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>

<ol>
	<li>Abercrombie, M., Ambrose, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+Microscope+Studies+of+Cell+Contacts+in+Tissue+Culture.">Interference Microscope Studies of Cell Contacts in Tissue Culture.</a> Experimental Cell Research. 15 (58), 332-345 (1958).</li><li>Coriell, L. L., McAllister, R. M., Wagner, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Criteria+for+Determining+Malignancy+in+Tissue-Culture+Cell+Lines+in+the+Albino+Rat.">Criteria for Determining Malignancy in Tissue-Culture Cell Lines in the Albino Rat.</a> New York Academy of Science. 5, 351-355 (1957).</li><li>Paul, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cancer+Cell+in+Vitro:+A+Review.">The Cancer Cell in Vitro: A Review.</a> Cancer Research. 22, 431-440 (1962).</li><li>Dean, J. L., 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=Therapeutic+response+to+CDK4/6+inhibition+in+breast+cancer+defined+by+ex+vivo+analysis+of+human+tumors.">Therapeutic response to CDK4/6 inhibition in breast cancer defined by ex vivo analysis of human tumors.</a> Cell Cycle. 11 (14), 2756-2761 (2012).</li><li>Bray, 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=Bcl-2+Modulation+to+Activate+Apoptosis+in+Prostate+Cancer.">Bcl-2 Modulation to Activate Apoptosis in Prostate Cancer.</a> Molecular Cancer Research. 7 (9), 1487-1496 (2009).</li><li>Quail, D. F., Joyce, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microenvironmental+regulation+of+tumor+progression+and+metastasis.">Microenvironmental regulation of tumor progression and metastasis.</a> Nature Medicine. 19 (11), 1423-1437 (2013).</li><li>Nakasone, E. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Tumor-Stroma+Interactions+during+Chemotherapy+Reveals+Contributions+of+the+Microenvironment+to+Resistance.">Imaging Tumor-Stroma Interactions during Chemotherapy Reveals Contributions of the Microenvironment to Resistance.</a> Cancer Cell. 21, 488-503 (1016).</li><li>Shi, Y., Hogue, J., Dixit, D., Koh, J., Olson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+genetic+studies+of+isolated+cells+from+parathyroid+tumors+reveal+the+complex+pathogenesis+of+parathyroid+neoplasia.">Functional and genetic studies of isolated cells from parathyroid tumors reveal the complex pathogenesis of parathyroid neoplasia.</a> PNAS. 111 (8), 3092-3097 (2014).</li><li>Vidal, S. 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=Isolation+of+cancer+stem+cells+from+human+prostate+cancer+samples.">Isolation of cancer stem cells from human prostate cancer samples.</a> J. Vis. Exp. (85), e51332 (2014).</li><li>Wanping, X., Neckers, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+the+Molecular+Chaperone+Heat+Shock+Protein+90+Provides+a+Multifaceted+Effect+on+Diverse+Cell+Signaling+of+Cancer+Cells.">Targeting the Molecular Chaperone Heat Shock Protein 90 Provides a Multifaceted Effect on Diverse Cell Signaling of Cancer Cells.</a> Clinical Cancer Research. 13, 1625-1629 (2007).</li><li>Moulick, 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=Affinity-based+proteomics+reveal+cancer-specific+networks+coordinated+by+Hsp90.">Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90.</a> Nature Chemical Biology. 7 (11), 818-826 (1038).</li><li>Alpaugh, M. L., Tomlinson, J. S., Ye, Y., Barsky, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationship+of+sialyl-Lewis(x/a)+underexpression+and+E-cadherin+overexpression+in+the+lymphovascular+embolus+of+inflammatory+breast+cancer.">Relationship of sialyl-Lewis(x/a) underexpression and E-cadherin overexpression in the lymphovascular embolus of inflammatory breast cancer.</a> American Journal of Pathology. 161 (2), 619-628 (2002).</li><li>Chu, K., Boley, K. M., Moraes, R., Barsky, S. H., Robertson, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+paradox+of+E-cadherin:+role+in+response+to+hypoxia+in+the+tumor+microenvironment+and+regulation+of+energy+metabolism.">The paradox of E-cadherin: role in response to hypoxia in the tumor microenvironment and regulation of energy metabolism.</a> Oncotarget. 4 (3), 446-462 (2013).</li><li>Vaira, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+model+of+organotypic+culture+for+pharmacodynamic+profiling+of+human+tumors.">Preclinical model of organotypic culture for pharmacodynamic profiling of human tumors.</a> PNAS. 107 (18), 8352-8356 (2010).</li><li>Centenera, M. M., Raj, G. V., Knudsen, K. E., Tilley, W. D., Butler, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+culture+of+human+prostate+tissue+and+drug+development.">Ex vivo culture of human prostate tissue and drug development.</a> Nature Reviews Urology. 10, 483-487 (2013).</li><li>Jhaveri, 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=Heat+shock+protein+90+inhibitors+in+the+treatment+of+cancer:+current+status+and+future+directions.">Heat shock protein 90 inhibitors in the treatment of cancer: current status and future directions.</a> Expert Opinion on Investigational Drugs. 5, 611-628 (2014).</li><li>Gerecitano, J. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+124-PU-H71PET+imaging+to+predict+intratumoral+concentration+in+patients+on+a+phase+I+trial+of+PU-H71.">Using 124-PU-H71PET imaging to predict intratumoral concentration in patients on a phase I trial of PU-H71.</a> Journal of Clinical Oncology. 31, 11076-11 (2013).</li><li>Yildiz-Aktas, I., Dabbs, D. J., Bhargava, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+cold+ischemic+time+on+the+immunohistochemical+evaluation+of+estrogen+receptor,+progesterone+receptor,+and+HER2+expression+in+invasive+breast+carcinoma.">The effect of cold ischemic time on the immunohistochemical evaluation of estrogen receptor, progesterone receptor, and HER2 expression in invasive breast carcinoma.</a> Modern Pathology. 25, 1098-1105 (2012).</li></ol>

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><tbody><tr><td>Vibratome Leica VT1000s</td><td>Leica</td><td>14047235613</td><td></td></tr><tr><td>UltraPure agarose</td><td>Invitrogen</td><td>16500500</td><td>Prepare 4% and 6% before use</td></tr><tr><td>Injector blade</td><td>Ted Pella</td><td>121-4</td><td></td></tr><tr><td>MEM with Penicillin&amp;nbsp; + Streptomycin</td><td>Media Core Facilities (MSKCC)</td><td></td><td>The media is prepared at Memorial Sloan Kettering Cancer Center&amp;nbsp;</td></tr><tr><td>Scalpel no. 10</td><td>Thermo Scientific</td><td>31-200-32</td><td></td></tr><tr><td>Disposable forceps</td><td>Cole-Parmer</td><td>84011182</td><td></td></tr><tr><td>Embedding mold</td><td>Electron Microscopy Science</td><td>70181</td><td></td></tr><tr><td>FBS (heat inactivated)</td><td>Gemini</td><td>100106</td><td></td></tr><tr><td>24 well plates</td><td>Corning</td><td>3524</td><td></td></tr><tr><td>Formalin (10%)</td><td>Sigma Diagnostics</td><td>SDHT501128&amp;nbsp;</td><td></td></tr><tr><td>16% Formaldehyde solution</td><td>Thermo Scientific</td><td>28908</td><td></td></tr><tr><td>Embedding microsettes</td><td>Simport</td><td>M503-2</td><td></td></tr><tr><td>Ethanol (70%)</td><td>Fisher Scientific</td><td>A405P-4</td><td></td></tr><tr><td>Waterbath</td><td>Fisher Scientific</td><td>15-462-2SQ</td><td></td></tr><tr><td>Microwave</td><td>General Electric</td><td>ModelJES2051DNBB</td><td></td></tr><tr><td>Adhesive (Ethyl Cyanoacrylate)</td><td>Sigma-Aldrich</td><td>E1505-5G</td><td></td></tr><tr><td>10 mm dishes</td><td>BD Falcon</td><td>353003</td><td></td></tr><tr><td>15 ml tubes</td><td>BD Falcon</td><td>352096</td><td></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

14.7K 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

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Vivez l&#39;imagerie des réponses de drogues dans le microenvironnement des tumeurs dans les modèles murins de cancer du sein

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Recherche

Médecine

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.

Génération de tumeurs pancréatiques orthopiques et caractérisation ex vivo de la cytotoxicité des cellules T infiltrantes

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

Recherche en cancérologie

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

Test de thérapies ciblées dans le cancer à l’aide de l’analyse structurale de l’altération de l’ADN et des Xénogreffes dérivées du patient

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Utilisation combinée des essais de métastases de veine de queue et de l'imagerie in vivo en temps réel pour quantifier la colonisation métastatique et le fardeau du cancer du sein dans les poumons

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

Mesurer la réponse en temps réel des médicaments dans les tranches de tissu tumoral organotypique

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

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

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

Transplantation orthotopique de tumeurs mammaires comme modèles précliniques pour le cancer du sein

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

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable preclinical models that can accurately predict patient responses. One of the disadvantages of existing preclinical models is the inability to contextually preserve the human tumor microenvironment (TME) and accurately represent intratumoral heterogeneity, thus limiting the clinical translation of data. By contrast, by representing the culture of live fragments of human tumors, the patient-derived explant (PDE) platform allows drug responses to be examined in a three-dimensional (3D) context that mirrors the pathological and architectural features of the original tumors as closely as possible. Previous reports with PDEs have documented the ability of the platform to distinguish chemosensitive from chemoresistant tumors, and it has been shown that this segregation is predictive of patient responses to the same chemotherapies. Simultaneously, PDEs allow the opportunity to interrogate molecular, genetic, and histological features of tumors that predict drug responses, thereby identifying biomarkers for patient stratification as well as novel interventional approaches to sensitize resistant tumors. This paper reports PDE methodology in detail, from collection of patient samples through to endpoint analysis. It provides a detailed description of explant derivation and culture methods, highlighting bespoke conditions for particular tumors, where appropriate. For endpoint analysis, there is a focus on multiplexed immunofluorescence and multispectral imaging for the spatial profiling of key biomarkers within both tumoral and stromal regions. By combining these methods, it is possible to generate quantitative and qualitative drug response data that can be related to various clinicopathological parameters and thus potentially be used for biomarker identification.

This paper describes methods for the generation, drug treatment, and analysis of patient-derived explants for assessing tumor drug responses in a live, patient-relevant, preclinical model system.

Explantations tumorales dérivées de patients en tant que plate-forme préclinique « vivante » pour prédire la résistance aux médicaments chez les patients

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and treatment response to targeted inhibitors in cancer. Pioneering work in gastrointestinal and pancreatic cancers has highlighted the promise of patient-derived organoids (PDOs) as a patient-proximate culture system, with an increasing number of models emerging. Similarly, work in other cancer types has focused on establishing organoid models and optimizing culture protocols. Notably, 3D cancer organoid models maintain the genetic complexity of original tumor specimens and thus translate tumor-derived sequencing data into treatment with genetically informed targeted therapies in an experimental setting. Further, PDOs might foster the evaluation of rational combination treatments to overcome resistance-associated adaptation of tumors in the future. The latter focuses on intense research efforts in non-small-cell lung cancer (NSCLC), as resistance development ultimately limits the treatment success of targeted inhibitors. An early assessment of therapeutically targetable mechanisms using NSCLC PDOs could help inform rational combination treatments. This manuscript describes a standardized protocol for the cell culture plate-based assessment of drug sensitivities to targeted inhibitors in NSCLC-derived 3D PDOs, with potential adaptability to combinational treatments and other treatment modalities.

This protocol describes a standardized evaluation of drug sensitivities to targeted signaling inhibitors in NSCLC patient-derived organoid models.

Profilage de la sensibilité aux thérapies ciblées dans les organoïdes dérivés de patients atteints de CPNPC mutants EGFR

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

Multiparametric Tumor Organoid Drug Screening Using Widefield Live-Cell Imaging for Bulk and Single-Organoid Analysis

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex vivo drug screenings. However, current adenosine triphosphate (ATP)-based drug screening assays do not capture the complexity of a drug response (cytostatic or cytotoxic) and intratumor heterogeneity that has been shown to be retained in PDTOs due to a bulk readout. Live-cell imaging is a powerful tool to overcome this issue and visualize drug responses more in-depth. However, image analysis software is often not adapted to the three-dimensionality of PDTOs, requires fluorescent viability dyes, or is not compatible with a 384-well microplate format. This paper describes a semi-automated methodology to seed, treat, and image PDTOs in a high-throughput, 384-well format using conventional, widefield, live-cell imaging systems. In addition, we developed viability marker-free image analysis software to quantify growth rate-based drug response metrics that improve reproducibility and correct growth rate variations between different PDTO lines. Using the normalized drug response metric, which scores drug response based on the growth rate normalized to a positive and negative control condition, and a fluorescent cell death dye, cytotoxic and cytostatic drug responses can be easily distinguished, profoundly improving the classification of responders and non-responders. In addition, drug-response heterogeneity can by quantified from single-organoid drug response analysis to identify potential, resistant clones. Ultimately, this method aims to improve the prediction of clinical therapy response by capturing a multiparametric drug response signature, which includes kinetic growth arrest and cell death quantification.

This protocol describes a semi-automated method for medium- to high-throughput organoid drug screenings and microscope-agnostic, automated image analysis software to quantify and visualize multiparametric, single-organoid drug responses to capture intratumor heterogeneity.

Criblage multiparamétrique de médicaments organoïdes tumoraux à l’aide de l’imagerie de cellules vivantes à grand champ pour l’analyse en vrac et mono-organoïde

Patient-derived tumor organoids (PDTOs) hold great promise for preclinical and translational research and predicting the patient therapy response from ex ...

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same pathway, are likely to fail. Emphasis must be placed upon the inhibition of pathways that are responsible for intrinsic and/or adaptive resistance to therapy. An active field of investigation is the development and testing of DNA repair inhibitors that promote the action of, and prevent resistance to, commonly used chemotherapy and radiotherapy. We used a novel protocol to evaluate the effectiveness of BRCA2 inhibition as a means to sensitize tumor cells to the DNA damaging drug cisplatin. Tumor cell metabolism (acidification and respiration) was monitored in real-time for a period of 72 hr to delineate treatment effectiveness on a minute by minute basis. In combination, we performed an assessment of metastatic frequency using a chicken embryo chorioallantoic membrane (CAM) model of extravasation and invasion. This protocol addresses some of the weaknesses of commonly used in vitro and in vivo methods to evaluate novel cancer therapy regimens. It can be used in addition to common methods such as cell proliferation assays, cell death assays, and in vivo murine xenograft studies, to more closely discriminate amongst candidate targets and agents, and select only the most promising candidates for further development.

Evaluating the Effectiveness of Cancer Drug Sensitization In Vitro and In Vivo

Here, real-time monitoring of tumor cell metabolism, combined with an in vivo chicken embryo chorio-allantoic membrane (CAM) model of metastasis, are used to evaluate novel anti-cancer targets/agents for their ability to sensitize tumor cells to DNA damaging chemotherapeutics.

Évaluation de l&#39;efficacité des médicaments contre le cancer Sensibilisation<em&gt; In Vitro</em&gt; Et<em&gt; In Vivo</em

Due to the high level of heterogeneity and mutations inherent in human cancers, single agent therapies, or combination regimens which target the same ...

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

Résumé

Explorer plus de vidéos

PLAYLIST