Name: lung tumor cell recruitment assay
Uploaded: 2019-02-26T12:30:00
Description: Watch this Scientific Journal Video about Lung Tumor Cell Recruitment Assay at JoVE.com

Toutes les procédures effectuées sur des souris ont été approuvées par Medical University de l’Animal Research Committee of Tokyo Women.
1. queue Injection dans la veine des cellules de poumon de Lewis carcinome (LLC)
<ol><li>Maintenir les cellules LLC en DMEM additionné de 10 % FCS, dans un humidifié 5 % CO2 incubateur à 37 ° C. Cellules doivent contenir un système d’expression de protéine fluorescente (p. ex., GFP).</li>
	<li>Éliminer les cellules de la ...

<ol>
	<li>Valastyan, S., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+metastasis:+molecular+insights+and+evolving+paradigms.">Tumor metastasis: molecular insights and evolving paradigms.</a> Cell. 147 (2), 275-292 (2011).</li><li>Kaplan, R. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VEGFR1-positive+haematopoietic+bone+marrow+progenitors+initiate+the+pre-metastatic+niche.">VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metastatic niche.</a> Nature. 438 (7069), 820-827 (2005).</li><li>Hiratsuka, S., Watanabe, A., Aburatani, H., Maru, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumour-mediated+upregulation+of+chemoattractants+and+recruitment+of+myeloid+cells+predetermines+lung+metastasis.">Tumour-mediated upregulation of chemoattractants and recruitment of myeloid cells predetermines lung metastasis.</a> Nature Cell Biology. 8 (12), 1369-1375 (2006).</li><li>Hiratsuka, 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=The+S100A8-serum+amyloid+A3-TLR4+paracrine+cascade+establishes+a+pre-metastatic+phase.">The S100A8-serum amyloid A3-TLR4 paracrine cascade establishes a pre-metastatic phase.</a> Nature Cell Biology. 10 (11), 1349-1355 (2008).</li><li>Tomita, T., Sakurai, Y., Ishibashi, S., Maru, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imbalance+of+Clara+cell-mediated+homeostatic+inflammation+is+involved+in+lung+metastasis.">Imbalance of Clara cell-mediated homeostatic inflammation is involved in lung metastasis.</a> Oncogene. 30 (31), 3429-3439 (2011).</li><li>Deguchi, A., 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=Serum+amyloid+A3+binds+MD-2+to+activate+p38+and+NF-&#954;B+pathways+in+a+MyD88-dependent+manner.">Serum amyloid A3 binds MD-2 to activate p38 and NF-&#954;B pathways in a MyD88-dependent manner.</a> Journal of Immunology. 191 (4), 1856-1864 (2013).</li><li>Erler, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia-induced+lysyl+oxidase+is+a+critical+mediator+of+bone+marrow+cell+recruitment+to+form+the+premetastatic+niche.">Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premetastatic niche.</a> Cancer Cell. 15, 35-44 (2009).</li><li>Huang, Y., 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=Pulmonary+vascular+destabilization+in+the+premetastatic+phase+facilitates+lung+metastasis.">Pulmonary vascular destabilization in the premetastatic phase facilitates lung metastasis.</a> Cancer Research. 69 (19), 7292-7237 (2009).</li><li>Sceneay, J., Smyth, M. J., M&#246;ller, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pre-metastatic+niche:+finding+common+ground.">The pre-metastatic niche: finding common ground.</a> Cancer Metastasis Reviews. 32 (3-4), 449-464 (2013).</li><li>Castle, J., Shaker, H., Morris, K., Tugwood, J. D., Kirwan, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significance+of+circulating+tumor+cells+in+breast+cancer:+A+review.">The significance of circulating tumor cells in breast cancer: A review.</a> The Breast. 23, 552-560 (2014).</li><li>Wolf, M. 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=Endothelial+CCR2+signaling+induced+by+colon+carcinoma+cells+enables+extravasation+via+the+JAK2-Stat5+and+p38MAPK+pathway.">Endothelial CCR2 signaling induced by colon carcinoma cells enables extravasation via the JAK2-Stat5 and p38MAPK pathway.</a> Cancer Cell. 22 (1), 91-105 (2012).</li><li>Hiratsuka, 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=Primary+tumours+modulate+innate+immune+signalling+to+create+pre-metastatic+vascular+hyperpermeability+foci.">Primary tumours modulate innate immune signalling to create pre-metastatic vascular hyperpermeability foci.</a> Nature Communications. 4, 1853 (2013).</li><li>Deguchi, A., 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=Eritoran+inhibits+S100A8-mediated+TLR4/MD-2+activation+and+tumor+growth+by+changing+the+immune+microenvironment.">Eritoran inhibits S100A8-mediated TLR4/MD-2 activation and tumor growth by changing the immune microenvironment.</a> Oncogene. 35 (19), 1445-1456 (2016).</li><li>Ieguchi, 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=ADAM12-cleaved+ephrin-A1+contributes+to+lung+metastasis.">ADAM12-cleaved ephrin-A1 contributes to lung metastasis.</a> Oncogene. 33 (17), 2179-2190 (2014).</li></ol>

Prétraitement des souris avec des anticorps, de drogues, une alimentation riche en graisses ou milieu conditionné par tumeur avant l’injection de cellules tumorales est possible. L’étape la plus difficile dans cet essai est l’injection dans la veine queue. Injection incomplète se traduit par des données non concluantes. La veine caudale C57BL/6 est particulièrement difficile à identifier, résultant en des injections a échoué. Placer les souris sur un coussin chauffant (37 ° C) aide à dilate la veine cau...

Métastase tumorale représente une mortalité élevée dans les maladies liées au cancer. Du point de vue des enquêtes au niveau moléculaire, les métastases peuvent être divisés en plusieurs étapes : initiation tumorale sur le site de la tumeur primitive, la croissance de la tumeur primitive et l’invasion en entourant les tissus, intravasation, circulation dans les vaisseaux sanguins , l’extravasation à des organes éloignés et re-croissance de la tumeur. Chaque étape consiste à différents ensembles de molécules/signaux1.
Études à ce jour ont porté sur les événements qui se produisent dans des organes éloignés avant de commencent aux cellules tumorales circulant dans le sang, qui est également connu comme le stade métastatique pre2,3,4,5, 6. Nos études de modèle de souris ont révélé que la phase pré métastatique facilite les métastases pulmonaires en créant des réactions inflammatoires à basse altitude et de longue durée dans les poumons. La phase pré métastatique est caractérisée par l’hyperperméabilité de themicrovasculature, le recrutement des cellules de moelle osseuse provenant (BMDC) et une augmentation des cytokines/chemokine-comme des molécules dont S100A8 et SAA33,4 . Il a été signalé que lysyl oxydase modifie la matrice extracellulaire dans les poumons pour recruter BMDCs7. Un autre rapport a révélé l’importance de la Angpt2, MMP3 et MMP10 dans la phase pré métastatique8. En d’autres termes, les interactions complexes entre les tumeurs primaires, la moelle osseuse et des organes distants doivent être compris et correctement modulée (par exemple, à l’aide de médicaments qui ciblent les protéines impliquées dans ces interactions) pour prévenir les métastases9 . Pour régler la phase pré métastatique, il abord convient visualisé et une évaluation des interventions diagnostiques ou thérapeutiques. Nous rapportons ici une analyse de recrutement de cellules de tumeur pulmonaire qui permet l’étude de la phase pré métastatique dans les poumons.
Les cellules tumorales injectées directement dans le vaisseau sanguin de la souris sont similaires à circulant les cellules tumorales de patients atteints de cancer, bien qu’il peut y avoir des différences entre les cellules et les cellules tumorales circulantes. Cellules tumorales circulantes eux-mêmes subissent certains changements caractéristiques lorsqu’ils entrent dans la circulation de la tumeur primaire. Néanmoins, les cellules cultivées peuvent former des tumeurs chez des souris immunodéficientes lorsqu’ils sont injectés dans la veine caudale. En outre, dans le système de modèle de souris, fluorescence étiqueté cellules tumorales peut être utilisé qui permettent le suivi de ces cellules dans le corps. Le comportement des cellules tumorales circulantes est critique car ils déterminent les conséquences ultimes du cancer patients10. Sang n’est pas un bon endroit pour la survie des cellules de tumeur2. Ils doivent échapper à l’attaque par le système immunitaire et ont besoin de signaux anti-apoptotiques pour empêcher l’apoptosis en raison du manque de support physique. Des cellules tumorales avec des capacités plus élevées d’initiation tumorale métastatique sites génèrent plusieurs nodules de tumeur. Si les cellules tumorales montrent tropisme d’organe spécifique, métastase doit être observée dans ces organes particuliers. Dans ce test, les premières étapes de la cascade métastatique (croissance de la tumeur primitive et invasion) ne sont pas inclus, et si l’accent est mis sur l’interaction entre la circulation de cellules tumorales et les cellules du stroma (cellules endothéliales, les cellules épithéliales et les cellules du sang). Dans des essais d’injection de cellules de tumeur classiques, les chercheurs doivent attendre que les nodules de tumeur atteignent une taille tangible pour détecter les métastases. Dans ce cas, le nombre exact de cellules tumorales dans les vaisseaux sanguins ne peut être quantifié. En outre, ce processus comporte une phase de repousse de tumeur, ce qui indique que les autres facteurs dans les cellules tumorales peuvent affecter le résultat.
Comptage étiqueté sous un stéréomicroscope de fluorescence des cellules tumorales est un processus simple. Le stéréomicroscope offre un large champ de vision afin que le poumon entier peut être scanné. Cytométrie en flux est également un outil utile qui donne instantanément un nombre précis de cellules tumorales dans les tissus. Compter les cellules fluorescentes dans les tissus sous un microscope confocal est également possible et affiche les photos plus précises des cellules de tumeur métastatique, mais il prend du temps et peut donner des résultats biaisés si seulement une zone sélectionnée est comptabilisée. Le but ultime de ce test est d’observer avec quelle facilité les cellules tumorales s’accumuler dans les poumons. Comme indiqué précédemment,3,4, si les poumons sont en phase métastatique préalable en raison de la signalisation inflammatoires de la tumeur primaire, les cellules tumorales injectées ont tendance à s’accumuler dans les poumons, ce qui implique des chances plus élevées de métastases pulmonaires. Autres conditions (polyarthrite rhumatoïde, l’asthme, etc.) et leur traitement avec des agents anti-inflammatoires (par exemple les anti-TNFα) peuvent atténuer les métastases pulmonaires. Par conséquent, nous concluons que ce test est un outil puissant pour évaluer la possibilité de métastases pulmonaires.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Collagenase</td>
			<td>Sigma</td>
			<td>C6885</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Sigma</td>
			<td>A7030</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>no calcium, no magnesium</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease I</td>
			<td>Sigma</td>
			<td>DN-25</td>
			<td>trace amount</td>
		</tr>
		<tr>
			<td>RBC lysis buffer</td>
			<td>Sigma</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>BD</td>
			<td>352340</td>
			<td>40 micrometer mesh</td>
		</tr>
		<tr>
			<td>Fluorescence labeling kit</td>
			<td>Sigma</td>
			<td>MIN26-KIT</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>0.22 &#956;m syringe filter</td>
			<td>sartorius</td>
			<td>17597K</td>
			<td></td>
		</tr>
		<tr>
			<td>O.C.T. compound</td>
			<td>Sakura Finetek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde Phosphate Buffer Solution</td>
			<td>Wako</td>
			<td>163-20145</td>
			<td></td>
		</tr>
	</tbody>
</table>

lung tumor cell recruitment assay

Afin d’étudier les mécanismes moléculaires qui régissent les métastases tumorales, divers essais à l’aide de la souris comme animal modèle ont été proposées. Nous démontrons ici, un simple test pour évaluer l’extravasation de cellules tumorales ou micrométastase. Dans ce test, les cellules tumorales ont été injectées dans la veine de la queue, et après une courte période, les poumons ont été disséqués et digérés pour compter les cellules tumorales marquées accumulés. Ce dosage saute l’étape initiale de l’invasion de la tumeur primitive dans le vaisseau sanguin et facilite l’étude des événements dans l’organe lointain où se produit la métastase tumorale. Le nombre de cellules injectées dans le vaisseau sanguin peut être optimisé afin d’observer un nombre limité de métastases. Il a été signalé que les cellules du stroma dans des organes éloignés contribuent à métastases. Ainsi, cet essai pourrait être un outil utile pour étudier les éventuels médicaments ou dispositifs pour la prévention des métastases tumorales.

Les poumons présentent plusieurs cellules de GFP-LLC 2 h après l’injection (Figure 1 a). Il est à noter que les taches fluorescentes également détectés dans le filtre rouge devraient être exclus du nombre nombre d’éléments. La grande majorité des cellules LLC disparaître des poumons 24 h après l’injection (Figure 1).
Pour confirmer le nombre de la GFP-...

Watch this Scientific Journal Video about Lung Tumor Cell Recruitment Assay at JoVE.com

Lung Tumor Cell Recruitment Assay

Analyse de recrutement de cellules de tumeur pulmonaire

Cet article décrit une méthode pour quantifier l’accumulation de cellules de tumeur dans les poumons dans un modèle animal de métastases tumorales.

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

Cette méthode peut aider à répondre aux questions clés dans le domaine de la biologie tumorale sur les mécanismes moléculaires de la métastase. Le principal avantage de cette technique est que le recrutement de cellules tumorales dans un organe éloigné peut être quantitate. Commencez par utiliser un réaccien de dissociation cellulaire non enzymatique pour enlever le carcinome pulmonaire de Lewis exprimant des protéines fluorescentes, ou LLC, cellules de leur récipient de culture selon les protocoles standard.Transférer la solution cellulaire résultante dans un tube conique de 15 millilitres avant la centrifugation, et la centrifugeuse laver les granulés deux fois en cinq millilitres de PBS par lavage. Après le deuxième lavage, filtrer les cellules à travers une passoire à cellules poreuses de 40 micromètres, et diluer la suspension à une fois 10 à la concentration de six cellules par millilitre. Chargez ensuite les cellules dans une seringue d’un millilitre munie d’une aiguille de calibre 29 et injectez 100 microlitres de cellules dans la veine arrière de chaque souris mâle de huit à dix semaines, C57-Black-6.Au point de terminaison expérimental approprié, utilisez des ciseaux pour enlever la peau de la surface ventrale de chaque souris, et coupez les côtes et le diaphragme pour exposer la cavité thoracique. Utilisez une aiguille d’infusion ailé de calibre 25 et un tube pour rincer 15 millilitres de PBS à travers le ventricule droit, et enlever le cœur et le thymus. Saisir la trachée avec des forceps, tirer vers le haut, disséquer les tissus conjonctifs autour de la trachée, pour récolter les poumons.Ensuite, lavez les poumons dans PBS, et détachez un lobe pour la visualisation des cellules fluorescentes in situ sous un microscope à fluorescence. Pour digérer le tissu pulmonaire, dés d’abord le lobe caudal avec des ciseaux, et placer les morceaux de poumon dans une seringue de 10 millilitres. Fermez la seringue avec le piston et aspirez cinq millilitres de solution de digestion dans la seringue, déplaçant le piston dans et hors de l’arbre de seringue jusqu’à ce que le tissu pulmonaire soit disséminé dans toute la solution.Distribuez le lisier pulmonaire dans un tube de 50 millilitres et digéz le tissu sur un shaker réciproque pendant 15 minutes à 37 degrés Celsius et 150 rpm. À la fin de l’incubation, triturer le tissu restant jusqu’à ce que les cellules pulmonaires soient complètement dispersées et remettre le tube au shaker pendant 30 minutes supplémentaires. Filtrez ensuite les cellules à travers une passoire poreuse de 40 micromètres et recueillez les cellules par centrifugation.Pour analyser les cellules pulmonaires isolées par cytométrie d’écoulement, résuspendez la pastille en deux millilitres de tampon de lyse de globule rouge pendant une minute à température ambiante, et recueillez les cellules par centrifugation. Puis centrifugez la pastille deux fois en cinq millilitres de PBS frais complétés par 1% d’albumine de sérum bovine par lavage. Après la deuxième centrifugation, resuspendez la pastille en un millilitre de PBS frais plus BSA, et filtrez les cellules à travers une nouvelle passoire pore de 40 micromètres pour la quantification des cellules tumorales par cytométrie d’écoulement.Les poumons présentent de nombreuses cellules GFP-LLC deux heures après l’injection, la grande majorité des cellules disparaissant des poumons dans les 24 heures. Notez que les taches fluorescentes détectées dans le filtre rouge doivent être exclues du nombre de cellules. Pour confirmer le nombre de GFP-LLC dans les poumons, l’un des lobes peut être utilisé pour l’analyse cytométrique de flux comme démontré.Tout en essayant cette procédure, il est important de se rappeler qu’une procédure de section congelée est une méthode plus précise pour observer l’emplacement exact des cellules tumorales.

Research

JoVE Journal

Cancer Research

Conditional Knockdown of Gene Expression in Cancer Cell Lines to Study the Recruitment of Monocytes/Macrophages to the Tumor Microenvironment

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, in the case that the KD of the protein of interest has a lethal effect on cells or the anticipated effect of the KD is time-dependent, unconditional KD methods are not appropriate. Conditional systems are more suitable in these cases and have been the subject of much interest. These include Ecdysone-inducible overexpression systems, Cytochrome P-450 induction system1, and the tetracycline regulated gene expression systems. 
The tetracycline regulated gene expression system enables reversible control over protein expression by induction of shRNA expression in the presence of tetracycline. In this protocol, we present an experimental design using functional Tet-ON system in human cancer cell lines for conditional regulation of gene expression. We then demonstrate the use of this system in the study of tumor cell-monocyte interaction.

This protocol serves as a scheme for setting up a functional Tet-ON system in cancer cell lines and its subsequent use, in particular for studying the role of tumor cell-derived proteins in recruitment of monocytes/macrophages to the tumor microenvironment.

Knockdown conditionnel de l’Expression génique dans les lignées de cellules cancéreuses pour étudier le recrutement des Monocytes/Macrophages pour le microenvironnement tumoral

siRNA and shRNA-mediated knock down (KD) methods of regulating gene expression are invaluable tools for understanding gene and protein function. However, ...

Recherche

Recherche en cancérologie

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

La mise en place d’un essai de colonisation poumon pour la visualisation de cellules de tumeur dans les tissus pulmonaires en circulation

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

Cellulaire et acellulaire poumon modèle pour étudier les métastases tumorales

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include rapid proliferation and pervasive invasion into the normal brain. Recurrence is thought to result from the presence of radio- and chemo-resistant brain tumor stem cells (BTSCs) that invade away from the initial cancerous mass and, thus, evade surgical resection. Hence, therapies that target BTSCs and their invasive abilities may improve the otherwise poor prognosis of this disease. Our group and others have successfully established and characterized BTSC cultures from GBM patient samples. These BTSC cultures demonstrate fundamental cancer stem cell properties such as clonogenic self-renewal, multi-lineage differentiation, and tumor initiation in immune-deficient mice. In order to improve on the current therapeutic approaches for GBM, a better understanding of the mechanisms of BTSC migration and invasion is necessary. In GBM, the study of migration and invasion is restricted, in part, due to the limitations of existing techniques which do not fully account for the in vitro growth characteristics of BTSCs grown as neurospheres. Here, we describe rapid and quantitative live-cell imaging assays to study both the migration and invasion properties of BTSCs. The first method described is the BTSC migration assay which measures the migration toward a chemoattractant gradient. The second method described is the BTSC invasion assay which images and quantifies a cellular invasion from neurospheres into a matrix. The assays described here are used for the quantification of BTSC migration and invasion over time and under different treatment conditions.

Here, we describe live-cell imaging techniques to quantitatively measure the migration and invasion of glioblastoma brain tumor stem cells over time and under multiple treatment conditions.

Épreuves d’imagerie de cellules vivantes pour étude glioblastome Brain Tumor Stem Cell Migration et Invasion

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

Digital Polymerase Chain Reaction Assay for la Variation génétique chez un Patient sporadiques de la polypose adénomateuse familiale en utilisant le Format de puce-in-a-tube

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Isolement des cellules souches mésenchymateuses du tissu de pulpe et co-culture avec les cellules cancéreuses afin d’étudier leurs Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

Détection en temps réel de dans l’apoptose des cellules tumorales in Vitro induite par CD8+ T Cells pour étudier les fonctions immunitaires de suppresseurs de tumeur-infiltration de cellules myéloïdes

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

Caractérisation semi-automatique De MD-L1 et recensement des cellules tumorales circulantes de patients atteints d'immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

In Vitro Assay to Study Tumor-macrophage Interaction

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a malignant brain tumor with no cure, has up to a half the tumor mass TAMs. TAMs can be pro-tumoral or anti-tumoral, depending on the activation of specific genes in the cells. Genetic mutations in the tumors, through regulating cytokine expression, can affect recruitment of TAMs to the tumor microenvironment. Here, we describe a quantitative cell-based assay to assess macrophage recruitment by the conditioned medium from the tumor cells. This assay uses the human macrophage cell line MV-4-11 to study macrophage attraction by the conditioned medium from glioblastoma, allowing for high reproducibility and low variability. Data generated with this assay can contribute to a better understanding of the interaction between the tumor and the tumor microenvironment. Similar assay can be used to assess interaction between the tumor cells and other immune cells, including T cells and natural killer (NK) cells.

This article represents a useful in vitro assay to evaluate the capability of conditioned medium from tumor cells to attract macrophages.

In Vitro Assay to Study Tumor-macrophage Interaction (en)

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

Détection de la progression des tumeurs pulmonaires chez les souris par imagerie par ultrasons

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...