Este trabajo fue apoyado por la fundación alemana de la investigación (WE 3548 / 4-1) y Roland-Ernst-Stiftung für Gesundheitswesen (1/14).

Los experimentos con animales presentados aquí fueron revisados ​​y permitidos de forma independiente por un Comité de Cuidado de Animales y Uso Animal institucional y gubernamental y fueron conducidos de acuerdo con las directrices de la Federación de Asociaciones de Ciencias de Animales de Laboratorio (FELASA). Se tomaron todas las medidas posibles para minimizar el sufrimiento, incluyendo anestesia y analgesia o, si es necesario, eutanasia prematura. 1. Preparación de células y organoides NOTA: Utilice un volumen de 20 μL con 100.000 células para cada inyección. Utilizar la matriz de la membrana basal (BMM) para evitar fugas y asegurar la inyección estandarizada. Para asegurar resultados reproducibles, realice ensayos de autenticación de líneas celulares ( por ejemplo , mediante perfiles STR) a intervalos regulares. <ol><li> Preparación de suspensiones celulares primarias a partir de tejido fresco NOTA: Siempre trabaje bajo condiciones estériles. La transferencia inmediata de losEl tejido resecado de la sala de operaciones al laboratorio sobre hielo es necesario para asegurar la alta viabilidad de las células. El bienestar del paciente y el tratamiento óptimo deben ser siempre la primera prioridad. Por lo tanto, las muestras de tejido para la investigación deben obtenerse de una manera que no interfiera con el posterior tratamiento patológico y estadificación del tumor resecado. En la mayoría de los casos, por lo tanto, es razonable tener las muestras para la investigación obtenida por un patólogo entrenado con el fin de garantizar la no interferencia con el diagnóstico patológico. <ol><li> Inmediatamente después de la extracción estéril de la muestra resecada (idealmente ~ 1 cm 3 ), coloque la muestra de tejido en un tubo de 50 mL previamente llenado con 20-30 mL de solución salina tamponada con fosfato (PBS). Guarde el tubo en hielo y transfiéralo inmediatamente al laboratorio. </li><li> Poner el tejido en una placa de Petri y lavar dos veces con un montón de PBS para eliminar la sangre restante. </li><li> Cortar el tejido en trozos pequeños (~ 2-4 mm) con una scaLpel ( por ejemplo , con una cuchilla # 20 o # 36). </li><li> Utilizar el disociador de acuerdo con las instrucciones del fabricante con el fin de disociar aún más el tejido tumoral a una suspensión de una sola célula. Alternativamente, utilice otros protocolos de digestión enzimática del tumor. </li><li> Contar las células de la suspensión de una sola célula resultante ( por ejemplo , en un contador de cuchillas o un hemocitómetro). </li><li> Centrifugar (5 min a 1.500 xg) y lavar la suspensión celular dos veces con PBS. </li><li> Resuspender las células en BMM a una concentración de 5 x 10 6 células / mL y mantenerlos en hielo. </li></ol></li><li> Preparación de líneas celulares para inyección <ol><li> Cultivar todas las líneas celulares de cáncer colorrectal en condiciones normales de cultivo (37 ° C, 5% CO 2 ) y prepararlas el día de la cirugía. </li><li> Cosechar las células de acuerdo con los protocolos de cultivo de células estándar, contar las células ( por ejemplo , en un contador de cuchara o un hemocitómetro) y calcular la demandaCantidad necesaria para todas las inyecciones dependiendo del número de animales a inyectar. </li><li> Prepare 3-5x del volumen realmente necesario para tener en cuenta las pérdidas de pipeteo y el volumen muerto de la jeringa de inyección. </li><li> Centrifugar (5 min a 1.500 g) y lavar la suspensión celular dos veces con PBS. </li><li> Resuspender las células en BMM a una concentración de 5 x 10 6 células / mL y mantenerlos en hielo. </li></ol></li><li> Preparación de los organoides NOTA: Todos los cultivos organoide 3D se cultivan en condiciones de cultivo estándar (37ºC, 5% CO 2 ). Ya se han publicado protocolos detallados para el cultivo de organoides. 18 , 19 , 20 Similar a las líneas celulares convencionales, se recomienda un volumen de 20 μL de BMM con 100.000 células por inyección. Los organoides se preparan el día de la cirugía como sigue: <ol><li> Preparar el siguiente medio (en los siguientesA DMEM / F12 +++): Medio de Eagle modificado de Dulbecco avanzado (DMEM) / F12 + 1% de HEPES (1M) + 1% de glutamina (200 mM) + 1% de penicilina / estreptomicina. </li><li> Pre-llenar tubos de 15 mL con 1 mL de DMEM / F12 +++. </li><li> Aspirar cuidadosamente los organoides de la superficie de la placa de cultivo y transferir el contenido de 3 a 5 pocillos en un tubo de 15 ml. </li><li> Cuidadosamente romper las organoides en trozos más pequeños por pipetearlos arriba y abajo con una pipeta de vidrio extendida. </li><li> Añadir 5 mL de DMEM / F12 +++ al tubo, seguido de centrifugación a 1.000 xg durante 5 min. </li><li> Después de la centrifugación, se retira el sobrenadante, se resuspende el sedimento en 600 μl de tampón de disociación enzimática y se transfiere la suspensión a un pocillo de una placa de 6 pocillos. </li><li> Incubar a 37 ° C durante 1-5 minutos y luego cuidadosamente pipetear la suspensión hacia arriba y hacia abajo con el fin de disolver los organoides. NOTA: Verificar la digestión a través del microscopio; Se completa cuando todos los clusters de células se han disociadoD a las células individuales. </li><li> Tras la digestión exitosa, agregar 1,4 ml de DMEM / F12 +++ para detener la digestión. Luego transferir todos los pocillos de organoides digeridos a un tubo de 50 ml. </li><li> Cuente las células y calcule la cantidad requerida para todas las inyecciones. </li><li> Prepare de 3 a 5 veces el volumen realmente necesario para tener en cuenta las pérdidas de pipeteado y el volumen muerto de la jeringa de inyección </li><li> Centrifugar (5 min a 1.500 xg) y lavar la suspensión celular dos veces con PBS. </li><li> Se resuspendieron las células en BMM a una concentración de 5 x 106 células / mL y se mantuvieron en hielo. </li></ol></li></ol> 2. Modelo de ratón ortotópico <ol><li> Preparación de los animales receptores para la cirugía NOTA: Utilice como destinatarios a los ratones NOD.Cg-Prkdc scid de 6 a 8 semanas de edad como ratones II2rg tm1Wjl / SzJ (NOD scid gamma, NSG). 21 NSG están entre los ratones más inmunocomprometidos, carecen de células maduras B, T y NK junto con otros múltiples inmunes.Los efectos. 21 Ellos crecen de forma fiable los tumores incluso si se inyectan bajo número de células y son muy propensos a la metástasis a distancia. Los ratones NSG son excelentes reproductores y pueden mantenerse en unidades convencionales libres de patógenos específicos (SPF). <ol><li> Antes de la primera incisión, inyectar 0,05 mg / kg de buprenorfina por vía subcutánea. </li><li> Utilice sevoflurano al 3-3.5% en volumen para anestesia general. Una pérdida del reflejo de pinza del dedo del pie indica suficiente anestesia. </li><li> Cubrir los ojos de los ratones anestesiados con pomada oftálmica para evitar la desecación de la córnea. </li><li> Detenga los ratones en posición supina sobre una mesa pequeña. </li><li> Afeitar el abdomen con una máquina de afeitar eléctrica (la crema depilatoria se puede utilizar alternativamente) y desinfectar con al menos 3 veces clorhexidine / yodo y alcohol del 70% alternativamente. </li><li> Cubrir el campo quirúrgico con cortinas estériles. NOTA: El uso de antibióticos perioperatorios es opcional y está sujeto a las pautas institucionales. </li></ol></li><li> Laparotomía de la línea media y la exposición del ciego <ol><li> Use tijeras (los bisturíes se pueden usar alternativamente) para hacer una pequeña incisión de la línea media (3 - 5 mm) de la piel en el bajo abdomen. Recoger la musculatura de la pared abdominal con fórceps y cuidadosamente incise con tijeras, abriendo así la cavidad abdominal. </li><li> Identificar y cuidadosamente exteriorizar el ciego con pinzas atraumáticas. Coloque la bolsa de terminación ciega del ciego en el abdomen apuntando cranealmente. </li><li> Una vez exteriorizado, mantenga el ceco húmedo utilizando hisopos salinos tibios en todo momento. </li></ol></li><li> Inyección ortotópica, cierre del abdomen y recuperación postoperatoria <ol><li> Para inyección intracecal, use una jeringa estándar de 1 ml con una cánula de 30 G. Monte esta jeringa en una bomba de microinyección, que a su vez se monta en un micromanipulador. </li><li> Cuidadosamente agarre la punta del ciego con fórceps atraumático y suavemente suavizarlo acariciándolo downwarD con un segundo conjunto de fórceps humedecido con solución salina caliente. </li><li> Coloque la cánula directamente sobre el ciego. </li><li> Realice los siguientes pasos bajo control visual con un microscopio quirúrgico binocular. </li><li> Coger con cuidado el ciego con dos pinzas atraumáticas en ambos extremos de la parte exteriorizada del ciego, estirarlo ligeramente y luego tirar lentamente sobre la cánula que se coloca paralelo y directamente encima. Es crucial no perforar toda la pared intestinal (inyectando así las células en el lumen), así como no perforar la serosa más allá del punto inicial de penetración, ya que esto conduciría a la fuga ya la diseminación peritoneal. <ol><li> Mueva el intestino hacia la cánula, no la cánula hacia el intestino. Sostenga y estire el intestino entre dos fórceps. Las manos del cirujano deben descansar sobre una superficie para reducir el temblor. </li></ol></li><li> Inyectar las células entre la serosa (visto como un revestimiento muy fino y translúcidoE intramurales) y la muscular. Por lo tanto, la cánula debe colocarse visualmente por encima de los vasos sanguíneos y por debajo de la delgada membrana translúcida. </li><li> Utilice un interruptor de pie para iniciar la inyección con el fin de reducir el temblor mientras la cánula está dentro de la pared intestinal. </li><li> Una vez que la cánula está en posición, iniciar la inyección. Utilice una duración de 20 s, resultando en un ajuste de 1 μl / s en la unidad de control de la bomba. NOTA: La inyección en o cerca de un vaso sanguíneo dañado conduce a diseminación intravascular directa ya metástasis a distancia y por lo tanto debe evitarse. </li><li> Después de completar la inyección, retire cuidadosamente la cánula tirando hacia atrás. </li><li> Coloque un hisopo seco debajo del ciego, y luego enjuague completamente el ciego con agua destilada con el fin de lisar las células filtradas y evitar así la difusión peritoneal artificial. </li></ol></li><li> Cierre del abdomen y recuperación postoperatoria <ol><li> Después de rInsing, devolver suavemente el ciego a la cavidad abdominal. </li><li> Cierre la pared abdominal con suturas corrientes de absorción rápida de 6-0. </li><li> Cierre la piel con clips para heridas quirúrgicas. </li><li> Coloque el ratón en un mapa de calefacción establecido a 38 ° C hasta que se ha recuperado completamente de la anestesia. </li><li> Observar de cerca el estado postoperatorio de los ratones durante las siguientes 48 h. En caso de malestar, tratar con 0,05 mg / kg de buprenorfina cada 12 h. </li><li> Vigilar a los ratones al menos una vez al día para detectar signos de angustia debido al crecimiento del tumor. </li></ol></li></ol> 3. Aislamiento de células tumorales circulantes de muestras de sangre entera NOTA: Obtenga sangre por punción cardiaca transcutánea en ratones anestesiados, seguida de eutanasia. Idealmente, se inyectan 1.000 μl de sangre en una jeringa precargada con 100 μl de un anticoagulante (ácido etilendiaminotetraacético (EDTA) o heparina). Utilizar un anti-humano-EpCAM (adhesión de células epitelialesMolécula) para identificar los CTC. Esto funciona muy bien si se usan líneas de células epiteliales humanas en el modelo de ratón. Para otros aparatos, pueden ser necesarios diferentes anticuerpos. <ol><li> Enriquecimiento de CTC: <ol><li> Pre-llene los tubos de 15 mL con 5 mL de medio de gradiente de densidad y transfiera cuidadosamente la sangre al tubo de 15 mL. </li><li> Centrifugar (30 min a 300 xg sin freno) y retirar cuidadosamente el sobrenadante superior. </li><li> Verter el resto en un nuevo tubo de 15 ml y centrifugar (15 min, 300 xg con freno). </li><li> Recuperar la interfase que contiene las células mononucleares (descarte el resto) y lavar las células dos veces con PBS. </li><li> Resuspender el sedimento en 200 μL de PBS / 1% de EDTA y añadir 4 μl de anticuerpo EpCAM. Incubar 20 min en hielo en la oscuridad. </li></ol></li><li> Tamizaje y selección <ol><li> Preparar el siguiente tampón (en lo siguiente denominado tampón de recogida): PBS + 10% de suero de ternera fetal (FCS) + 1% de penicilina / STreptomicina (1%) + 0,8% de EDTA. </li><li> Utilice un lápiz PAP para dibujar un círculo de ~ 1 cm en una placa de Petri estéril de 6 cm (evita que el líquido se disperse en el plato) y pipetee 700 μl de tampón de recogida en este círculo. </li><li> Añadir 50 μl de suspensión celular al tampón de recogida de 700 μl dentro del círculo. Compruebe la densidad de las células con el microscopio. Dividir la muestra en platos diferentes si es demasiado densa. </li><li> Espere a que las células se establezcan (~ 5 min). </li><li> Muestre la gota de la suspensión celular para las células teñidas (= EpCAM-positivas). </li><li> Una vez que se encuentra una célula, recoger la célula con el micromanipulador y ponerlo en 50 μ l de buffer dependiendo de la intención de análisis aguas abajo ( por ejemplo , RNA extracción de amortiguación o medio de cultivo). </li><li> Dependiendo de los análisis de aguas abajo, aislar las células EpCAM negativas y / o el medio como controles negativos. </li><li> Proceder con análisis de aguas abajo ( por ejemplo , cultivo celular, PCR). </li></ol></li></ol>

<ol>
	<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics,+2016.">Cancer statistics, 2016.</a> CA Cancer J Clin. 66 (1), 7-30 (2016).</li><li>Weitz, J., Koch, M., Debus, J., H&#246;hler, T., Galle, P. R., B&#252;chler, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorectal+cancer.">Colorectal cancer.</a> Lancet. 365 (9454), 153-165 (2005).</li><li>Sch&#246;lch, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells+of+colorectal+cancer.">Circulating tumor cells of colorectal cancer.</a> Cancer Cell Microenviron. 1 (5), (2014).</li><li>Steinert, G., Sch&#246;lch, S., Koch, M., Weitz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+and+significance+of+circulating+and+disseminated+tumour+cells+in+colorectal+cancer.">Biology and significance of circulating and disseminated tumour cells in colorectal cancer.</a> Langenbecks Arch Surg. 397 (4), 535-542 (2012).</li><li>Bork, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prognostic+relevance+of+minimal+residual+disease+in+colorectal+cancer.">Prognostic relevance of minimal residual disease in colorectal cancer.</a> World J Gastroenterol. 20 (30), 10296-10304 (2014).</li><li>Bork, U., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumour+cells+and+outcome+in+non-metastatic+colorectal+cancer:+a+prospective+study.">Circulating tumour cells and outcome in non-metastatic colorectal cancer: a prospective study.</a> Br J Cancer. 112 (8), 1306-1313 (2015).</li><li>Rahbari, N. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmental+differences+of+circulating+tumor+cells+in+colorectal+cancer.">Compartmental differences of circulating tumor cells in colorectal cancer.</a> Ann Surg Oncol. 19 (7), 2195-2202 (2012).</li><li>Rahbari, N. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+Spread+Emerging+From+Liver+Metastases+of+Colorectal+Cancer:+Does+the+Seed+Leave+the+Soil+Again?.">Metastatic Spread Emerging From Liver Metastases of Colorectal Cancer: Does the Seed Leave the Soil Again?.</a> Ann Surg. 263 (2), 345-352 (2016).</li><li>Rahbari, N. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meta-analysis+shows+that+detection+of+circulating+tumor+cells+indicates+poor+prognosis+in+patients+with+colorectal+cancer.">Meta-analysis shows that detection of circulating tumor cells indicates poor prognosis in patients with colorectal cancer.</a> Gastroenterology. 138 (5), 1714-1726 (2010).</li><li>Steinert, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+Escape+and+Survival+Mechanisms+in+Circulating+Tumor+Cells+of+Colorectal+Cancer.">Immune Escape and Survival Mechanisms in Circulating Tumor Cells of Colorectal Cancer.</a> Cancer Res. 74 (6), 1694-1704 (2014).</li><li>Sharpless, N. E., Depinho, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mighty+mouse:+genetically+engineered+mouse+models+in+cancer+drug+development.">The mighty mouse: genetically engineered mouse models in cancer drug development.</a> Nat Rev Drug Discov. 5 (9), 741-754 (2006).</li><li>Roper, J., Hung, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Priceless+GEMMs:+genetically+engineered+mouse+models+for+colorectal+cancer+drug+development.">Priceless GEMMs: genetically engineered mouse models for colorectal cancer drug development.</a> Trends Pharmacol Sci. 33 (8), 449-455 (2012).</li><li>Sch&#246;lch, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiotherapy+combined+with+TLR7/8+activation+induces+strong+immune+responses+against+gastrointestinal+tumors.">Radiotherapy combined with TLR7/8 activation induces strong immune responses against gastrointestinal tumors.</a> Oncotarget. 6 (7), 4663-4676 (2015).</li><li>Sch&#246;lch, S., Rauber, C., Weitz, J., Koch, M., Huber, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLR+activation+and+ionizing+radiation+induce+strong+immune+responses+against+multiple+tumor+entities.">TLR activation and ionizing radiation induce strong immune responses against multiple tumor entities.</a> Oncoimmunology. 4 (11), e1042201 (2015).</li><li>Sch&#246;lch, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells+exhibit+stem+cell+characteristics+in+an+orthotopic+mouse+model+of+colorectal+cancer.">Circulating tumor cells exhibit stem cell characteristics in an orthotopic mouse model of colorectal cancer.</a> Oncotarget. 7 (19), 27232-27242 (2016).</li><li>Nanduri, L. K., Garc&#237;a, S., Weitz, J., Sch&#246;lch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+Models+of+Colorectal+Cancer-Derived+Circulating+Tumor+Cells.">Mouse Models of Colorectal Cancer-Derived Circulating Tumor Cells.</a> Med Chem (Los Angeles). 6 (7), 497-499 (2016).</li><li>van Noort, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Drug+Candidates+for+the+Treatment+of+Metastatic+Colorectal+Cancer+through+Global+Inverse+Gene-Expression+Profiling.">Novel Drug Candidates for the Treatment of Metastatic Colorectal Cancer through Global Inverse Gene-Expression Profiling.</a> Cancer Res. 74 (20), 5690-5699 (2014).</li><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+expansion+of+epithelial+organoids+from+human+colon,+adenoma,+adenocarcinoma,+and+Barrett's+epithelium.">Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.</a> Gastroenterology. 141 (5), 1762-1772 (2011).</li><li>Boj, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+models+of+human+and+mouse+ductal+pancreatic+cancer.">Organoid models of human and mouse ductal pancreatic cancer.</a> Cell. 160 (1-2), 324-338 (2015).</li><li>Gao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+cultures+derived+from+patients+with+advanced+prostate+cancer.">Organoid cultures derived from patients with advanced prostate cancer.</a> Cell. 159 (1), 176-187 (2014).</li><li>Ito, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NOD/SCID/gamma(c)(null)+mouse:+an+excellent+recipient+mouse+model+for+engraftment+of+human+cells.">NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells.</a> Blood. 100 (9), 3175-3182 (2002).</li><li>Simon, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+phenotypic+and+genomic+analysis+of+C57BL/6J+and+C57BL/6N+mouse+strains.">A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains.</a> Genome Biol. 14 (7), R82 (2013).</li><li>Kalish, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C57BL/6N+Mice+Are+More+Resistant+to+Ehrlich+Ascites+Tumors+Than+C57BL/6J+Mice:+The+Role+of+Macrophage+Nitric+Oxide.">C57BL/6N Mice Are More Resistant to Ehrlich Ascites Tumors Than C57BL/6J Mice: The Role of Macrophage Nitric Oxide.</a> Med Sci Monit Basic Res. 21, 235-240 (2015).</li><li>Tseng, W., Leong, X., Engleman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+mouse+model+of+colorectal+cancer.">Orthotopic mouse model of colorectal cancer.</a> J Vis Exp. (10), e484 (2007).</li><li>Roper, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combination+PI3K/MEK+inhibition+promotes+tumor+apoptosis+and+regression+in+PIK3CA+wild-type,+KRAS+mutant+colorectal+cancer.">Combination PI3K/MEK inhibition promotes tumor apoptosis and regression in PIK3CA wild-type, KRAS mutant colorectal cancer.</a> Cancer Lett. 347 (2), 204-211 (2014).</li><li>Coffee, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concomitant+BRAF+and+PI3K/mTOR+blockade+is+required+for+effective+treatment+of+BRAF(V600E)+colorectal+cancer.">Concomitant BRAF and PI3K/mTOR blockade is required for effective treatment of BRAF(V600E) colorectal cancer.</a> Clin Cancer Res. 19 (10), 2688-2698 (2013).</li><li>Belmont, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Resistance+to+dual+blockade+of+the+kinases+PI3K+and+mTOR+in+KRAS-mutant+colorectal+cancer+models+results+in+combined+sensitivity+to+inhibition+of+the+receptor+tyrosine+kinase+EGFR.">Resistance to dual blockade of the kinases PI3K and mTOR in KRAS-mutant colorectal cancer models results in combined sensitivity to inhibition of the receptor tyrosine kinase EGFR.</a> Sci Signal. 7 (351), ra107 (2014).</li><li>Hung, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+mouse+model+for+sporadic+and+metastatic+colon+tumors+and+its+use+in+assessing+drug+treatment.">Development of a mouse model for sporadic and metastatic colon tumors and its use in assessing drug treatment.</a> Proc Natl Acad Sci USA. 107 (4), 1565-1570 (2010).</li><li>Wang, F., Johnson, R. L., Snyder, P. W., DeSmet, M. L., Fleet, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Inducible,+Large-Intestine-Specific+Transgenic+Mouse+Model+for+Colitis+and+Colitis-Induced+Colon+Cancer+Research.">An Inducible, Large-Intestine-Specific Transgenic Mouse Model for Colitis and Colitis-Induced Colon Cancer Research.</a> Dig Dis Sci. 61 (4), 1069-1079 (2016).</li><li>Xue, Y., Johnson, R., Desmet, M., Snyder, P. W., Fleet, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+transgenic+mouse+for+colorectal+cancer+research+with+intestinal+cre+expression+limited+to+the+large+intestine.">Generation of a transgenic mouse for colorectal cancer research with intestinal cre expression limited to the large intestine.</a> Mol Cancer Res. 8 (8), 1095-1104 (2010).</li><li>Tetteh, P. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+an+inducible+colon-specific+Cre+enzyme+mouse+line+for+colon+cancer+research.">Generation of an inducible colon-specific Cre enzyme mouse line for colon cancer research.</a> Proc Natl Acad Sci USA. 113 (42), 11859-11864 (2016).</li></ol>

Los autores no tienen nada que revelar.

A pesar de su actividad preclínicamente probada en los modelos de ratones subcutáneos, la gran mayoría de los nuevos compuestos fallan en ensayos clínicos y nunca llegan a la clínica. 11 Esta insuficiencia evidente de modelos subcutáneos de ratón para simular con precisión los patrones de la biología y de crecimiento de los tumores ha conducido al desarrollo de modelos ortotópicos ratón basa en la inyección de las células tumorales directamente en el órgano original. <p class="j...

El cáncer colorrectal (CCR) es una de las causas más comunes de muerte por cáncer en los países occidentales. 1 Mientras que el tumor primario a menudo puede ser resecado, la aparición de metástasis distantes empeora drásticamente el pronóstico y a menudo conduce a la muerte. 2 , 3 El correlato biológico de la metástasis son las células tumorales circulantes (CTC), que se separan del tumor, sobreviven en la circulación, se adhieren al epitelio en el órgano diana, invaden el órgano y en última instancia, crecen a nuevas lesiones. 4 Aunque se sabe que los CTC son de relevancia pronóstica, 5 , 6 , 7 , 8 , 9 su biología sólo se entiende en parte como resultado de su extrema rareza en la CRC. 10 Los modelos de ratón son un poderoso tOol para estudiar diversos aspectos de la biología del cáncer. Los modelos clásicos de tumores subcutáneos se producen mediante inyección subcutánea de células tumorales en ratones receptores, que pueden ser inmunocompetentes (si se utilizan células tumorales murinas singénicas) o inmunodeficientes. Los modelos de tumor subcutáneo son baratos y producen datos rápidamente; Su crecimiento tumoral de punto final puede ser medido fácilmente y no invasivamente. Sin embargo, el 88% de los nuevos compuestos que han demostrado actividad antitumoral en estos modelos fallan en ensayos clínicos. 11 Esto es en parte debido a las diferencias entre especies entre humanos y ratones; Sin embargo, una gran parte de este fracaso se debe al bajo valor predictivo de los modelos de ratones subcutáneos. Los modelos de ratones ortotópicos, en los que las células tumorales se inyectan en el órgano de origen y, por tanto, crecen en su microambiente original, se utilizan cada vez más en la investigación del cáncer. 11 , 12 , </sUp&gt; 13 , 14 Los modelos ortotópicos no sólo simulan las condiciones locales de crecimiento tumoral; Como resultado del sitio anatómicamente correcto del crecimiento tumoral, los modelos de ratón ortotópico también permiten la simulación realista de la metástasis y por lo tanto se usan para estudiar la biología del CTC 8 , 15 , 16 o su respuesta a diferentes tratamientos en la CRC. 13 , 17 Una de las principales desventajas de los modelos de ratones ortotópicos es su complejidad técnica. Dependiendo del órgano en el que se van a inyectar las células, la curva de aprendizaje hasta que el experimentador es capaz de inducir tumores reproducibles es bastante larga. Esto se aplica especialmente a los modelos de cáncer colorrectal, ya que las células tumorales necesitan ser inyectadas en la pared intestinal, lo que a menudo produce perforación, pérdida de células tumorales o pérdida endoluminal de células tumorales. Esto unEl objetivo de este artículo es describir el método de preparación celular a partir de muestras de tejido primario, líneas celulares 2D y cultivo organoide 3D y su inyección en el ciego de ratones. La técnica descrita aquí conduce a tumores muy uniformes y, dependiendo de la biología tumoral de la línea celular utilizada para inyección, la formación reproducible de metástasis a distancia y CTC en los ratones receptores. 15

<table border="0" cellpadding="0" cellspacing="0" width="1294">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:421px;">Cell culture Media and Components</td>
			<td style="width:297px;"></td>
			<td style="width:119px;"></td>
			<td style="width:457px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Advanced DMEM F12</td>
			<td style="width:297px;">Invitrogen</td>
			<td style="width:119px;">12634010</td>
			<td style="width:457px;">DMEM/ F12 +++ medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">HEPES (1 M)</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">15630056</td>
			<td style="width:457px;">DMEM/ F12 +++ medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Glutamax-I Supplement (200 mM)</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">35050038</td>
			<td style="width:457px;">DMEM/ F12 +++ medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Penicillin/Streptomycin (PenStrep)</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">15140122</td>
			<td style="width:457px;">DMEM/ F12 +++ medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">DMEM</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">61965026</td>
			<td>basic medium of 2D cell lines (DMEM/10%FCS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Fetal Calf Serum (FCS)</td>
			<td style="width:297px;">BIOCHROM AG</td>
			<td style="width:119px;">S 0115</td>
			<td>basic medium of 2D cell lines (DMEM/10%FCS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">TrypLE Express enzymatic dissociation buffer</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">12604021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Matrigel basement membrane matrix (BMM, phenol red free)</td>
			<td style="width:297px;">CORNING B.V. Life Sciences</td>
			<td style="width:119px;">356231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">14190169</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Trypsin-EDTA (0,25%, Phenol-Red)</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">25200072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">6-/48-well plates with lid</td>
			<td style="width:297px;">CORNING</td>
			<td style="width:119px;">3516/3548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">cell culture flask 75cm&#178;, 250 mL</td>
			<td style="width:297px;">VWR International GmbH</td>
			<td style="width:119px;">734-2066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">cell culture flask 150cm&#178;, 600 mL</td>
			<td style="width:297px;">Corning B.V. Life Sciences</td>
			<td style="width:119px;">355001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:421px;">Eppendorf tubes 1,5 mL / 2 mL</td>
			<td style="width:297px;">Sarstedt AG &#38; Co.</td>
			<td style="width:119px;">72.706.400/ 72.695.400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">15 ml, 50 ml centrifuge tubes</td>
			<td style="width:297px;">Greiner-Bio-One GmbH</td>
			<td style="width:119px;">188271/227270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">TC10 Counting Slides (for TC20 Counting Machine)</td>
			<td style="width:297px;">Bio-Rad Laboratories GmbH</td>
			<td style="width:119px;">1450016</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:421px;">Pasteur pipettes (glass, 150 mm)</td>
			<td style="width:297px;">Fisher Scientific GmbH</td>
			<td style="width:119px;">11546963/ FB50251</td>
			<td>thinly pulled by using a bunsen burner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gentleMACS Dissociator</td>
			<td style="width:297px;">Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td>for primary tumor tissue preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACSmix Tube Rotator</td>
			<td style="width:297px;">Miltenyi Biotec</td>
			<td>130-090-753</td>
			<td>for primary tumor tissue preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gentleMACS C Tubes</td>
			<td style="width:297px;">Miltenyi Biotec</td>
			<td>130-093-237</td>
			<td>for primary tumor tissue preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human Tumor Dissociation Kit</td>
			<td style="width:297px;">Miltenyi Biotec</td>
			<td>130-095-929</td>
			<td>for primary tumor tissue preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon 70&#181;m Cell Strainer</td>
			<td style="width:297px;">Corning B.V. Life Sciences</td>
			<td>352350</td>
			<td>for primary tumor tissue preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Name</td>
			<td style="width:297px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:421px;">Surgical Equipment</td>
			<td style="width:297px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Sevoflurane</td>
			<td style="width:297px;">AbbVie Germany GmbH &#38; Co. KG</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Medical oxygen</td>
			<td style="width:297px;">Air Liquide Medical GmbH</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Buprenorphine</td>
			<td style="width:297px;">Temgesic</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Bepanthen - opthalmic ointment</td>
			<td style="width:297px;">Bayer Vital GmbH</td>
			<td style="width:119px;">10047757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Normal saline 0.9% (E154)</td>
			<td style="width:297px;">Serumwerk Bernburg AG</td>
			<td style="width:119px;">10013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Aqua ad injectabilia</td>
			<td style="width:297px;">Braun</td>
			<td style="width:119px;">235144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">1 mL Syringe (without dead volume) - Injekt-F SOLO</td>
			<td style="width:297px;">Braun/neoLab</td>
			<td style="width:119px;">194291661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">30G injection needle</td>
			<td style="width:297px;">BECTON DICKINSON</td>
			<td style="width:119px;">304000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">cellulose swabs</td>
			<td style="width:297px;">Lohmann &#38; Rauscher Deutschland</td>
			<td style="width:119px;">13356</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Micro-Adson Forceps</td>
			<td style="width:297px;">FST - Fine Science Tools</td>
			<td style="width:119px;">11018-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Iris Scissor - ToughCut</td>
			<td style="width:297px;">FST - Fine Science Tools</td>
			<td style="width:119px;">14058-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Olsen-Hegar Needle Holder</td>
			<td style="width:297px;">FST - Fine Science Tools</td>
			<td style="width:119px;">12002-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">AutoClip Kit</td>
			<td style="width:297px;">FST - Fine Science Tools</td>
			<td style="width:119px;">12020-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">PDS Z1012H 6/0 C1 (surgical suture)</td>
			<td style="width:297px;">Johnson &#38; Johnson Medical GmbH</td>
			<td style="width:119px;">Z1012H</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:421px;">Table Top Research Anesthesia Machine w/O2 Flush and a Sevoflurane Vaporizer</td>
			<td style="width:297px;">Parkland Scientific</td>
			<td style="width:119px;">V3000PS/PK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">UltraMicro Pump with Micro4 Controller</td>
			<td style="width:297px;">World Precision Instruments</td>
			<td style="width:119px;">UMP3-4</td>
			<td>equipment for highly controlled orthotopic injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Footswitch for SYS-Micro4 Controller</td>
			<td style="width:297px;">World Precision Instruments</td>
			<td style="width:119px;">15867</td>
			<td>equipment for highly controlled orthotopic injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Three-axis Manual Micromanipulator</td>
			<td style="width:297px;">World Precision Instruments</td>
			<td style="width:119px;">M325</td>
			<td>equipment for highly controlled orthotopic injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Magnetic Stand for Micromanipulator</td>
			<td style="width:297px;">World Precision Instruments</td>
			<td style="width:119px;">M10</td>
			<td>equipment for highly controlled orthotopic injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Steel Base Plate for M10 Magnetic Stand</td>
			<td style="width:297px;">World Precision Instruments</td>
			<td style="width:119px;">5479</td>
			<td>equipment for highly controlled orthotopic injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Hot Plate 062</td>
			<td style="width:297px;">Labotect</td>
			<td style="width:119px;">13854</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Isis - Hair shaver</td>
			<td style="width:297px;">AESCULAP - Braun</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Binocular Surgical Microscope</td>
			<td style="width:297px;">Parkland Scientific</td>
			<td style="width:119px;">VS-2Z</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Name</td>
			<td style="width:297px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:421px;">CTC isolation</td>
			<td style="width:297px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">EDTA</td>
			<td style="width:297px;">Roth</td>
			<td>8040.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Density gradient medium &#8211; Ficoll</td>
			<td>StemCell - Lymphoprep</td>
			<td>7801</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:421px;">Alexa Fluor 488 anti-human CD326 (EpCAM) Antibody clone 9C4</td>
			<td>BioLegend</td>
			<td>324210</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:421px;">Alexa Fluor 488 anti-mouse CD326 (EpCAM) Antibody clone G8.8</td>
			<td>BioLegend</td>
			<td style="width:119px;">118210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dish, &#248; 60 x 15 mm, 21 cm&#178;, Vent</td>
			<td>Greiner bio-one</td>
			<td>628102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence Cell Culture Microscope</td>
			<td style="width:297px;">Leica</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transferman 4r Micromanipulator</td>
			<td style="width:297px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTram Air</td>
			<td style="width:297px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td style="width:457px;">aspiration pump connected to the micromanipulator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dmz Universal Microelectrode Puller</td>
			<td style="width:297px;">Dagan Corporation</td>
			<td style="width:119px;"></td>
			<td>required for the manufacturing of micro capillaries for single cell aspiration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prism Glass Capillaries</td>
			<td style="width:297px;">Dagan Corporation</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">PAP pen</td>
			<td style="width:297px;">Abcam</td>
			<td style="width:119px;">ab2601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">14190169</td>
			<td>picking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Fetal Calf Serum (FCS)</td>
			<td style="width:297px;">BIOCHROM AG</td>
			<td style="width:119px;">S 0115</td>
			<td>picking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Penicillin/Streptomycin (PenStrep)</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">15140122</td>
			<td>picking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">EDTA</td>
			<td style="width:297px;">Roth</td>
			<td>8040.1</td>
			<td>picking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Name</td>
			<td style="width:297px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:421px;">Immunohistochemistry</td>
			<td style="width:297px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Purified anti-human CD326 (EpCAM) antibody clone 9C4</td>
			<td>BioLegend</td>
			<td style="width:119px;">324201</td>
			<td>EpCAM immunohistochemistry (cf, fig 2C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">HRP rabbit anti-mouse IgG</td>
			<td style="width:297px;">Abcam</td>
			<td style="width:119px;">ab97046</td>
			<td>EpCAM immunohistochemistry (cf, fig 2C)</td>
		</tr>
	</tbody>
</table>

isolation of circulating tumor cells in an orthotopic mouse model of colorectal cancer

A pesar de las ventajas de la fácil aplicabilidad y rentabilidad, los modelos de ratón subcutáneo tienen limitaciones severas y no simulan con precisión la biología tumoral y la diseminación de células tumorales. Se han introducido modelos de ratones ortotópicos para superar estas limitaciones; Sin embargo, estos modelos son técnicamente exigentes, especialmente en órganos huecos como el intestino grueso. Con el fin de producir tumores uniformes que crecen de forma fiable y metástasis, las técnicas estandarizadas de preparación e inyección de células tumorales son críticas. Hemos desarrollado un modelo de ratón ortotópico de cáncer colorrectal (CRC) que desarrolla tumores muy uniformes y puede ser utilizado para estudios de biología de tumores, así como ensayos terapéuticos. Se inyectan células tumorales de tumores primarios, líneas de células bidimensionales (2D) o organoides tridimensionales (3D) en el ciego y, dependiendo del potencial metastásico de las células tumorales inyectadas, forman tumores altamente metastáticos. En adición,Los CTC se pueden encontrar regularmente. Aquí describimos la técnica de preparación de células tumorales tanto de líneas celulares 2D como de organoides 3D, así como el tejido tumoral primario, las técnicas quirúrgicas y de inyección, así como el aislamiento de CTCs de los ratones portadores de tumores y los consejos actuales para la resolución de problemas.

La generación exitosa y reproducible de tumores colorrectales en este modelo depende en gran medida de una inyección precisa de las células sin derrames o fugas. Si esto se consigue, este modelo es extremadamente fiable y rara vez da lugar a la diseminación peritoneal artificial. La cinética de crecimiento de los tumores, así como sus patrones de diseminación dependen de la biología de los organoides y células utilizados. 15 Mientras que las células HCT1...

Watch this Scientific Journal Video about Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer at JoVE.com

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Describimos el establecimiento de tumores colorrectales ortotópicos mediante la inyección de células tumorales o organoides en el ciego de ratones y el subsiguiente aislamiento de células tumorales circulantes (CTC) de este modelo.

Aislamiento de células tumorales circulantes en un modelo de ratón ortotópico de cáncer colorrectal

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

isolation-circulating-tumor-cells-an-orthotopic-mouse-model

Research

JoVE Journal

Cancer Research

9.9K vistas.  Technische Universität Dresden.  Describimos el establecimiento de tumores colorrectales ortotópicos mediante la inyección de células tumorales o organoides en el ciego de ratones y el subsiguiente aislamiento de células tumorales circulantes (CTC) de este modelo. 

Video: Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Fluorescente modelo de ratón ortotópico de cáncer de páncreas

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

Investigación

Investigación sobre el cáncer

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

Murino modelo experimental de desarrollo del tumor original y la metástasis peritoneal<em&gt; vía</em&gt; Ortotópico La inoculación con células de carcinoma de ovario

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

Capture and Release of Viable Circulating Tumor Cells from Blood

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip for downstream analysis and/or culture. The strategy employs the use of a novel Parylene C membrane slot pore microfilter to capture CTC and a coating of poly (N-iso-propylacrylamide) (PIPAAm) for thermoresponsive viable release of the captured CTC. The capture of live cells is enabled by leveraging the design of a slot pore geometry with specific dimensions to reduce the shear stress typically associated with the filtration process. While the microfilter exhibits a high capture efficiency, the release of these cells is non-trivial. Typically, only a small percentage of cells are released when techniques such as reverse flow or cell scraping are used. The strong adhesion of these epithelial cancer cells to the Parylene C membrane is attributable to non-specific electrostatic interaction. To counteract this effect, we employed the use of PIPAAm coating and exploited its thermal responsive interfacial properties to release the cells from the filter. Blood is first filtered at room temperature. Below 32 &#176;C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 &#176;C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells.

A protocol to utilize a poly(N-iso-propylacrylamide) (PIPAAm) coated microfilter for effective capture and thermoresponsive release of viable circulating tumor cells (CTC) is presented. This method allows capture of CTC from patients&#39; blood and subsequent release of viable CTC for downstream off-chip culture, analyses and characterization.

Captura y Liberación de viables células tumorales circulantes de la sangre

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip ...

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

Un Modelo de tumor y el tumor murino ortotópico de vejiga Sistema de Detección

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

Un modelo de ratón de ingeniería genética de cáncer colorrectal esporádico

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In particular, endothelial cells (EC) constituting the inner surface of vessels, directly participate in oxygen delivery, nutrient supply, and removal of waste products to and from tumors, and are thereby prominently involved in the constitution of the tumor microenvironment (TME). Tumor endothelial cells (TECs) can be used as cellular biosensors of the intratumoral microenvironment established by communication between tumor and stromal cells. TECs also serve as targets of therapy. Accordingly, in culture these cells allow studies on mechanisms of response or resistance to anti-angiogenic treatment. Recently, it was found that TECs isolated from human colorectal carcinoma (CRC) exhibit memory-like effects based on the specific TME they were derived from. Moreover, these TECs actively contribute to the establishment of a specific TME by the secretion of different factors. For example, TECs in a prognostically favorable Th1-TME secrete the anti-angiogenic tumor-suppressive factor secreted protein, acidic and rich in cysteine-like 1 (SPARCL1). SPARCL1 regulates vessel homeostasis and inhibits tumor cell proliferation and migration. Hence, cultures of pure, viable TECs isolated from human solid tumors are a valuable tool for functional studies on the role of the vascular system in tumorigenesis. Here, a new up-to-date protocol for the isolation of primary EC from the normal colon as well as CRC is described. The technique is based on mechanical and enzymatic tissue digestion, immunolabeling, and fluorescence activated cell sorting (FACS)-sorting of triple-positive cells (CD31, VE-cadherin, CD105). With this protocol, viable TEC or normal endothelial cell (NEC) cultures could be isolated from colon tissues with a success rate of 62.12% when subjected to FACS-sorting (41 pure EC cultures from 66 tissue samples). Accordingly, this protocol provides a robust approach to isolate human EC cultures from normal colon and CRC.

Tumor endothelial cells are important determinants of the tumor microenvironment and the course of the disease. Here, a protocol for the isolation of pure and viable endothelial cells from human colorectal carcinoma and normal colon to be used in drug testing and pathogenesis research is described.

Aislamiento de células endoteliales humanas de Colon Normal y el Carcinoma colorrectal - un protocolo mejorado

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful biomarkers for clinical practice. Here, we present a protocol to extract, reverse transcribe and evaluate the expression levels of circulating miRNAs from plasma samples of patients with colorectal cancer (CRC). microRNAs (miRNAs) are a class of non-coding RNAs of 18-25 nucleotides in length that regulate the expression of target genes at the translational level and play an important role, including that of pro- and anti-angiogenic function, in the physiopathology of various organs. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis and treatment decision-making and monitoring. Circulating miRNA extraction was performed using a rapid and effective method that involves both organic and column-based methodologies. For miRNA retrotranscription, we used a multi-step procedure that considers polyadenylation at 3&#39; and the ligation of an adapter at 5&#39; of the mature miRNAs, followed by random miRNA pre-amplification. We selected a 24-miRNA custom panel to be tested by quantitative real-time polymerase chain reaction (qRT-PCR) and spotted the miRNA probes on array custom plates. We performed qRT-PCR plate runs on a real-time PCR System. Housekeeping miRNAs for normalization were selected using GeNorm software (v. 3.2). Data were analyzed using Expression suite software (v 1.1) and statistical analyses were performed. The method proved to be reliable and technically robust and could be useful to evaluate biomarker levels in liquid samples such as plasma and/or serum.

We present a protocol to evaluate the expression levels of circulating microRNA (miRNAs) in plasma samples from cancer patients. In particular, we used a commercially available kit for circulating miRNA extraction and reverse transcription. Finally, we analyzed a panel of 24 selected miRNAs using real-time pre-spotted probe custom plates.

Detección de un Panel personalizado de microARN circulantes en pacientes con cáncer colorrectal metastásico

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Micromanipulación de células tumorales circulantes para análisis molecular descendente y evaluación potencial metastásica

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional (3D) tumor architecture and low relevance to human cancer. Researchers have recently developed a 3D in vitro cancer model referred to as tumor organoids that can mimic the characteristics of a native tumor in a culture dish. Here, experimental procedures are described in detail for the establishment of bladder tumor organoids from a carcinogen-induced murine bladder tumor, including culture, passage, and maintenance of the resulting 3D tumor organoids in vitro. In addition, protocols to manipulate the established bladder tumor organoid lines for genetic engineering using lentivirus-mediated transduction are described, including optimized conditions for the efficient introduction of new genetic elements into tumor organoids. Finally, the procedure for orthotopic transplantation of bladder tumor organoids into the wall of the murine bladder for further analysis is laid out. The methods described in this article can facilitate the establishment of an in vitro model for bladder cancer for the development of better therapeutic options.

This protocol provides detailed experimental steps to establish a three-dimensional in vitro culture of bladder tumor organoids derived from carcinogen-induced murine bladder cancer. Culture methods including passaging, genetic engineering, and orthotopic transplantation of tumor organoids are described.

Cultivo, manipulación y trasplante ortotópico de organoides tumorales de la vejiga de ratón

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

Un modelo ortotópico de ratón reseccional de cáncer de páncreas

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...