Name: an oncogenic hepatocyte-induced orthotopic mouse model of hepatocellular cancer arising in the setting of hepatic inflammation and fibrosis
Uploaded: 2019-09-12T13:00:00
Description: Watch this Scientific Journal Video about An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis at JoVE.com

Este trabajo cuenta con el apoyo de NIH/NCI R01 CA164335-01A1 (K. F. Staveley-O'Carroll, PI) y NIH/NCI R01CA208396 (Mark Kester, Guangfu Li, Kevin F. Staveley-O'Carroll).

NOTA: Todo el procedimiento, incluidos los sujetos de animales, ha sido aprobado por la IACUC de la Universidad de Missouri. Todos los ratones recibieron atención humana de acuerdo con los criterios descritos en la "Guía para el cuidado y uso de animales de laboratorio". El siguiente procedimiento para el aislamiento celular y la inoculación se debe realizar en una campana. Todos los artistas deben usar el equipo de protección personal estándar para el manejo de los ratones y tejidos.
1. In...

<ol>
	<li>O'Connor, S., Ward, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocellular+carcinoma+-+United+States.">Hepatocellular carcinoma - United States.</a> Morbidity and Mortality Weekly Report. 59, 517-520 (2010).</li><li>Petrick, J., Kelly, S., Altekruse, S., McGlynn, K., Rosenberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Future+of+Hepatocellular+Carcinoma+Incidence+in+the+United+States+Forecast+Through+2030.">Future of Hepatocellular Carcinoma Incidence in the United States Forecast Through 2030.</a> Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 34, 1787-1794 (2016).</li><li>Greten, T., Lai, C., Li, G., Staveley-O'Carroll, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+and+Immune-based+Therapies+for+Hepatocellular+Carcinoma.">Targeted and Immune-based Therapies for Hepatocellular Carcinoma.</a> Gastroenterology. 156, 510-524 (2019).</li><li>Llovet, J., Zucman-Rossi, J., Pikarsky, E., Sangro, B., Schwartz, M., Sherman, M., Gores, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocellular+Carcinoma.">Hepatocellular Carcinoma.</a> Nature reviews. Disease primers. 2, 16018 (2016).</li><li>Ding, X., 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=Precision+medicine+for+hepatocellular+carcinoma:+driver+mutations+and+targeted+therapy.">Precision medicine for hepatocellular carcinoma: driver mutations and targeted therapy.</a> Oncotarget. 8, 55715-55730 (2017).</li><li>Colombo, M., Maisonneuve, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlling+liver+cancer+mortality+on+a+global+scale:+still+a+long+way+to+go.">Controlling liver cancer mortality on a global scale: still a long way to go.</a> Journal of Hepatology. 67, 216-217 (2017).</li><li>Llovet, J., Burroughs, A., Bruix, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocellular+carcinoma.">Hepatocellular carcinoma.</a> Lancet. 362, 1907-1917 (2003).</li><li>Parkin, D., Bray, F., Ferlay, J., Pisani, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+world+cancer+burden:+Globocan+2000.">Estimating the world cancer burden: Globocan 2000.</a> International Journal of Cancer. 94, 153-156 (2001).</li><li>Thomas, M., Zhu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepocellular+carcinoma:+the+need+for+progress.">Hepocellular carcinoma: the need for progress.</a> Journal of Clinical Oncology. 23, 2892-2899 (2005).</li><li>Llovet, J., Bruix, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+targeted+therapies+in+hepatocellular+carcinoma.">Molecular targeted therapies in hepatocellular carcinoma.</a> Hepatology. 48, 1312-1327 (2008).</li><li>Bruix, J., Sherman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+hepatocellular+carcinoma:+an+update.">Management of hepatocellular carcinoma: an update.</a> Hepatology. 53, 1020-1022 (2011).</li><li>Pang, T., Lam, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+management+of+hepatocellular+carcinoma.">Surgical management of hepatocellular carcinoma.</a> World Journal of Hepatology. 7, 245-252 (2015).</li><li>Llovet, 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=Design+and+endpoints+of+clinical+trials+in+hepatocellular+carcinoma.">Design and endpoints of clinical trials in hepatocellular carcinoma.</a> Journal of the National Cancer Institution. 100, 698-711 (2008).</li><li>Mueller, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+immunology+and+immunotherapy.+Realizing+the+promise.+Introduction.">Cancer immunology and immunotherapy. Realizing the promise. Introduction.</a> Science. 348, 54-55 (2015).</li><li>Gajewski, T., Schreiber, H., Fu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+and+adaptive+immune+cells+in+the+tumor+microenvironment.">Innate and adaptive immune cells in the tumor microenvironment.</a> Nature Immunology. 14, 1014-1022 (2013).</li><li>Ribas, A., Wolchok, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+cancer+immunotherapy+and+targeted+therapy.">Combining cancer immunotherapy and targeted therapy.</a> Current Opinion in Immunology. 25, 291-296 (2013).</li><li>Postow, M., Callahan, M., Wolchok, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+checkpoint+blockade+in+cancer+therapy.">Immune checkpoint blockade in cancer therapy.</a> Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology. 33, 1974-1982 (2015).</li><li>Gao, 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=VISTA+is+an+inhibitory+immune+checkpoint+that+is+increased+in+ipilimumab+therapy+in+patients+with+prostate+cancer.">VISTA is an inhibitory immune checkpoint that is increased in ipilimumab therapy in patients with prostate cancer.</a> Nature Medicine. 23, 551-555 (2017).</li><li>Hahn, A., Gill, D., Pal, S., Agarwal, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+future+of+immune+checkpoint+cancer+therapy+after+PD-1+and+CTLA-4.">The future of immune checkpoint cancer therapy after PD-1 and CTLA-4.</a> Immunotherapy. 9, 681-692 (2017).</li><li>Remon, J., Besse, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+checkpoint+inhibitors+in+first-line+therapy+of+advanced+non-small+cell+lung+cancer.">Immune checkpoint inhibitors in first-line therapy of advanced non-small cell lung cancer.</a> Current Opinion in Oncology. 29, 97-104 (2017).</li><li>Kudo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+checkpoint+blockade+in+hepatocellular+carcinoma:+2017+update.">Immune checkpoint blockade in hepatocellular carcinoma: 2017 update.</a> Liver Cancer. 6, 1-12 (2017).</li><li>Kudo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+checkpoint+inhibition+in+hepatocellular+carcinoma:+Basics+and+ongoing+clinical+trials.">Immune checkpoint inhibition in hepatocellular carcinoma: Basics and ongoing clinical trials.</a> Oncology. 92, 50-62 (2017).</li><li>Breous, E., Thimme, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+of+immunotherapy+for+hepatocellular+carcinoma.">Potential of immunotherapy for hepatocellular carcinoma.</a> Journal of Hepatology. 54, 830-834 (2011).</li><li>Sprinzl, M., Galle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facing+the+dawn+of+immunotherapy+for+hepatocellular+carcinoma.">Facing the dawn of immunotherapy for hepatocellular carcinoma.</a> Journal of Hepatology. 59, 9-10 (2013).</li><li>Liu, D., Staveley-O'Carroll, K., Li, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune-based+therapy+clinical+trials+in+hepatocellular+carcinoma.">Immune-based therapy clinical trials in hepatocellular carcinoma.</a> Journal of Clinical Cell Immunology. 6, 376 (2015).</li><li>Greten, T., Wang, X., Korangy, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+concepts+of+immune+based+treatments+for+patients+with+HCC:+from+basic+science+to+novel+treatment+approaches.">Current concepts of immune based treatments for patients with HCC: from basic science to novel treatment approaches.</a> Gut. 64, 842-848 (2015).</li><li>Koster, B., de Gruijl, T., van den Eertwegh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+and+future+challenges+in+immune+checkpoint+inhibitory+cancer+treatment.">Recent developments and future challenges in immune checkpoint inhibitory cancer treatment.</a> Current Opinion in Oncology. 27, 482-488 (2015).</li><li>Johnson, D., Sullivan, R., Menzies, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+checkpoint+inhibitors+in+challenging+populations.">Immune checkpoint inhibitors in challenging populations.</a> Cancer. 123, 1904-1911 (2017).</li><li>Li, H., 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=Programmed+cell+death-1+(PD-1)+checkpoint+blockade+in+combination+with+an+mTOR+inhibitor+restrains+hepatocellular+carcinoma+growth+induced+by+hepatoma+cell-intrinsic+PD-1.">Programmed cell death-1 (PD-1) checkpoint blockade in combination with an mTOR inhibitor restrains hepatocellular carcinoma growth induced by hepatoma cell-intrinsic PD-1.</a> Hepatology. 66, 1920-1933 (2017).</li><li>Heindryckx, F., Colle, I., van Vlierberghe, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+mouse+models+for+hepatocellular+carcinoma+research.">Experimental mouse models for hepatocellular carcinoma research.</a> International Journal of Experimental Pathology. 90, 367-386 (2009).</li><li>Qi, X., 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+inCVAX,+in+situ+cancer+vaccine,+and+its+immune+response+in+mice+with+hepatocellular+cancer.">Development of inCVAX, in situ cancer vaccine, and its immune response in mice with hepatocellular cancer.</a> Journal of Clinical and Cellular Immunology. 7, 438 (2016).</li><li>Qi, X., 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+radiofrequency+ablation+platform+in+a+clinically+relevant+murine+model+of+hepatocellular+cancer.">Development of a radiofrequency ablation platform in a clinically relevant murine model of hepatocellular cancer.</a> Cancer Biology and Therapy. 16, 1812-1819 (2015).</li><li>Liu, 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=Sunitinib+represses+regulatory+T+cells+to+overcome+immunotolerance+in+a+murine+model+of+hepatocellular+cancer.">Sunitinib represses regulatory T cells to overcome immunotolerance in a murine model of hepatocellular cancer.</a> Oncoimmunology. 7, 1372079 (2017).</li><li>Staveley-O'Carroll, K., 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=In+vivo+ligation+of+CD40+enhances+priming+against+the+endogenous+tumor+antigen+and+promotes+CD8++T+cell+effector+function+in+SV40+T+antigen+transgenic+mice.">In vivo ligation of CD40 enhances priming against the endogenous tumor antigen and promotes CD8+ T cell effector function in SV40 T antigen transgenic mice.</a> Journal of Immunology. 171, 697-707 (2003).</li><li>Avella, 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=Regression+of+established+hepatocellular+carcinoma+is+induced+by+chemoimmunotherapy+in+an+orthotopic+murine+model.">Regression of established hepatocellular carcinoma is induced by chemoimmunotherapy in an orthotopic murine model.</a> Hepatology. 55, 141-152 (2012).</li><li>Li, 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=Successful+chemoimmunotherapy+against+hepatocellular+cancer+in+a+novel+murine+model.">Successful chemoimmunotherapy against hepatocellular cancer in a novel murine model.</a> Journal of Hepatology. 66, 75-85 (2017).</li><li>Li, 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=Nanoliposome+C6-ceramide+increases+the+anti-tumor+immune+response+and+slows+growth+of+liver+tumors+in+ice.">Nanoliposome C6-ceramide increases the anti-tumor immune response and slows growth of liver tumors in ice.</a> Gastroenterology. 154, 1024-1036 (2018).</li><li>Held, 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=T+antigen+expression+and+tumorigenesis+in+transgenic+mice+containing+a+mouse+major+urinary+protein/SV40+T+antigen+hybrid+gene.">T antigen expression and tumorigenesis in transgenic mice containing a mouse major urinary protein/SV40 T antigen hybrid gene.</a> EMBO Journal. 8, 183-191 (1989).</li><li>Hanahan, D., Weinber, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallmarks+of+cancer:+the+next+generation.">Hallmarks of cancer: the next generation.</a> Cell. 144, 646-674 (2011).</li></ol>

Con este protocolo, hemos establecido un modelo murino fiable y reproducible de HCC que imita la iniciación y progresión humana de HCC. Clínicamente, muchos factores de riesgo inducen sucesivamente lesiones hepáticas, fibrosis hepática, cirrosis y la etapa final del HCC. En nuestro protocolo, la inyección IP de CCl4 se utiliza primero para producir fibrosis hepática en ratones de tipo salvaje, lo que permite que los posteriores hepatocitos oncogénicos formen los tumores en el entorno de la fibrosis hep...

El cáncer hepatocelular (HCC) es el tipo de cáncer que aumenta más rápidamente en los Estados Unidos (EE.UU.)1,2,3. Cada año, aproximadamente 850.000 nuevos casos se diagnostican4,5 y 700.000 pacientes mueren a causa de esta enfermedad letal6,7,8,9,10 , lo que la convierte en la segunda causa más alta de muerte relacionada con el cáncer en todo el mundo. El manejo de HCC incluye resección quirúrgica, trasplante, ablación, quimioembolización o terapias sistémicas, como sorafenib11. El diagnóstico y el manejo tempranos con resección quirúrgica o trasplante tienen el mayor beneficio de supervivencia global4. Desafortunadamente, la mayoría de los pacientes se presentan en una etapa posterior y requieren tratamiento con ablación, quimioembolización o sorafenib12. Sorafenib, un inhibidor receptor de tirosina quinasa (RTKI), fue aprobado por la Administración de Alimentos y Medicamentos en 2008 como la única terapia de medicamentos sistémicos disponible para el tratamiento de HCC no resecable. Aunque el fármaco sólo proporciona un aumento modesto en la supervivencia global, de 7.9 a 10.7 meses13, proporcionó una nueva estrategia terapéutica que podría ser utilizado para manejar HCC.
Manipular el sistema inmunitario para eliminar los cánceres establecidos es un campo de rápido crecimiento en la investigación del cáncer14. Los estudios de punto de control inmune han avanzado considerablemente el desarrollo de fármacos inmunoterapéuticos en el tratamiento del cáncer15,16. La FDA aprobó el uso de anticuerpos (Abs) contra el antígeno t-linfocitos T citotóxico 4 (CTLA-4), la proteína de muerte celular programada 1 (PD-1), y su ligando PD-L1 para el tratamiento del melanoma, el cáncer de pulmón, el cáncer de cabeza y cuello, y el cáncer de vejiga17, 18 , 19 , 20. Los ensayos clínicos de monoterapia o terapia combinada con uno o varios anticuerpos contra PD-1, PD-L1 o CTLA-4 para el tratamiento de la HCC avanzada están en curso21,22,23,y algunos ensayos han mostrado resultados favorables. En 2017, la FDA otorgó la aprobación acelerada para el anticuerpo anti-PD-1 para tratar a los pacientes con HCC, que son resistentes a sorafenib, pero la tasa de respuesta general de esta terapia es de sólo 14.3%. Otras estrategias no se han traducido en la práctica clínica en este momento24,25. Superar la tolerancia inmune profunda inducida por tumores para mejorar la terapia de punto de control inmune26; predecir la eficacia de la terapia de punto de control inmune; prevenir eventos adversos relacionados con el sistema inmunitario; optimizar la ruta de administración, la dosis y la frecuencia; y encontrar combinaciones efectivas de terapias27,28,29 siguen siendo tareas extremadamente desafiantes.
Hay varios enfoques convencionales utilizados para inducir HCC en modelos de ratón actualmente y se utilizan dependiendo de la pregunta de investigación particular del investigador30. Los modelos de ratón HCC inducidos químicamente con compuestos genotóxicos imitan la neoplasia maligna inducida por lesiones. Los modelos de xenoinjerto a través de la implantación ectópica u ortotópica de líneas celulares HCC son adecuados para la detección de fármacos. Varios ratones modificados genéticamente han sido diseñados para investigar la fisiopatología del HCC. Los ratones transgénicos que expresan genes virales, oncogenes y/o factores de crecimiento permiten la identificación de las vías implicadas en la hepatocarcinogénesis. Debido a las limitaciones inherentes, estos modelos no recapitulan las características inmunitarias típicas que se ven en el HCC humano, lo que ha impedido significativamente el esclarecimiento de los mecanismos subyacentes y el desarrollo de estrategias inmunoterapéuticas innovadoras14 ,15. Recientemente hemos creado un modelo murino clínicamente relevante. Este nuevo modelo no sólo imita la iniciación y progresión humana de HCC, sino que también refleja las características más típicas de la enfermedad humana, incluida la disfunción inmune. Hemos caracterizado sus características biológicas e inmunológicas. Aprovechando este novedoso modelo, hemos explorado diversas estrategias inmunoterapéuticas para tratar HCC31,32,33,34,35,36, 37. Esta plataforma única nos permite estudiar mecanismos de inmunotolerancia inducida por tumores y desarrollar estrategias terapéuticas de prueba de concepto para HCC hacia la posterior traducción clínica.

<table border="0" cellpadding="0" cellspacing="0" style="width:848px;" width="847">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:215px;">Anesthesia machine</td>
			<td style="width:123px;">VETEQUIP</td>
			<td style="width:344px;">IMPAC6</td>
			<td style="width:167px;">anesthesia machine for surgery</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Butterfly needle</td>
			<td>BD</td>
			<td>8122963</td>
			<td>Needle used for liver perfusion</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">C57BL/6 mice</td>
			<td>Jackson Lab</td>
			<td>000664&#160;</td>
			<td>mice used in prototol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carprofen</td>
			<td>CRESCENT CHEMICAL</td>
			<td>20402</td>
			<td>carprofen for pain release</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell Strainer&#160;</td>
			<td>CORNING</td>
			<td>REF 431751</td>
			<td>Cell strainer, 70&#181;m, for hepatocytes isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>Allegra X-30R</td>
			<td>centrifuge for cell isolation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clips&#160;</td>
			<td>Teleflex Medical</td>
			<td>REF 523700</td>
			<td>Titanium Clips for spleen</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope</td>
			<td>Zeiss</td>
			<td>Primovert&#160;</td>
			<td>microscope for cell observation</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Mtd2 mice</td>
			<td>N/A</td>
			<td></td>
			<td>Gift from Dr. William A Held at roswell Park Cancer Institute in 2002, maintained in our lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Needle</td>
			<td>BD</td>
			<td>REF 305109</td>
			<td>BD precisionglide needle, 27G x 1/2 (0.4mm x 13mm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Suture</td>
			<td>ETHICON</td>
			<td>J303H</td>
			<td>coated VICRYL suture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SV40 T Ag antibody</td>
			<td>Abcam</td>
			<td>ab16879</td>
			<td>anti-SV40 T-antigen antibody for IHC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe</td>
			<td>BD</td>
			<td>REF 309626</td>
			<td>1 mL TB syringe for cell injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan blue</td>
			<td>SIGMA</td>
			<td>T 8154</td>
			<td>Trypan blue solution for cell viability test</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wound clips</td>
			<td>Reflex</td>
			<td>reflex9, Part. No. 201-1000</td>
			<td>stainless steel wound clips for wound close</td>
		</tr>
	</tbody>
</table>

an oncogenic hepatocyte-induced orthotopic mouse model of hepatocellular cancer arising in the setting of hepatic inflammation and fibrosis

La ausencia de un modelo animal clínicamente relevante que aborde las características inmunitarias típicas del cáncer hepatocelular (HCC) ha impedido significativamente el esclarecimiento de los mecanismos subyacentes y el desarrollo de estrategias inmunoterapéuticas innovadoras. Para desarrollar un modelo animal ideal recapitulando HCC humano, los ratones machos inmunocompetentes C57BL/6J reciben primero una inyección de tetracloruro de carbono (CCl4) para inducir fibrosis hepática, luego reciben hepatocitos oncogénicos histológicamente normales de machojoven joven Ratones transgénicos de antígeno SV40 T (TAg) (MTD2) por inoculación intraesplénica (ISPL). Los andrógenos generados en ratones machoreceptores en la pubertad inician la expresión de TAg bajo el control de un promotor específico del hígado. Como resultado, los hepatocitos transferidos se convierten en células cancerosas y forman masas tumorales en el entorno de la fibrosis/cirrosis hepática. Este novedoso modelo imita la iniciación y progresión humana de hCC en el contexto de la fibrosis/cirrosis hepática y refleja las características más típicas de la HCC humana, incluida la disfunción inmune.

Los hepatocitos oncogénicos aislados de ratones transgénicos tag(Figura 2) fueron sembrados en el hígado de ratones de tipo salvaje por inyección intraesplénica(Figura 3). Los hepatocitos trasplantados crecieron con éxito y de forma fiable tumores ortotópicos de HCC(Figura 4) con antígeno específico tumoral SV40 TAg (Figura 5) en el entorno de inflamación hepática y fibrosis (Figura 1).
<p class="jove_content" fo:keep-toge...

Watch this Scientific Journal Video about An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis at JoVE.com

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

Aquí, describimos el desarrollo de un modelo murino clínicamente relevante de cáncer de hígado recapitulando las características inmunitarias típicas del cáncer hepatocelular (HCC).

Un modelo de ratón ortotópico inducido por hepatocitos oncogénicos que surge en el ajuste de la inflamación hepática y la fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

El uso de un modelo para desarrollar un modelo de masa de cáncer hepatocelular podría ayudar a las personas a dilucidar su inmunotolerencia inducida por tumores y a la inmunosuencia de tumores específicos. El tumor ortotópico que se desarrolla con la técnica de fibrosis hepática imita las características clínicas del cáncer de hígado humano. Este método podría proporcionar una herramienta útil en el estudio inmunológico del cáncer hepatocelular.La demostración visual de este método puede ilustrar los pasos más difíciles técnicamente, como la inyección intraesplénica, que son esenciales para una ejecución exitosa de este protocolo. A continuación, sujeta manualmente a un macho, de seis a ocho semanas de edad de edad 6 ratón de lado dorsal hacia arriba. Utilice exfoliantes alternativos de 70% de etanol y Betadina para limpiar el lugar de inyección tres veces.A continuación, entregue 160 microlitros de tetracloruro de carbono al ratón mediante inyección intraperitoneal y devuelva al animal a su jaula de origen. Después de confirmar la falta de respuesta al pellizco del dedo del pie, en un ratón transgénico de antígeno simiano de cinco meses de edad, coloque el ratón en la posición supina y asegure las extremidades con cinta adhesiva. Hacer una incisión de laparotomía de línea media a lo largo de la longitud de la linea alba, lo suficientemente grande como para proporcionar una exposición adecuada del hígado, y desplazar los intestinos hacia la izquierda.Disecciona sobre el hígado para exponer la vena cava inferior. Inducir una abrazadera de arteria para ligar el vaso. Utilice una aguja de mariposa para obtener acceso intravenoso a la vena porta y asegurar manualmente el catéter.Cambiando cuidadosamente las jeringas para cada solución, entregando con éxito 15 mililitros de solución uno, 15 mililitros de 0.75%solución de colagenasa dos, y 15 mililitros de solución dos sin colagenasa a través del catéter, a un aproximado de 8,9 mililitros de solución por minuto de flujo. Al final de la perfusión, cosecha la masa tumoral en un tubo cónico de 50 mililitros que contiene de 10 a 15 mililitros de PBS. Excóstate del tumor del próximo ratón MTD2 como se acaba de demostrar.Usa tijeras para cortar los hígados en trozos más pequeños. Transfiera los tejidos a un nuevo recipiente de PBS fresco para extraer el resto de la sangre del tejido antes de transferir las piezas de tejido a un tubo cónico de 50 mililitros que contenga cinco mililitros de medio RPMI completo. Mince las muestras de hígado lavado hasta que las piezas pueden caber fácilmente a través de la punta de una pipeta de cinco mililitros.Añadir suficiente medio al tubo para alcanzar un volumen final de 30 mililitros. Utilice una pipeta de cinco mililitros para triturar los fragmentos de tejido, antes de filtrar la solución a través de un colador de 70 micrómetros en un nuevo tubo de 50 mililitros. Lavar el colador varias veces con un medio completo.Ajuste el volumen final a 50 mililitros con un medio completo adicional. Gire rápidamente la suspensión por centrifugación hasta un máximo de 50 veces g antes de detener la centrífuga y decantar el sobrenadante. A continuación, resuspender el pellet en 20 mililitros de PBS para el conteo.Para la inyección intraesplej de los hepatocitos MTD2, cargue una jeringa de un mililitro equipada con una aguja de calibre 27 por cada animal receptor con 220 microlitros de hepatocitos por ratón. Confirme la falta de respuesta a la pellizca del dedo del pie en un animal anestesiado tratado con tetracloruro de carbono. Comenzando justo debajo del músculo de la columna vertebral, a continuación, hacer una incisión de un centímetro en el flanco izquierdo paralelo a la costilla decimotercera del extremo dorsal y utilizar fórceps de punta roma para exteriorizar el bazo.Sujeta el bazo con dos clips de titanio de tamaño medio entre la arteria esplénica y la vena, y entrega 200 microlitros de células en el polo inferior del bazo. Recorte la rama inferior del pediculo con un clip de tamaño medio y corte el bazo entre los dos clips colocados inicialmente. Retire el polo inferior del bazo que se inyectó directamente con las células tumorales y utilice 3-0 poliglactin 910 sutura interrumpido para cerrar la capa muscular interna.A continuación, utilice clips de herida de acero esterilizado para cerrar la capa externa de la piel y administrar por vía subcutánea cinco miligramos por kilogramo de carprofeno. Después del aislamiento de ratones transgénicos de antígeno T como se ha demostrado, se pueden observar hepatocitos y células infiltrantes tumorales. Las células suelen demostrar una viabilidad de casi el 100% después del procesamiento.Después de la inoculación intrasplénica en animales tratados con tetracloruro de carbono de tipo salvaje, los hepatocitos trasplantados crecen con éxito y de forma fiable tumores de carcinoma hepatocelular ortotópicos que expresan el antígeno simiano específico del tumor 40 T. La preparación de nuestra población viable y pura de hepatocitos a partir de la masa MTD2 y la administración precisa de la inyección intraesplénica son importantes para el éxito del procedimiento. Aunque nuestro equipo ha probado la inoculación de hepatocitos en ratones receptores a través de la inyección intravascular e intraperitoneal, estos otros métodos no fueron capaces de inducir carcinoma hepatocelular ortotópico.Este modelo proporciona una plataforma única que permite la investigación de la vigilancia inmune y la tolerancia en sus etapas tempranas y posteriores de la progresión tumoral. Tenga cuidado al usar el tetracloruro de carbono, ya que es tóxico.

an-oncogenic-hepatocyte-induced-orthotopic-mouse-model-hepatocellular

Research

JoVE Journal

Cancer Research

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

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

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

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 ...

Immunophenotyping of Orthotopic Homograft (Syngeneic) of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly its immune-components, is vital to the prognosis and prediction of treatment outcomes, especially those of immunotherapy. TME immune-components are composed of different subsets of tumor-infiltrating immune cells assessable by multi-color FACS. Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest malignances lacking good treatment options, thus an urgent and unmet medical need. One important reason for its non-responsiveness to various therapies (chemo-, targeted, I/O) has been its abundant TME, consisting of fibroblasts and leukocytes that protect tumor cells from these therapies. Orthotopically implanted PDAC is believed to more accurately recapture the TME of human pancreatic cancers than conventional subcutaneous (SC) models.
Homograft tumors (KPC) are transplants of mouse spontaneous PDAC originating from genetically engineered KPC-mice (KrasG12D/+/P53-/-/Pdx1-Cre) (KPC-GEMM). The primary tumor tissue is cut into small fragments (~2 mm3) and transplanted subcutaneously (SC) to the syngeneic recipients (C57BL/6, 7&#8211;9 weeks old). The homografts were then surgically orthotopically transplanted onto the pancreas of new C57BL/6 mice, along with SC-implantation, which reached tumor volumes of 300&#8211;1,000 mm3 by 17 days. Only tumors of 400&#8211;600 mm3 were harvested per approved autopsy procedure and cleaned to remove the adjacent non-tumor tissues. They were dissociated per protocol using a tissue dissociator into single-cell suspensions, followed by staining with designated panels of fluorescently-labeled antibodies for various markers of different immune cells (lymphoid, myeloid and NK, DCs). The stained samples were analyzed using multi-color FACS to determine numbers of immune cells of different lineages, as well as their relative percentage within tumors. The immune profiles of orthotopic tumors were then compared to those of SC tumors. The preliminary data demonstrated significantly elevated infiltrating TILs/TAMs in tumors over the pancreas, and higher B-cell infiltration into orthotopic rather than SC tumors.

Immunophenotyping of Orthotopic Homograft Syngeneic of Murine Primary KPC Pancreatic Ductal Adenocarcinoma by Flow Cytometry

The experimental procedure on the immunophenotyping of murine orthotopic PDAC homografts aims at profiling the tumor immuno-microenvironment. Tumors are orthotopically implanted via surgery. Tumors of 200&#8211;600 mm3 in size were harvested and dissociated to prepare single-cell suspensions, followed by multi-immune marker FACS analysis using different fluorescently-labeled antibodies.

Immunophenotyping de Orthotopic la homoplastia (Syngeneic) de Adenocarcinoma Ductal pancreática KPC primaria murinas por citometría de flujo

Homograft (syngeneic) tumors are the workhorse of today&#39;s immuno-oncology (I/O) preclinical research. The tumor microenvironment (TME), particularly ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Estudio del triple cáncer de mama negativo con el modelo de cáncer de mama ortotópico

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

We describe a protocol for the ultrasound guided implantation of murine-derived pancreatic ductal adenocarcinoma cell lines directly into the native tumor site. This approach resulted in pancreatic tumors detectable by ultrasound scanning within 2-4 weeks of injection, and significantly reduced the proportion tumor cell seeding on the peritoneal wall as compared to surgical orthotopic implantation.

Ultrasonido-Guiado Implantación Ortotópica de Adenocarcinoma Ductal Pancreático Murino

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

Generación de tumores pancreáticos ortotópicos y caracterización ex vivo de la citotoxicidad de células T infiltrantes en tumores

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

Evaluación In Vitro de la Transformación Oncogénica en Células Epiteliales Mamarias Humanas

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

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 ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

Un modelo esferoide tridimensional para investigar la interacción tumor-estroma en el carcinoma hepatocelular

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1&#945; expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG).

Imágenes PET/MR no invasivas en un modelo ortotópico de ratón de carcinoma hepatocelular

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...