Name: pre-clinical orthotopic murine model of human prostate cancer
Uploaded: 2016-08-29T14:00:00
Description: Watch this Scientific Journal Video about Pre-clinical Orthotopic Murine Model of Human Prostate Cancer at JoVE.com

We thank Dr. Roger Erickson for his support and assistance with the preparation of the manuscript. This work was supported by the National Cancer Institute at the National Institutes of Health through grant numbers RO1CA160079, RO1CA138642, UO1CA184966 and VA funded program project number 1P1 BX001604.

Protocoles pour toutes les procédures impliquant des animaux doivent être examinés et approuvés par un comité de protection et d&#39;utilisation institutionnelle des animaux (IACUC). Suivre les procédures approuvées officiellement pour le soin et l&#39;utilisation des animaux de laboratoire. L&#39;injection intra-prostatique nécessite une intervention chirurgicale ouverte abdominale et les animaux doivent être conservés dans un environnement exempt d&#39;agents pathogènes avec une pièce désignée de chirurg...

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Ce manuscrit décrit une procédure détaillée pour établir un modèle de xénogreffe de souris humaine orthotopique de cancer de la prostate. Ce modèle a été mis en place par implantation directe de la lignée cellulaire du cancer de la prostate humain PC3M-Luc-C6 dans les lobes prostatiques dorsale de souris immunodéprimées. Les tumeurs ont été autorisés à se développer au cours de l&#39;expérience. La croissance tumorale a été surveillée toutes les semaines par un système d&#39;imagerie par biolumine...

Le cancer de la prostate est la deuxième cause la plus fréquente de décès par cancer (9%) chez les hommes aux États-Unis, à côté d&#39; un cancer du poumon et des bronches (28%) 1. Selon des données récentes, on estime que 220 ​​800 cas de cancer de la prostate nouvellement diagnostiqués et 27 540 décès auront lieu en 2015 1. Le taux de survie relative à cinq ans du début de cancer de la prostate stade est&gt; 99% tandis que celle de la maladie métastatique avancé est seulement 28% 1. Un défi majeur pour le traitement de la maladie métastatique avancé est le manque de compréhension des mécanismes moléculaires qui sous-tendent la propension de cette maladie à métastaser à d&#39;autres organes, en particulier à l&#39;os, qui est un site fréquent pour le cancer de la prostate. Par conséquent, il existe un besoin évident d&#39;étudier la composition moléculaire de ces tumeurs de la prostate afin de développer des schémas thérapeutiques efficaces contre la progression à avancé 2,3 de la maladie métastatique. Prostate tumeurs présentent high hétérogénéité biologique sans une voie bien définie à la progression. Métastases se produisent souvent sans indication préalable de l&#39; invasivité tumorale 4. Cette hétérogénéité clinique est attribuée à la diversité moléculaire du cancer de la prostate. Comprendre la composition moléculaire de ces tumeurs mortelles est la clé pour concevoir de meilleures stratégies diagnostiques et thérapeutiques pour cette maladie. Par conséquent, la recherche sur le cancer de la prostate est actuellement axé sur la compréhension et la prévention des métastases. Modèles de souris pré-cliniques in vivo offrent une variété d&#39;options pour comprendre les mécanismes moléculaires de la progression du cancer de la prostate à la maladie métastatique avancé. En outre, ces modèles sont importants pour les évaluations précliniques de nouvelles stratégies thérapeutiques contre cette maladie. Les modèles animaux les plus couramment utilisés comprennent des modèles transgéniques de souris, l&#39;injection de queue veineuse, l&#39;implantation intra-cardiaque et des modèles de souris orthotopique humains. Les études transgéniques sont consumi de tempsng et la corrélation du développement du cancer de la prostate chez les souris avec celle de l&#39; homme ont montré une variabilité 11. Dans les modèles spontanés de souris métastatiques, les cellules sont injectées directement dans la circulation et bien, ils ont le temps de réponse rapide, ils ne peuvent pas être utilisés pour étudier la tumeur primaire ou les premières étapes de la cascade métastatique 5. des modèles de xénogreffes orthotopiques ont la limitation du développement de lésions métastatiques osseuses, le site commun de métastases du cancer de la prostate. Néanmoins, le modèle orthotopique humain du cancer de la prostate de xénogreffe de souris est bien caractérisé et largement utilisé pour étudier les événements moléculaires du développement de la tumeur primaire, diaphonie entre tumeur et microenvironnement d&#39;organes, de la phase initiale de la maladie métastatique et l&#39; utilisation de médicaments expérimentaux pour une intervention thérapeutique 6 , 7,8-11.

<table border="0" cellpadding="0" cellspacing="0" width="732">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">PC3 prostate cancer cell line&#160;</td>
			<td style="width:188px;">ATCC</td>
			<td style="width:111px;">CRL-1435</td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimum Essential Medium (MEM)&#160;</td>
			<td>GIBCO,Life Technology</td>
			<td>11095-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>GIBCO,Life Technology</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>GIBCO,Life Technology</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeocin</td>
			<td>Invitrogen,Life Technology</td>
			<td>R250-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin&#160;</td>
			<td>GIBCO,Life Technology</td>
			<td>25300-54</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IVIS&#160;</td>
			<td>Xenogen-Caliper</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin Syringes (300ul, 28.5g)</td>
			<td>Becton Dickinson</td>
			<td>309300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mice</td>
			<td>Charles River Laboratories, Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alcohol Swabs</td>
			<td>MEDEquip Depot</td>
			<td>326895 BD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PVP Iodine Prep Pad</td>
			<td>MEDEquip Depot</td>
			<td>C12400PDI</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical CatGut Chromic Suture</td>
			<td>Demetech</td>
			<td>CC224017F0P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel</td>
			<td>Corning</td>
			<td>354248</td>
			<td></td>
		</tr>
	</tbody>
</table>

pre-clinical orthotopic murine model of human prostate cancer

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information about this disease. The human orthotopic prostate cancer xenograft mouse model provides a useful alternative approach for understanding the specific interactions between genetically and molecularly altered tumor cells, their organ microenvironment, and for evaluation of efficacy of therapeutic regimens. This is a well characterized model designed to study the molecular events of primary tumor development and it recapitulates the early events in the metastatic cascade prior to embolism and entry of tumor cells into the circulation. Thus it allows elucidation of molecular mechanisms underlying the initial phase of metastatic disease. In addition, this model can annotate drug targets of clinical relevance and is a valuable tool to study prostate cancer progression. In this manuscript we describe a detailed procedure to establish a human orthotopic prostate cancer xenograft mouse model.

Après l&#39; implantation orthotopique de cellules PC3M-Luc-C6 dans le lobe de la prostate postérieure, les souris ont été hebdomadaire imagés en utilisant un système d&#39;imagerie par bioluminescence d&#39;animaux vivants pour contrôler la colonisation des cellules et la croissance des tumeurs au cours de l&#39; expérience (Figure 5A - B). Quantification du signal bioluminescent a indiqué que les cellules PC3M-Luc-C6 colonisé avec succès les...

Watch this Scientific Journal Video about Pre-clinical Orthotopic Murine Model of Human Prostate Cancer at JoVE.com

Pre-clinical Orthotopic Murine Model of Human Prostate Cancer

Prostate cancer is the second most common cause of cancer-related deaths in the United States. An orthotopic cancer model provides a useful approach to understand the biology of prostate cancer and to evaluate the efficacy of therapeutic regimens. This protocol describes detailed steps necessary to establish an orthotopic prostate cancer mouse model.

Pré-clinique Orthotopique modèle murin de cancer de la prostate humaine

To study the multifaceted biology of prostate cancer, pre-clinical in vivo models offer a range of options to uncover critical biological information ...

The overall goal of this procedure is to establish a mouse orthotopic prostate cancer xenograft model that provides a useful approach to studying the molecular events of primary tumor development in the early events of the metastatic cascade. This model can help answer key questions regarding the spastic interactions between genetically and molecularly altered tumor cells in the micro-environment of the growing tumor. It can also help in the pre-clinical evaluation of new therapeutic approaches against the human prostate cancer.The main advantage of this technique over the spontaneous metastatic models are that it promotes the study of the molecular events of primary tumor development and also the recapitulation of the initial phase of metastasis prior to entry and embolism of tumor cells into the circulation. Orthotopic model of prostate cancer is one of the best pre-clinical models to understand how prostate cancer cells metastasize to different organs. Most prostate cancer patients die of metastasis, and therefore this model is very important to investigate the molecular mechanisms of metastasis.Using this model we can design better strategies for the treatment of metastasis by using neural compounds, those are specific to metastatic pathways. Demonstrating this procedure will be Melissa Colden, a grad student and Dr.Varahram Shahryari, a research associate from our laboratory. Before beginning the procedure, wash the tumor cell culture of interest with PBS and incubate the cells with 2 milliliters of 05 percent trypsin for 3 to 5 minutes in the cell culture incubator.When the cells have detached, stop the reaction with 5 milliliters of complete medium and count the cells by trypan blue exclusion. Dilute the cells to a 2.5 times 10 to the 5 cells per 10 microliters of medium concentration, and mix the cell suspension with 10 microliters of basement membrane-like extracellular matrix extract. Hold the cells on ice, then place an anesthetized six to eight week old immunocompromised male experimental mouse in the supine position on a sterile heating pad and confirm a lack of response to toe pinch.Apply ointment to the animals eyes and remove the hair from the lower abdomen. Disinfect the surgical area with 10 percent povidone iodine scrub, followed by 70 percent ethanol about 1 to 2 centimeters above the penis sheath, and about 2 to 3 centimeters below the bottom of the rib cage. Use a pair of fine forceps to lift 2 millimeters of skin above the preputial gland.Using a scalpel, make a 1 centimeter mid-line incision through the skin, followed by a 1 centimeter incision through the muscle layer. Locate the yellow, light brown spherical bladder directly below the incision. Then use the fine forceps to grip the bladder and to lift the organ up and out of the body cavity toward the penis sheath to expose the seminal vesicles.With a wet cotton swab in each hand, carefully externalize the vesicles one at a time, and lay them top down on the outer surface of the abdomen with the bladder in the middle. Next, use the cotton swabs to gently tilt back the seminal vesicles at the point of insertion near the bladder neck towards the penis sheath so that the two dorsal prostate lobes are clearly visible. Now use a micro-pipette to re-suspend the cells in a single-cell suspension and label them with luciferin.Aspirate 25 microliters of the cells into a 0.5 cc syringe equipped with a 28.5 gauge needle and transfer the mouse under a dissecting microscope. Then, insert the syringe needle into a dorsal prostatic lobe and slowly inject 20 microliters of the cell suspension until a bulla formation is observed. When all of the cells have been administered, retract the needle, pressing lightly on the injection site with a cotton swab for a few seconds to prevent leakage.Then use the swabs to carefully return the vesicles to the body cavity one at a time, followed by the bladder. After placing the organs back into the body cavity, suture the muscle layer first in an interrupted pattern with absorbable 4-0 chromic cat gut sutures, followed by the skin closure with non-absorbable 4-0 nylon surgical sutures. The skin can also be pulled together and closed with surgical clamps to close the incision completely.Then, immediately image the animal, and return the mouse to a clean cage under a warming lamp with monitoring until it is fully recovered. Following the orthotopic implantation of luciferin-labeled prostate cancer cells into the posterior prostatic lobe, as just demonstrated, weekly imaging using live animal bioluminescence imaging reveals a successful colonization of the prostate lobes by the tumor cells. Indeed, an increase in the bioluminescence over time is indicative of the enlargement of the primary tumor over the course of the experiment, confirming the effectiveness of this technique in non-invasive, radiographic, fluorescent or luminescent imaging for tumor growth and metastatic lesion monitoring.While attempting this procedure, it is important to remember that intra-prostatic injection requires open abdominal surgery. So, it should be performed in a pathogen-free environment using proper aseptic surgical techniques. Working with human cancer cells can be extremely hazardous and precautions, such as wearing personal protective equipment, and working within biosafety level two containment should always be taken while performing this procedure.This model paved the way for the transplant of histologically intact tumor fragments to examine the effects of potential drug therapies on tumor growth or original lymph node metastasis. After watching this video, you should have a good understanding of how to implant cancer cells into the dorsal prostatic lobes for the establishment of an orthotopic human prostate cancer xenograft mouse model.

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