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

Tutte le procedure eseguite con i topi sono state approvate da Medical University animale ricerca comitato di Tokyo femminile.
1. iniezione della vena della coda del polmone di Lewis (LLC) delle cellule di Carcinoma
<ol><li>Mantenere le cellule LLC in DMEM completati con 10% FCS, in un umidificata 5% CO2 incubatore a 37 ° C. Le cellule devono contenere un sistema di espressione della proteina fluorescente (ad es., GFP).</li>
	<li>Rimuovere le cellule dalla piastra di col...

<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>

Pre-trattamento dei topi con anticorpi, farmaci, una dieta ricca di grassi o medio del tumore-condizionato prima dell'iniezione delle cellule del tumore è possibile. Il passo più difficile in questo dosaggio è l'iniezione della vena della coda. Iniezione incompleta provoca dati inconcludenti. La vena della coda in C57BL/6 è particolarmente difficile da identificare, con conseguente iniezioni non riuscite. Ponendo i mouse su un rilievo di riscaldamento (° C 37) aiuta a dilata la vena di coda affinché iniezione diven...

Metastasi del tumore rappresenta alta mortalità nelle malattie legate al cancro. Dal punto di vista delle indagini a livello molecolare, metastasi può essere diviso in più fasi: l'inizio del tumore al luogo primario del tumore, la crescita del tumore primario e invasione nei circostanti tessuti, intravasation, circolazione attraverso i vasi sanguigni , lo stravaso in organi distanti e la ricrescita del tumore. Ogni fase prevede diversi insiemi di molecole/segnali1.
Gli studi fin qui sono stati interessati con eventi che si verificano in organi distanti, prima le cellule tumorali circolanti nel sangue, che è anche conosciuto come la fase pre-metastatico2,3,4,5, 6. I nostri studi di modello del mouse ha rivelato che la fase di pre-metastatica facilita la metastasi del polmone con la creazione di lunga durata e basso livello le risposte infiammatorie nei polmoni. La fase di pre-metastatica è caratterizzata da hyperpermeability di themicrovasculature, reclutamento di cellule di derivazione midollare (BMDC) e il upregulation di citochine/chemochine-come molecole tra cui S100A8 e SAA33,4 . È stato segnalato che lisil ossidasi consente di modificare la matrice extracellulare nei polmoni per reclutare BMDCs7. Un altro rapporto ha rivelato l'importanza della Angpt2, MMP3 e MMP10 nella fase pre-metastatico8. In altre parole, le intricate interazioni tra tumori primari, midollo osseo e organi remoti devono essere capito e correttamente modulata (ad esempio, utilizzando farmaci mirati proteine coinvolte in queste interazioni) per prevenire futura metastasi9 . Per regolare la fase di pre-metastatica, in primo luogo dovrebbe essere visualizzato e valutato per interventi diagnostici o terapeutici. Qui, segnaliamo un'analisi di reclutamento polmonare del tumore delle cellule che consente lo studio della fase pre-metastatico nei polmoni.
Le cellule del tumore iniettate direttamente nel vaso sanguigno del mouse sono simili a fare circolare le cellule del tumore in malati di cancro, anche se ci possono essere differenze tra cellule coltivate e cellule tumorali circolanti. Cellule tumorali circolanti stessi subire alcuni cambiamenti caratteristici quando entrano in circolazione dal tumore primario. Tuttavia, le cellule coltivate possono formare tumori in topi immunodeficienti quando vengono iniettati nella vena caudale. Inoltre, nel sistema modello del mouse, può essere utilizzato fluorescenza etichettato le cellule del tumore che consentono il rilevamento di queste cellule nel corpo. Il comportamento di cellule tumorali circolanti è critico che determinano le estreme conseguenze in cancro pazienti10. Sangue non è un buon posto per la sopravvivenza del tumore le cellule2. Hanno bisogno di fuggire da attacco da sistema immunitario dell'ospite e hanno bisogno di segnali anti-apoptotici per prevenire l'apoptosi dovuto una mancanza di supporto fisico. Le cellule del tumore con maggiore abilità dell'inizio del tumore ai siti metastatici generano più noduli del tumore. Se le cellule tumorali mostrano tropismo organo specifico, metastasi dovrebbe essere osservata in quegli organi particolari. In questa analisi, non sono inclusi i passaggi iniziali della cascata metastatica (crescita primaria del tumore e l'invasione), e così il focus è sull'interazione fra le cellule del tumore e le cellule stromali (cellule endoteliali, cellule epiteliali e le cellule del sangue) di circolazione. Nelle analisi di iniezione delle cellule del tumore convenzionali, i ricercatori devono attendere fino a quando i noduli del tumore crescono a una dimensione tangibile per rilevare la metastasi. In questo caso, il numero esatto delle cellule del tumore nel vaso sanguigno non può essere quantificato. Inoltre, questo processo include una fase di ricrescita del tumore, che indica che altri fattori nelle cellule del tumore possono influenzare il risultato.
Conteggio etichettato le cellule del tumore sotto un microscopio stereoscopico di fluorescenza è un processo semplice. Lo stereomicroscopio fornisce un ampio campo di vista in modo che l'intero polmone può essere scansionato. Citometria a flusso è anche un utile strumento che dà immediatamente un numero preciso di cellule del tumore nel tessuto. Contando le cellule fluorescenti nel tessuto sotto un microscopio confocale è anche possibile e visualizza le immagini più precise delle cellule del tumore metastatico, ma esso richiede tempo e può dare risultati parziale se solo un'area selezionata viene conteggiata. L'obiettivo finale di questo test è quello di osservare come facilmente le cellule del tumore si accumulano nei polmoni. Come riferito precedentemente3,4, se i polmoni sono in fase pre-metastatico a causa della segnalazione infiammatoria dal tumore primario, le cellule tumorali iniettate sono inclini ad accumularsi nei polmoni, così che implica una maggiore probabilità di metastasi del polmone. Altre condizioni (artrite reumatoide, asma, ecc.) e il loro trattamento con agenti anti-infiammatori (ad esempio anti-TNFα) può attenuare la metastasi del polmone. Quindi, possiamo concludere che questo test è un potente strumento per valutare la possibilità della metastasi del polmone.

<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

Per studiare i meccanismi molecolari che regolano la metastasi del tumore, sono stati proposti vari saggi usando il mouse come un animale di modello. Qui, dimostriamo un semplice test per valutare lo stravaso delle cellule di tumore o micrometastasi. In questa analisi, le cellule del tumore sono state iniettate attraverso la vena caudale, e dopo un breve periodo, i polmoni erano dissecati e digeriti per contare le cellule del tumore con etichetta accumulato. Questo test salta il passaggio iniziale dell'invasione del tumore primario nel vaso sanguigno e facilita lo studio di eventi in organi distanti dove si verifica la metastasi del tumore. Il numero di cellule iniettate nel vaso sanguigno può essere ottimizzato per osservare un numero limitato di metastasi. È stato segnalato che le cellule stromal in organi distanti contribuiscano alla metastasi. Così, questo test potrebbe essere uno strumento utile per esplorare il potenziale terapeutici farmaci o dispositivi per la prevenzione della metastasi del tumore.

I polmoni presentano molte cellule GFP-LLC 2 h dopo l'iniezione (Figura 1A). Dovrebbe essere notato che le macchie fluorescenti anche rilevate nel filtro rosso devono essere escluso dal conteggio del numero di cellulare. La maggior parte delle cellule LLC scomparsa dai polmoni 24 h dopo l'iniezione (Figura 1).
Per confermare il numero della GFP-LLC nei polmoni, uno dei ...

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

Lung Tumor Cell Recruitment Assay

Analisi di reclutamento delle cellule di tumore del polmone

Questo manoscritto descrive un metodo per quantificare l'accumulo di cellule del tumore nei polmoni in un modello animale della metastasi del tumore.

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

Questo metodo può aiutare a rispondere a domande chiave nel campo della biologia tumorale sui meccanismi molecolari della metastasi. Il principale vantaggio di questa tecnica è che il reclutamento di cellule tumorali all'interno di un organo remoto può essere quantificato. Inizia usando un reagente di dissociazione cellulare non enzimatica per rimuovere il carcinoma polmonare lewis che esprime proteine fluorescenti, o LLC, dalle loro cellule di coltura secondo protocolli standard.Trasferire la soluzione cellulare risultante in un tubo conico da 15 millilitri prima della centrifugazione e centrifugare lavare il pellet due volte in cinque millilitri di PBS per lavaggio. Dopo il secondo lavaggio, filtrare le cellule attraverso un filtro cellulare a poro di 40 micrometri e diluire la sospensione a una volta per 10 alle sei cellule per concentrazione di millilitro. Quindi caricare le cellule in una siringa da un millilitro dotata di un ago calibro 29 e iniettare 100 microlitri di cellule nella vena di coda di ogni topo maschio di otto-10 settimane, C57-Black-6.All'endpoint sperimentale appropriato, utilizzare le forbici per rimuovere la pelle dalla superficie ventrale di ogni topo e tagliare le costole e il diaframma per esporre la cavità toracica. Utilizzare un ago per infusione alato calibro 25 e tubi per lavare 15 millilitri di PBS attraverso il ventricolo destro e rimuovere il cuore e il timo. Afferrando la trachea con le forcelle, tirare su, sezionando i tessuti connettivi intorno alla trachea, per raccogliere i polmoni.Quindi lavare i polmoni in PBS e staccare un lobo per la visualizzazione delle cellule fluorescenti in situ al microscopio a fluorescenza. Per digerire il tessuto polmonare, prima dadi il lobo caudale con le forbici e posiziona i pezzi polmonari in una siringa da 10 millilitri. Chiudere la siringa con lo stantuffo e aspirare cinque millilitri di soluzione di digestione nella siringa, spostando lo stantuffo in entrata e in uscita dall'albero della siringa fino a quando il tessuto polmonare non viene diffuso in tutta la soluzione.Erogare il liquame polmonare in un tubo da 50 millilitri e digerire il tessuto su uno shaker reciproco per 15 minuti a 37 gradi Celsius e 150 giri/min. Alla fine dell'incubazione, triturare il tessuto rimanente fino a quando le cellule polmonari non sono completamente disperse e restituire il tubo allo shaker per altri 30 minuti. Quindi filtrare le cellule attraverso un colino poro di 40 micrometri e raccogliere le cellule mediante centrifugazione.Per analizzare le cellule polmonari isolate per citometria a flusso, rimescolare il pellet in due millilitri di tampone di lisi dei globuli rossi per un minuto a temperatura ambiente e raccogliere le cellule per centrifugazione. Quindi centrifugare il pellet due volte in cinque millilitri di PBS fresco integrato con l'1% di albumina di siero bovino per lavaggio. Dopo la seconda centrifugazione, rimescolare il pellet in un millilitro di PBS fresco più BSA e filtrare le cellule attraverso un nuovo colino poro di 40 micrometri per la quantificazione delle cellule tumorali per citometria a flusso.I polmoni presentano molte cellule GFP-LLC due ore dopo l'iniezione, con la stragrande maggioranza delle cellule che scompaiono dai polmoni entro 24 ore. Si noti che tutte le macchie fluorescenti rilevate all'interno del filtro rosso devono essere escluse dal conteggio delle celle. Per confermare il numero di GFP-LLC nei polmoni, uno dei lobi può essere utilizzato per l'analisi citometrica del flusso come dimostrato.Durante il tentativo di questa procedura, è importante ricordare che una procedura di sezione congelata è un metodo più accurato per osservare la posizione esatta delle cellule tumorali.

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.

Atterramento condizionale dell'espressione genica in linee cellulari del cancro per studiare il reclutamento dei monociti/macrofagi al microambiente tumorale

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

Ricerca

Ricerca sul cancro

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.

L'istituzione di un'analisi di colonizzazione del polmone per la visualizzazione di cellule del tumore nei tessuti polmonari di circolazione

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.

Modello del polmone acellulare e cellulari per lo studio di metastasi del tumore

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.

Analisi Live Cell Imaging Studio Glioblastoma cerebrale tumore cellule staminali migrazione e invasione

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.

Reazione a catena della polimerasi digitale per la variazione genetica in un paziente di poliposi adenomatosa familiare sporadica utilizzando il formato di Chip-in-a-tubo

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.

Isolamento di cellule staminali mesenchimali da tessuto della polpa e co-coltura con cellule tumorali per studiare le loro interazioni

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.

Rilevazione in tempo reale nell'apoptosi delle cellule tumorali in Vitro indotta da CD8+ T cellule per studiare funzioni immuni soppressive di tumore-infiltrazione di cellule mieloidi

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.

Caratterizzazione semiautomatica PD-L1 ed enumerazione delle cellule tumorali circolanti da pazienti affetti da cancro del polmone a cellule non piccole mediante immunofluorescenza

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 Analisi per studiare l'interazione tumora-macrophage

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.

Rilevamento della progressione del tumore del polmone nei topi mediante 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 ...