones de bolsa &quot;coldspot&quot;, las células de desmontaje de nTSG se deslaminan desde el lado basal y sufren apoptosis. Por el contrario, como &quot;hotspot&quot; bisagra células poseen una red de estructuras robusto citoesqueleto en sus lados basales, nTSG-knockdown células delaminate desde el lado apical del epitelio e iniciar tumorigenic sobrecrecimiento [ 16] . Por lo tanto, el análisis de los fenotipos tumorales en discos imaginal requiereLa susceptibilidad específica de la región a estímulos tumorigénicos. En este sentido, se describe un protocolo para inducir la formación de tumores neoplásicos en el disco Drosophila imaginal discos utilizando el GAL4-UAS- RNAi sistema por el cual nTSG-knockdown células se generan en el epitelio del disco de ala normal. Aunque estos sistemas experimentales son útiles para estudiar las primeras etapas del cáncer, no se ha descrito claramente un método claro de clasificación para evaluar las etapas de progresión tumoral en epitelios discales imaginarios. Por lo tanto, también proponemos un método de diagnóstico para clasificar los fenotipos clonales pro-tumorales inducidos en el epitelio del disco ala en tres categorías: hiperplasia (acumulación de un número excesivo de células de aparición normal con aumento de la proliferación), displasia (tejido premaligno compuesto de anormalmente aparente Células), y neoplasia (tumor benigno o maligno compuesto de células que tienen un aspecto anormal y un patrón de proliferación anormal).

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS)</td>
			<td>Wako</td>
			<td>162-19321</td>
			<td></td>
		</tr>
		<tr>
			<td>TritonX-100</td>
			<td>Wako</td>
			<td>168-11805</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Wako</td>
			<td>064-00406</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>normal goat serum</td>
			<td>Sigma</td>
			<td>G6767</td>
			<td></td>
		</tr>
		<tr>
			<td>mounting medium, Vectashield</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>mouse-anti-Dlg 4F3</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>4F3 anti-discs large, RRID:AB_528203</td>
			<td>dilute in PBTG, 1:40</td>
		</tr>
		<tr>
			<td>mouse-anti-MMP1</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>3A6B4, RRID:AB_579780</td>
			<td>3 mixed 1:1:1 and dilute in PBTG, 1:40</td>
		</tr>
		<tr>
			<td>mouse-anti-MMP1</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>3B8D12, RRID:AB_579781</td>
			<td>3 mixed 1:1:1 and dilute in PBTG, 1:40</td>
		</tr>
		<tr>
			<td>mouse-anti-MMP1</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>5H7B11, RRID:AB_579779</td>
			<td>3 mixed 1:1:1 and dilute in PBTG, 1:40</td>
		</tr>
		<tr>
			<td>mouse-anti-atubulin</td>
			<td>Developmental Studies Hybridoma Bank</td>
			<td>AA4.3, RRID:AB_579793</td>
			<td>dilute in PBTG, 1:100</td>
		</tr>
		<tr>
			<td>Alexa Fluor 546 Phalloidin</td>
			<td>Molecular probes</td>
			<td>A22283</td>
			<td>dilute in PBS, 1:40</td>
		</tr>
		<tr>
			<td>goat anti-mouse IgG antibody, Alexa Fluor 546</td>
			<td>Molecular probes</td>
			<td>A11030</td>
			<td>dilute in PBTG, 1:400</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Fly strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sd-Gal4</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#8609</td>
			<td>recombined with UAS-EGFP</td>
		</tr>
		<tr>
			<td>upd-Gal4</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#26796</td>
			<td>recombined with UAS-EGFP</td>
		</tr>
		<tr>
			<td>UAS-lgl-RNAi</td>
			<td>Vienna Drosophila RNAi center</td>
			<td>#51247</td>
			<td></td>
		</tr>
		<tr>
			<td>UAS-scrib-RNAi</td>
			<td>Vienna Drosophila RNAi center</td>
			<td>#105412</td>
			<td></td>
		</tr>
		<tr>
			<td>UAS-RasV12</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#64196</td>
			<td></td>
		</tr>
		<tr>
			<td>UAS-Yki3SA</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#28817</td>
			<td></td>
		</tr>
		<tr>
			<td>hsFLP</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#6</td>
			<td></td>
		</tr>
		<tr>
			<td>Act&#62;CD2&#62;GAL4 (flip-out GAL4)</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#4780</td>
			<td>recombined with UAS-EGFP</td>
		</tr>
		<tr>
			<td>UAS-EGFP</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#5428</td>
			<td>X chromosome</td>
		</tr>
		<tr>
			<td>UAS-EGFP</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#6658</td>
			<td>third chromosome</td>
		</tr>
		<tr>
			<td>UAS-Dicer2</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#24650</td>
			<td>second chromosome</td>
		</tr>
		<tr>
			<td>UAS-Dicer2</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>#24651</td>
			<td>third chromosome</td>
		</tr>
		<tr>
			<td>vkg-GFP</td>
			<td>Morin et al. 2001</td>
			<td></td>
			<td>GFP protein trap</td>
		</tr>
	</tbody>
</table>

induction and diagnosis of tumors in drosophila imaginal disc epithelia

En las primeras etapas del cáncer, las células mutantes transformadas muestran anormalidades citológicas, comienzan un crecimiento excesivo no controlado y alteran progresivamente la organización tisular. Drosophila melanogaster ha surgido como un modelo experimental popular en el sistema de biología del cáncer para estudiar los mecanismos genéticos y celulares de la tumorigénesis. En particular, las herramientas genéticas para los discos imaginarios de Drosophila (desarrollo de epitelios en larvas) permiten la creación de células pro tumorales transformadas dentro de un tejido epitelial normal, situación similar a las etapas iniciales del cáncer humano. Sin embargo, un estudio reciente de tumorigénesis en discos imaginarios de alas de Drosophila demostró que la iniciación tumoral depende de la citoarquitectura intrínseca del tejido y del microambiente local, lo que sugiere que es importante considerar la susceptibilidad específica de la región a los estímulos tumorigénicos en la evaluación de fenotipos tumorales en imaginal Discos Para facilitar el análisis fenotípico del progreso tumoralEn discos imaginal, aquí se describe un protocolo para experimentos genéticos utilizando el sistema GAL4-UAS para inducir tumores neoplásicos en discos imaginal de ala. Además, hemos introducido un método de diagnóstico para clasificar los fenotipos de las lesiones clonales inducidas en el epitelio imaginal, como un método de clasificación clara para discriminar las diversas etapas de la progresión tumoral (como la hiperplasia, displasia o neoplasia) no se había descrito anteriormente. Estos métodos podrían aplicarse ampliamente al análisis clonal de fenotipos tumorales en diversos órganos de Drosophila .

Para demostrar la formación de tumores neoplásicos inducidos experimentalmente por RNAi mediada nTSG-knockdown en Drosophila ala imaginal discos, tres diferentes GAL4 conductores se utilizaron para expresar UAS-RNAi para lgl o scrib : (1) sd-GAL4, que impulsa fuerte UAS expresión en el Bolsa de ala y expresión suave en las regiones de bisagra ( Figura 2 y Figura 3A ); (2)...

Watch this Scientific Journal Video about Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia at JoVE.com

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

El análisis de clones mosaicos en el epitelio discal imaginario de Drosophila es un poderoso sistema modelo para estudiar los mecanismos genéticos y celulares de la tumorigénesis. Aquí se describe un protocolo para inducir tumores en Drosophila ala imaginal discos utilizando el sistema GAL4-UAS, e introducir un método de diagnóstico para clasificar los tumores fenotipos.

Inducción y diagnóstico de tumores en<em&gt; Drosophila</em&gt; Imaginal Disc Epitelio

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue ...

induction-diagnosis-tumors-drosophila-imaginal-disc

Research

JoVE Journal

Cancer Research

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

14.8K vistas.  SOKENDAI.  El análisis de clones mosaicos en el epitelio discal imaginario de Drosophila es un poderoso sistema modelo para estudiar los mecanismos genéticos y celulares de la tumorigénesis. Aquí se describe un protocolo para inducir tumores en Drosophila ala imaginal discos utilizando el sistema GAL4-UAS, e introducir un método de diagnóstico para clasificar los tumores fenotipos. 

Video: Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

Siguiente Generación de secuenciación para la detección de mutaciones procesable en tumores sólidos y líquidos

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

Investigación

Investigación sobre el cáncer

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

los<em&gt; Drosophila</em&gt; Imaginal modelo de tumor de disco: visualización y cuantificación de la expresión génica y la invasividad tumoral Uso Genético Mosaicos

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

Desarrollo de un modelo humano de preclínico de osteoclastogénesis de monocitos de sangre periférica cultivadas conjuntamente con líneas de células de cáncer de mama

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

Establecimiento de un cultivo primario de Sarcoma de tejidos blandos derivados del paciente

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

Enriquecimiento y caracterización de los microambientes Tumor inmunes y no inmunes en tumores murinos subcutáneos establecidos

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

Modelo murino de tumores hepáticos metastásicos en el escenario de la lesión por reperfusión de isquemia

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Obtaining Cancer Stem Cell Spheres from Gynecological and Breast Cancer Tumors

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and recurrent disease. This population can be identified by surface markers, enzymatic activity and a functional profile. These approaches per se are limited, due to phenotypic heterogeneity and CSC plasticity. Here, we update the sphere-forming protocol to obtain CSC spheres from breast and gynecological cancers, assessing functional properties, CSC markers and protein expression. The spheres are obtained with single-cell seeding at low density in suspension culture, using a semi-solid methylcellulose medium to avoid migration and aggregates. This profitable protocol can be used in cancer cell lines but also in primary tumors. The tridimensional non-adherent suspension culture thought to mimic the tumor microenvironment, particularly the CSC-niche, is supplemented with epidermal growth factor and basic fibroblast growth factor to ensure CSC signaling. Aiming for robust identification of CSC, we propose a complementary approach, combining functional and phenotypic evaluation. Sphere-forming capacity, self-renewal and sphere projection area establish CSC functional properties. Additionally, characterization comprises flow cytometry evaluation of the markers, represented by CD44+/CD24- and CD133, and Western blot, considering ALDH. The presented protocol was also optimized for primary tumor samples, following a sample digestion procedure, useful for translational research.

The aim of this methodology is to identify cancer stem cells (CSC) in cancer cell lines and primary human tumor samples with the sphere-forming protocol, in a robust manner, using functional assays and phenotypic characterization with flow cytometry and Western blot.

Obtención de esferas de células madre de cáncer de cáncer de tumores ginecológicos y de cáncer de mama

Cancer stem cells (CSC) are a small population with self-renewal and plasticity which are responsible for tumorigenesis, resistance to treatment and ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Trasplante ortotópico de tumores de mama como modelos preclínicos para el cáncer de mama

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Quail Chorioallantoic Membrane - A Tool for Photodynamic Diagnosis and Therapy

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties make it an excellent in vivo experimental model to study angiogenesis, tumor growth, drug delivery systems, or photodynamic diagnosis (PDD) and photodynamic therapy (PDT). At the same time, this model addresses the requirement for the replacement of experimental animals with a suitable alternative. Ex ovo cultivated embryo allows easy substance application, access, monitoring, and documentation. The most frequently used is chick CAM; however, this article describes the advantages of the Japanese quail CAM as a low-cost and high-throughput model. Another advantage is the shorter embryonic development, which allows higher experimental turnover. The suitability of quail CAM for PDD and PDT of cancer and microbial infections is explored here. As an example, the use of the photosensitizer hypericin in combination with lipoproteins or nanoparticles as a delivery system is described. The damage score from images in white light and changes in fluorescence intensity of the CAM tissue under violet light (405 nm) was determined, together with analysis of histological sections. The quail CAM clearly showed the effect of PDT on the vasculature and tissue. Moreover, changes like capillary hemorrhage, thrombosis, lysis of small vessels, and bleeding of larger vessels could be observed. Japanese quail CAM is a promising in vivo model for photodynamic diagnosis and therapy research, with applications in studies of tumor angiogenesis, as well as antivascular and antimicrobial therapy.

The chorioallantoic membrane (CAM) of the avian embryo is a very useful and applicable tool for various areas of research. A special ex ovo model of Japanese quail CAM is suitable for photodynamic treatment investigation.

Membrana corioalantoidea de codorniz: una herramienta para el diagnóstico fotodinámico y la terapia

The chorioallantoic membrane (CAM) of an avian embryo is a thin, extraembryonic membrane that functions as a primary respiratory organ. Its properties ...

High-Resolution Ultrasonography for the Analysis of Orthotopic ATC Tumors in a Genetically Engineered Mouse Model

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes significantly. Genetically engineered murine models that mimic ATC&#39;s progression may help researchers to study treatments for this disease. Crossing three different genotypes of mice, a TPO-cre/ERT2; BrafCA/wt; Trp53&#916;ex2-10/&#916;ex2-10&#160;transgenic ATC model was developed. The ATC murine model was induced by an intraperitoneal injection of tamoxifen with overexpression of BrafV600E and deletion of Trp53, and the tumors were generated within about 1 month. High-resolution ultrasound was applied to investigate the tumor initiation and progression, and the dynamic growth curve was obtained by measuring the tumor sizes. Compared to magnetic resonance imaging (MRI) and computed tomography scanning, ultrasound has advantages in observing the ATC murine model, such as being noninvasive, portable, in real-time, and without radiation exposure. High-resolution ultrasound is suitable for dynamic and multiple measurements. However, ultrasonographic examination of the thyroid in mice requires relevant anatomical knowledge and experience. This article provides a detailed procedure for utilizing high-resolution ultrasound to scan tumors in the transgenic ATC model. Meanwhile, ultrasonic parameter adjustment, ultrasound scanning skills, anesthesia and recovery of the animals, and other elements that need attention during the process are listed.

The present protocol describes high-frequency ultrasonography for visualizing the entire mouse thyroid gland and monitoring the growth of anaplastic thyroid carcinoma.

Ultrasonografía de alta resolución para el análisis de tumores ATC ortotópicos en un modelo de ratón modificado genéticamente

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes ...