The authors would like to thank the MSKCC Tissue Procurement Service Team (TPS), specifically, Maria Corazon Mariano, Priscilla McNeil, Anas Idelbi, Daniel Navarrete and Katrina Allen, in all of their efforts in the successful pursuit of this project and funding from the following sources: 5 R21 CA158609-02 and the Conquer Cancer Foundation and the Breast Cancer Research Foundation. In addition, the authors would like to thank Eric Cottington PhD, Vice President of the Office of Research and Project Administration, the Office of Technology Development, Research Outreach and Compliance and RTM Information Systems Support, in the support of the submission of this manuscript.

REMARQUE: l&#39;obtention de tissus des patients a été autorisé par l&#39;examen institutionnel (CISR) -approved des échantillons biologiques et des protocoles cliniques (numéros de protocole 09-121 et 11-041, respectivement) au Memorial Sloan Kettering Cancer Center. 1. tissus achats <ol><li> Achat de patients tumeur primaire / métastases NOTE: A ce jour, ce protocole a été effectuée sur les types enlevés chirurgicalement pancréatiques, gastriques et tumeurs du sein, ainsi que, les métastases de lymphome. <ol><li> Diriger l&#39;équipe chirurgicale de livrer l&#39;échantillon par courrier ou par système de tube pneumatique au département de pathologie dans étanche et stérile sac d&#39;échantillons de plastique étanche dans le conteneur anti-éclaboussures stérile. Diriger l&#39;équipe chirurgicale pour transporter les échantillons à l&#39;état frais sans formol ou fixateur. </li><li> Diriger le pathologiste ou un pathologiste assistant de récolter les échantillons frais en utilisant une technique stérile, qui incl&#39;utilisation de gants de ludes et des instruments stériles sous une hotte à flux laminaire. </li><li> Noter le temps de la récolte de l&#39;échantillon d&#39;un marché, qui est tenue rigoureuse de moins de 30 minutes de la fin de la procédure chirurgicale. En tenant compte des effets de l&#39;ischémie froide, ne pas prendre des échantillons de pièces de résection chirurgicale avec un temps d&#39;ischémie froide&gt; 30 min 18. </li><li> Diriger le personnel Département de Pathologie de retirer un échantillon de tumeurs primaires d&#39;environ 0,5 cm3 à 1,0 cm 3 sous une hotte à flux laminaire afin de maintenir un environnement stérile. Lorsque cela est possible, choisir un tissu tumoral à partir de la périphérie de la lésion d&#39;indice pour éviter la nécrose centrale (mort cellulaire) franc potentiel. NOTE: Le tissu nécrotique peut être grossièrement reconnaissable par l&#39;un des critères suivants: perte de la couleur ou la pâleur des tissus; perte de force dans laquelle les tissus nécrosés est douce et friable; une ligne de démarcation nette entre le tissu nécrotique et viable. </li><li> Diriger le pathologier ou le pathologiste assistant pour fournir les tissus périphériques qui est en excès (rejets chirurgicale) après prélèvement immédiat de la tumeur pour une évaluation diagnostique. Placer l&#39;échantillon dans un tube à centrifuger conique de 15 ml stérile contenant environ 5 ml de milieu essentiel minimum (MEM) contenant 1% d&#39;antibiotiques (pénicilline et streptomycine). </li><li> Si elle est disponible (par exemple, les ganglions lymphatiques axillaires de la queue de l&#39;échantillon ayant subi une mastectomie), se procurer un échantillon nettement positifs des ganglions lymphatiques associés de la même taille (0,5 cm3 à 1,0 cm 3) et comparer à la réponse aux médicaments de la tumeur primitive. Comme les échantillons de tumeurs primaires, de placer l&#39;échantillon de ganglion lymphatique associé à un tube à centrifuger conique de 15 ml stérile contenant environ 5 ml de milieu essentiel minimum (MEM) contenant 1% d&#39;antibiotiques (pénicilline et streptomycine). </li><li> Si la taille de permis de spécimens chirurgicaux (par exemple, l&#39;échantillon de mastectomie), supprimer (éloigné de la tumeur primaire) un échantillon de tissu normal (c&#39;est-à-parenchyme normale dense / fibreux sein) et le placer dans un tube à centrifuger conique de 15 ml stérile contenant environ 5 ml de milieu essentiel minimum (MEM) contenant 1% d&#39;antibiotiques à des fins de transfert. Après le transfert des équipements de laboratoire, transférer l&#39;échantillon «normal» à un, gel et magasin cryovial «de rupture» dans un congélateur à -80 ° C pour les analyses moléculaires futures. </li><li> Placez tous les échantillons sur la glace mouillée et transporter immédiatement à l&#39;espace de laboratoire ex vivo sectionnement de tissu frais. Ne section de l&#39;échantillon normal, mais plutôt stocker dans un congélateur à -80 ° C pour les analyses moléculaires futures. Transférer une partie de la tumeur primaire et les métastases associée à un, gel et magasin cryovial «de rupture» dans un congélateur à -80 ° C pour les analyses moléculaires futures. </li></ol></li><li> Le prélèvement de tissu à partir d&#39;études cliniques (par exemple, base facultative biopsies (CNB)) NOTE: Nous obtenons les tissus tumoraux de patientsinscrits sur les protocoles cliniques qui donnent leur consentement à subir une CNB d&#39;une métastase de la tumeur accessible avant le début de la thérapie. CNB, à ce jour, ont été réalisées sur des patients atteints sein, le lymphome de la zone marginale, lymphome à cellules du manteau, paragangliome métastatique, col de l&#39;utérus (cellules squameuses) et des ovaires. <ol><li> Ont le personnel clinique de radiologie interventionnelle ou département concerné effectuent un noyau biopsie à l&#39;aiguille de pré-traitement et le spécimen dans un tube de 15 ml à centrifuger conique stérile contenant environ 5 ml de milieu essentiel minimum (MEM) contenant 1% d&#39;antibiotiques (pénicilline et la streptomycine). </li><li> La biopsie sur la glace mouillée et transporter immédiatement à l&#39;espace de laboratoire ex vivo sectionnement de tissu frais. </li></ol></li></ol> 2 Préparation du matériel pour les paramètres de sectionnement et Vibratome <ol><li> Préparation du matériel pour la coupe <ol><li> Faire une solution d&#39;agarose à 4% par addition de 4 g de agarose de 100 ml de PBS. Autoclave (paramètres du cycle: liquides, 121 ° C, 30 min, 15 livres par pouce carré), la solution avant la première utilisation. Pour les utilisations ultérieures, placez le 4% d&#39;agarose dans un micro-ondes pour liquéfier (température élevée des intervalles de 30 s) avant chaque utilisation et laisser refroidir de manière appropriée à ~ 25 ° C (c&#39;est à dire, chaud au toucher) conscient que agarose se solidifie à température ambiante. </li><li> Utilisez les 4% d&#39;agarose comme un média enrobage de tissus non-permanent. Lorsque le 4% d&#39;agarose a suffisamment refroidi et est encore à l&#39;état liquide, avec des pinces stériles placer l&#39;échantillon de tissu dans un moule et versez l&#39;intégration dans les 4% d&#39;agarose. </li><li> Placer l&#39;échantillon de tissu à proximité (~ 2 mm de distance), mais pas directement sur la paroi de la surface de coupe du moule enrobage. L&#39;agarose 4% va commencer à se solidifier rapidement, donc positionner de manière appropriée l&#39;échantillon de tissu rapidement. Pendant la coupe, gardez le 4% d&#39;agarose dans un bain-marie à 55 ° pour rester en solution. </li><li> Pour une plaque de 24 puits pour l&#39;analyse de l&#39;article, ajouter 1 ml of milieu complet (c.-à-MEM + 10% de sérum foetal bovin + 1% d&#39;antibiotique) à chaque puits. Conservez la plaque de 24 puits à température ambiante (~ 25 ° C) pendant toute la durée de la coupe. </li></ol></li><li> Préparation des paramètres Vibratome <ol><li> Utilisation du microtome vibrant, régler la vitesse à laquelle la lame se rapproche de l&#39;échantillon [0 (0,00 mm / s) à 10 (2,5 mm / s)] et la fréquence de la lame [0 (0 Hz) et 10 (100 Hz) ]. Ajustez les paramètres en fonction de la texture du tissu (c&#39;est à dire, la densité / fermeté). </li><li> Pour une tumeur du sein primaire de carcinome canalaire invasif, ajuster les paramètres à 6 et 3, respectivement la vitesse et la fréquence. Pour le tissu tumoral moins dense / entreprise, réduire la vitesse et augmenter la fréquence en conséquence. </li><li> Réglez l&#39;épaisseur de coupe dans les limites de l&#39;instrument (1 - 999 um). Pour ces analyses, définir l&#39;épaisseur de coupe à 200 um. </li><li> Placer l&#39;échantillon d&#39;agarose-intégré à l&#39;aide d&#39;une fine couche de colle liquide sur le disque de tha échantillont est immergé dans un réservoir de médias qui est enfermé dans la glace mouillée. </li></ol></li></ol> 3. tissus sectionnement, traitement et analyses <ol><li> Retirez les sections de précision tranché immédiatement après la coupe à l&#39;aide de pinces stériles et les placer dans une plaque multi-puits avec 1 ml de milieu complet. Lorsque les sections suffisantes sont acquises pour une réponse et des analyses corrélatives, placer la plaque multi-puits pour la réponse au traitement dans un incubateur de culture de tissu fixé à des conditions de culture de tissu standard. </li><li> Prendre plusieurs sections de contrôle que les serre-livres sections traitées en vue de confirmer la réponse dose-dépendante par rapport à des objets en culture. Effectuer tissu coupe rapidement (~ 30 - 40 min), bien en deçà des effets néfastes de temps froid ischémique (~ 2 - 4 heures) 18. Effectuer le traitement des sections uniquement après une période d&#39;acclimatation de 1 heure. </li><li> Après la période d&#39;acclimatation d&#39;une heure, ajouter des doses croissantes (thérapeutiquement pertinent; <em.&gt; Par exemple, 0 - 10 uM concentrations) de drogue vers les sections et la maison dans des conditions de culture de tissu standard pour la période de traitement. Pour cette étude, utiliser 24 et / ou 48 périodes d&#39;évaluation de h. Toutefois, des coupes de tissus de contrôle (véhicule seulement) restent viables, comme testé dans cette étude, pour des périodes allant jusqu&#39;à et y compris les périodes d&#39;évaluation de 96 h. </li><li> Après le traitement, supprimer toutes les sections dans des conditions stériles et le formol fix en plaçant la coupe de tissu dans un tube contenant soit 10% de formaline tamponnée au lieu et à TA O / N ou à un tube contenant du paraformaldéhyde 4% (dilution de 16% de paraformaldéhyde à partir d&#39;un flacon avec du PBS à une concentration finale de 4%) et le mettre à la température ambiante pendant 2 heures. <ol><li> Fixer des coupes de tissus plus grandes (par exemple., ~ 6 mm x 4 mm) 200 um d&#39;épaisseur sur 10% de formol tamponné O / N à la température ambiante. Fixer sections plus petites (par ex., 1 mm x 1 mm) de 200 um d&#39;épaisseur dans du paraformaldéhyde 4% pendant 2 heures à température ambiante. REMARQUE: Pour une pénétration efficace de fixateur le volume de fixative convient 20X le volume du tissu. Etant donné que ce protocole conduit à de très petites tailles (par exemple des tissus, des tissus de 6 mm 3 de volume = 6 pi), pour garantir l&#39;utilisation d&#39;une fixation appropriée ml de fixatif pour toutes les tailles de tissu. </li><li> Après fixation ont les échantillons de tissus inclus en paraffine par le personnel du ministère de pathologie. Section spécimen blocs de paraffine sur des lames chargées d&#39;être hématoxyline et éosine teinté et évalué par immunohistochimie (par exemple, l&#39;apoptose). </li></ol></li></ol>

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No conflicts of interest disclosed.

biologistes du cancer font face à des défis importants en essayant de développer des stratégies thérapeutiques efficaces. Test de médicaments en développement sur ​​des lignées cellulaires de cancer établis ne peut pas refléter avec précision en réponse vivo et in vivo sur des modèles PDX sont laborieux et très coûteux. Compte tenu de ce qui précède, l&#39;application de techniques ex vivo de tumeurs primaires du patient 14, 15 est maintenant positionn?...

Développement de traitements efficaces contre le cancer s&#39;est avéré être extrêmement difficile. lignes de cancer des cellules et des explants de tumeur - ainsi que les xénogreffes ont été utilisées dans la recherche sur le cancer pour plus d&#39;un demi siècle 1,2,3. A ce jour, l&#39;analyse moléculaire de la sensibilité aux médicaments et la résistance dans les deux lignées de cellules cancéreuses établies et des xénogreffes provenant de patients (PDX) est indispensable. Cependant, le test de composés sur des lignées cellulaires de cancer établies n&#39;est pas souvent prédictifs de l&#39;efficacité in vivo, et correspondant à des études in vivo chez les animaux, en particulier dans les modèles PDX, est très coûteux et prend du temps. Les limites de ces systèmes modèles, à savoir l&#39;incapacité d&#39;informer sur l&#39;influence du micro-environnement natif dans la progression tumorale et la réponse à des stratégies thérapeutiques, a conduit le domaine de la recherche afin de développer des méthodes supplémentaires pour compléter ces analyses. Récente, une attention accrue est accordée à une analyse ex vivo de son tour maladeou explants 4, 5 en raison de la plus grande compréhension que la réponse thérapeutique du cancer n&#39;est pas exclusif à la composition moléculaire inhérente des cellules cancéreuses mais est fortement influencée par le microenvironnement des cellules tumorales 6, 7 une fonction qui ne peut être récapitulé par des méthodes de culture traditionnelles et / ou PDX. analyse ex vivo dans le contexte ci-dessus (c.-à-., l&#39;influence de la tumeur adjacente entourant cellule microenvironnement) l&#39;évaluation des sections tumeur / métastases primaires viables implique, plutôt que ex vivo l&#39;analyse d&#39;isolats cellulaires 8, 9. Nous rapportons ici sur une technique ex vivo (c.-à-., Sections de précision tranches fraîches tissus) des deux tumeurs primaires de patients et métastases associées (par exemple, les ganglions lymphatiques) qui informe fidèlement réponse (IC50), les effets hors-cible et permet de moléculaire analyse des mécanismes de résistance et de rétroaction. En outre, une analyse corrélative de therapeUTIC sensibilité / résistance en fonction de biomarqueurs et profil d&#39;expression génique peut être réalisée dans un effort pour identifier les patients les plus susceptibles de répondre à la drogue expérimentale d&#39;intérêt (c.-à-., la réponse médicamenteuse élevée correspond à des patients en particulier le profil biologique). Application de la technique et l&#39;évaluation ex vivo dans un mode multi-paramètre est un mouvement vers la sélection des patients et l&#39;amélioration globale des résultats cliniques. Analyse ex vivo de la réponse au traitement pourrait devenir un outil standard dans le développement préclinique et clinique de traitements contre le cancer et est envisagée comme une étape vers une approche de médecine personnalisée dans les stratégies de développement thérapeutique.

<table border="0" cellpadding="0" cellspacing="0" width="598">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:96px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vibratome Leica VT1000s</td>
			<td style="width:96px;">Leica</td>
			<td style="width:119px;">14047235613</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">UltraPure agarose</td>
			<td style="width:96px;">Invitrogen</td>
			<td style="width:119px;">16500500</td>
			<td style="width:167px;">Prepare 4% and 6% before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Injector blade</td>
			<td style="width:96px;">Ted Pella</td>
			<td style="width:119px;">121-4</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">MEM with Penicillin&#160; + Streptomycin</td>
			<td style="width:96px;">Media Core Facilities (MSKCC)</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">The media is prepared at Memorial Sloan Kettering Cancer Center&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scalpel no. 10</td>
			<td style="width:96px;">Thermo Scientific</td>
			<td style="width:119px;">31-200-32</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disposable forceps</td>
			<td style="width:96px;">Cole-Parmer</td>
			<td style="width:119px;">84011182</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Embedding mold</td>
			<td style="width:96px;">Electron Microscopy Science</td>
			<td style="width:119px;">70181</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS (heat inactivated)</td>
			<td style="width:96px;">Gemini</td>
			<td style="width:119px;">100106</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">24 well plates</td>
			<td style="width:96px;">Corning</td>
			<td style="width:119px;">3524</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Formalin (10%)</td>
			<td style="width:96px;">Sigma Diagnostics</td>
			<td style="width:119px;">SDHT501128&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">16% Formaldehyde solution</td>
			<td style="width:96px;">Thermo Scientific</td>
			<td style="width:119px;">28908</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Embedding microsettes</td>
			<td style="width:96px;">Simport</td>
			<td style="width:119px;">M503-2</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethanol (70%)</td>
			<td style="width:96px;">Fisher Scientific</td>
			<td style="width:119px;">A405P-4</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Waterbath</td>
			<td style="width:96px;">Fisher Scientific</td>
			<td style="width:119px;">15-462-2SQ</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microwave</td>
			<td style="width:96px;">General Electric</td>
			<td style="width:119px;">ModelJES2051DNBB</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Adhesive (Ethyl Cyanoacrylate)</td>
			<td style="width:96px;">Sigma-Aldrich</td>
			<td style="width:119px;">E1505-5G</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10mm dishes</td>
			<td style="width:96px;">BD Falcon</td>
			<td style="width:119px;">353003</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">15ml tubes</td>
			<td style="width:96px;">BD Falcon</td>
			<td style="width:119px;">352096</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

ex vivo treatment response of primary tumors and/or associated metastases for preclinical and clinical development of therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Pour cette étude, la technique ex vivo a été utilisé dans une analyse de corrélation thérapeutique sensibilité / résistance d&#39;un inhibiteur de la protéine de choc thermique 90 (Hsp90i). Dans une évaluation préclinique de cette Hsp90i, la tumeur primaire de cancer du sein, un ER + carcinome canalaire invasif (IDC), et associé métastase des ganglions lymphatiques ont été analysés ex vivo de la réponse au traitement &#160; (Figure 1). Plusieurs 200 um...

Watch this Scientific Journal Video about Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics at JoVE.com

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Lignées de cellules cancéreuses établies et les xénogreffes ont été le pilier de la recherche sur le cancer au cours des dernières décennies. Cependant, des données récentes suggèrent que la réponse thérapeutique est fortement influencée par le micro-environnement des cellules tumorales. Par conséquent, nous avons développé une analyse ex vivo des échantillons de tumeurs primaires à des fins de développement de médicaments.

La réponse au traitement ex vivo de tumeurs primaires et / ou métastases associées préclinique et développement clinique de la thérapeutique

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

ex-vivo-treatment-response-primary-tumors-andor-associated-metastases

Research

JoVE Journal

Medicine

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

14.6K vues.  Memorial Sloan Kettering Cancer Center.  Lignées de cellules cancéreuses établies et les xénogreffes ont été le pilier de la recherche sur le cancer au cours des dernières décennies. Cependant, des données récentes suggèrent que la réponse thérapeutique est fortement influencée par le micro-environnement des cellules tumorales. Par conséquent, nous avons développé une analyse ex vivo des échantillons de tumeurs primaires à des fins de développement de médicaments. 

Vidéo: Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Thermal Ablation for the Treatment of Abdominal Tumors

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ablation procedure, a thin applicator is guided into the target tumor under imaging guidance. Energy is then applied to the tissue until temperatures rise to cytotoxic levels (50-60 &deg;C). Various energy sources are available to heat biological tissues, including radiofrequency (RF) electrical current, microwaves, laser light and ultrasonic waves. Of these, RF and microwave ablation are most commonly used worldwide.
During RF ablation, alternating electrical current (~500 kHz) produces resistive heating around the interstitial electrode. Skin surface electrodes (ground pads) are used to complete the electrical circuit. RF ablation has been in use for nearly 20 years, with good results for local tumor control, extended survival and low complication rates1,2. Recent studies suggest RF ablation may be a first-line treatment option for small hepatocellular carcinoma and renal-cell carcinoma3-5. However, RF heating is hampered by local blood flow and high electrical impedance tissues (eg, lung, bone, desiccated or charred tissue)6,7. Microwaves may alleviate some of these problems by producing faster, volumetric heating8-10. To create larger or conformal ablations, multiple microwave antennas can be used simultaneously while RF electrodes require sequential operation, which limits their efficiency. Early experiences with microwave systems suggest efficacy and safety similar to, or better than RF devices11-13.
Alternatively, cryoablation freezes the target tissues to lethal levels (-20 to -40 &deg;C). Percutaneous cryoablation has been shown to be effective against RCC and many metastatic tumors, particularly colorectal cancer, in the liver14-16. Cryoablation may also be associated with less post-procedure pain and faster recovery for some indications17. Cryoablation is often contraindicated for primary liver cancer due to underlying coagulopathy and associated bleeding risks frequently seen in cirrhotic patients. In addition, sudden release of tumor cellular contents when the frozen tissue thaws can lead to a potentially serious condition known as cryoshock 16.
Thermal tumor ablation can be performed at open surgery, laparoscopy or using a percutaneous approach. When performed percutaneously, the ablation procedure relies on imaging for diagnosis, planning, applicator guidance, treatment monitoring and follow-up. Ultrasound is the most popular modality for guidance and treatment monitoring worldwide, but computed tomography (CT) and magnetic resonance imaging (MRI) are commonly used as well. Contrast-enhanced CT or MRI are typically employed for diagnosis and follow-up imaging.

A thermal tumor ablation procedure is described. The entire procedure is detailed, including pretreatment planning and imaging studies, anesthesia, adjuvant techniques to facilitate a percutaneous approach, imaging guidance of the ablation device to the tumor, thermal treatment, post-treatment care and follow-up.

Ablation thermique pour le traitement des tumeurs abdominales

Percutaneous thermal ablation is an emerging treatment option for many tumors of the abdomen not amenable to conventional treatments. During a thermal ...

Recherche

Médecine

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

Surveillance des métastases tumorales et les lésions ostéolytiques avec bioluminescence et Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

A Novel haute résolution<em&gt; In vivo</em&gt; Technique d&#39;imagerie pour étudier la réponse dynamique des structures intracrâniennes à la croissance tumorale et de thérapeutique

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Adaptation des semi-automatique de circulation des cellules tumorales (CTC) Essais pour les applications cliniques et précliniques de recherche

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Un préclinique modèle murin de métastases hépatiques

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

Isolated Hepatic Perfusion as a Treatment for Liver Metastases of Uveal Melanoma

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic agent melphalan. Briefly, the procedure starts with the setup of a percutaneous veno-venous bypass from the femoral vein to the external jugular vein. Via a laparotomy, catheters are then inserted into the proper hepatic artery and the caval vein. The portal vein and the caval vein, both supra- and infrahepatically, are then clamped. The arterial and venous catheters are connected to a heart lung machine and the liver is perfused with melphalan (1 mg/kg body weight) for 60 min. This way it is possible to locally perfuse the liver with a high dose of a chemotherapeutic agent, without leakage to the systemic circulation.
In previous studies including patients with isolated liver metastases of uveal melanoma, an overall response rate of 33-100% and a median survival between 9 and 13 months, have been reported. The aim of this protocol is to give a clear description of how to perform the procedure and to discuss IHP as a treatment option for liver metastases of uveal melanoma.

Here, we present a protocol how to perform an isolated liver perfusion (IHP) with melphalan and we also discuss IHP as a treatment option for liver metastases of uveal melanoma.

Perfusion hépatique isolée comme un traitement pour les métastases hépatiques de mélanome uvéal

Isolated hepatic perfusion (IHP) is a procedure where the liver is surgically isolated and perfused with a high concentration of the chemotherapeutic ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Génération de microtumeurs Utilisation 3D Biogel humain Système de culture et les cellules de glioblastome patients dérivés pour Kinomic Profilage et Testing Réponse Drug

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&#946; and Tau Neuropathology

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. The paradigm includes tests for spontaneous alternation in a Y-maze, novel object recognition, and limb clasping. These tests were selected because they: 1) interrogate function of cognitive or motor domains and the correlate neural circuitry relevant to the human disease state, 2) have clearly defined endpoints, 3) have easily implementable quality control checks, 4) can be run in a moderate throughput format, and 5) require little intervention by the investigator. These methods are designed for investigators looking to screen compounds for activity in short-term and working memory tasks, or functional motor behaviors associated with Alzheimer's disease mouse models. The methods described here use behavioral tests that engage a number of different brain regions including hippocampus and various cortical areas. Investigators that desire cognitive tests that specifically assess cognition mediated by a single brain region could use these techniques to supplement other behavioral tests.

Assessment of Spontaneous Alternation, Novel Object Recognition and Limb Clasping in Transgenic Mouse Models of Amyloid-&amp;#946; and Tau Neuropathology

Described here is a staged, behavioral screening approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional motor behaviors in transgenic mouse models of &#946;-amyloidosis and tauopathy. These methods are optimized to screen compounds for activity in short-term and working memory tasks.

Évaluation de l&#39;alternance spontanée, reconnaissance récente d&#39;objets et assemblage des membres dans les modèles de souris transgéniques de la neuropathologie amyloïde-β et tau

Here we describe a staged, behavioral testing approach that can be used to screen for compounds that exhibit in vivo efficacy on cognitive and functional ...

Treatment of Liver Metastases Using an Internal Target Volume Method for Stereotactic Body Radiotherapy

The prognosis of patients with metastatic cancers has improved in the past decades due to effective chemotherapy and oligometastatic surgery. For inoperable patients, local ablation therapies, such as stereotactic body radiotherapy (SBRT), can provide effective local tumor control with minimal toxicity. Because of its high precision and accuracy, SBRT delivers a higher radiation dose per fraction, is more effective, and targets smaller irradiation volumes than does conventional radiotherapy. In addition, steep dose gradients from target lesions to surrounding normal tissues are achieved using SBRT; thus, SBRT provides more effective tumor control and exhibits fewer side effects than conventional radiotherapy. The use of SBRT is prevalent for treating intracranial lesions (known as stereotactic radiosurgery); however, it is now also used for treating spinal and adrenal metastases. Because of advancements in image-guided assistance and respiratory motion management, several studies have investigated the use of SBRT for treating lung or liver tumors, which move as a patient breathes. The results of these studies have suggested that SBRT favorably controls tumors in the case of moving lesions.
Four-dimensional computed tomography (4D-CT) with an abdominal compressor (AC) is clinically convenient for effective respiratory motion management. Because this method is noninvasive and allows free breathing, its use reduces complications. Furthermore, patients consider this method convenient. Moreover, it is considered more efficient than other methods of respiratory motion management by physicians and therapists. The use of 4D-CT with an AC for treating pulmonary lesions has also been widely investigated, and the technique is gaining acceptance for treating hepatic lesions. However, the protocols for using 4D-CT with an AC for treating hepatic lesions are different from those used for treating pulmonary lesions. In this article, we describe a new protocol for SBRT with 4D-CT and an AC for treating liver metastases.

Stereotactic body radiotherapy (SBRT) requires rigorous accuracy and precision for delivering high radiation doses per fraction to small treatment volumes to improve tumor control and simultaneously reduce toxicity. Herein, we present a noninvasive and clinically convenient respiratory motion management protocol for SBRT for liver metastases.

Traitement des métastases hépatiques en utilisant une méthode de Volume cible interne pour la radiothérapie stéréotaxique corps

The prognosis of patients with metastatic cancers has improved in the past decades due to effective chemotherapy and oligometastatic surgery. For ...

Contractions of Human-iPSC-derived Cardiomyocyte Syncytia Measured with a Ca-sensitive Fluorescent Dye in Temperature-controlled 384-well Plates

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac physiology and pharmacology. Various methods have been proposed to record this spontaneous activity and to evaluate drug effects, but many of these methods suffer from limited throughput and/or physiological relevance. We developed a high-throughput screening system to quantify the effects of exogenous compounds on hiPSC-CM's beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader. We describe how to prepare the cell plates and the compound plates and how to run the automated assay to achieve high sensitivity and reproducibility. We also describe how to transform and analyze the fluorescence data to provide reliable measures of drug effects on spontaneous rhythm. This assay can be used in drug discovery programs to guide chemical optimization away from, or toward, compounds affecting human cardiac function.

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells are useful models of human cardiac physiology and pharmacology. Here we present a high-throughput screening system to quantify the effects of exogenous compounds on beating frequency, using a Ca-sensitive fluorescent dye and a temperature-controlled imaging multi-well plate reader.

Contractions de syncytiums Cardiomyocyte iPSC-anthropique mesurées avec un colorant Fluorescent Ca sensible dans le contrôle de la température des plaques 384 puits

Spontaneously contracting syncytia of cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CM) are a useful model of human cardiac ...