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

Alle procedures uitgevoerd met muizen werden goedgekeurd door het dier onderzoek Comité van Tokyo vrouwen Medizinische Universität.
1. staart veneuze injectie van Lewis Lung Carcinoma (LLC) cellen
<ol><li>Handhaven van LLC cellen in DMEM aangevuld met 10% FCS, in een bevochtigde 5% CO2 incubator bij 37 ° C. Cellen moeten bevatten een fluorescerend eiwit expressie systeem (b.v., GFP).</li>
	<li>Cellen verwijderen uit het cultuur schotel met behulp van een niet-enzymatis...

<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>

Voorbehandeling van muizen met antilichamen, drugs, een vetrijke dieet of tumor-geconditioneerd medium vóór tumor cel injectie is mogelijk. De moeilijkste stap in deze test is de staart veneuze injectie. Onvolledige injectie leidt tot twijfelachtige gegevens. De ader van de staart in C57BL/6 is bijzonder moeilijk te identificeren, wat resulteert in mislukte injecties. Plaatsen van de muizen op een verwarming pad (37 ° C) verwijden helpt de staart ader zodat injectie gemakkelijker wordt.
Dez...

Tumor metastase rekeningen voor de hoge sterfte in kanker-gerelateerde ziekten. Vanuit het oogpunt van onderzoek op moleculair niveau, metastase kan worden onderverdeeld in meerdere stappen: tumor inleiding op de primaire tumor site, de groei van de primaire tumor en de invasie in de omringende weefsels, intravasation, omloop door de bloedvaten , extravasation op verre organen en tumor uitgroei. Elke stap impliceert verschillende soorten moleculen/signalen1.
Studies tot op heden zijn bezorgd met gebeurtenissen die plaatsvinden in verre organen voordat de tumorcellen circuleren in het bloed, die ook bekend staat als de pre metastatische fase2,3,4,5, 6. Onze muis model studies is gebleken dat de vooraf metastatische fase Long metastase vergemakkelijkt door het creëren van duurzame en low-level inflammatoire reacties in de longen. De vooraf metastatische fase wordt gekenmerkt door hyperpermeability van themicrovasculature, werving van afgeleid beenmergcellen (BMDC), en opregulatie van cytokine/chemokine-achtige moleculen met inbegrip van S100A8 en SAA33,4 . Er werd gemeld dat lysyl-oxidase wijzigt de extracellulaire matrix in de longen te werven BMDCs7. Een ander rapport is gebleken dat het belang van Angpt2, MMP3 en MMP10 in de vooraf metastatische fase8. Met andere woorden, de complexe interacties tussen primaire tumoren, beenmerg en externe organen moeten worden begrepen en correct gemoduleerd (bijvoorbeeld door het gebruik van drugs die gericht zijn op eiwitten die betrokken zijn in deze interacties) om te voorkomen dat toekomstige metastase9 . Voor het regelen van de vooraf metastatische fase, moet eerst worden gevisualiseerd en voor diagnostische of therapeutische interventies geëvalueerd. Wij rapporteren hier, een Long tumor cel aanwerving assay waarmee de studie van de vooraf metastatische fase in de longen.
Tumorcellen direct geïnjecteerd in het bloedvat van de muis zijn vergelijkbaar met de circulerende tumorcellen bij kankerpatiënten, hoewel er verschillen tussen gekweekte cellen zijn kunnen en circulerende tumorcellen. Circulerende tumorcellen ondergaan zich enkele karakteristieke wijzigingen bij het invoeren van verkeer van de primaire tumor. Gekweekte cellen vormen echter kunnen tumoren in immunodeficiëntie muizen wanneer ze worden geïnjecteerd in de ader van de staart. Daarnaast, in de muis modelsysteem, kan fluorescentie label tumorcellen worden gebruikt waarmee het volgen van deze cellen in het lichaam. Het gedrag van de circulerende tumorcellen is cruciaal omdat zij de uiteindelijke gevolgen van kanker patiënten10 bepalen. Bloed is niet een goede plek om te overleven van tumor cellen2. Ze moeten ontsnappen uit de aanval door het immuunsysteem van de gastheer en anti-apoptotic signalen om te voorkomen dat apoptosis als gevolg van een gebrek aan fysieke ondersteuning. Tumorcellen met hogere capaciteiten van tumor inleiding op metastatische sites genereren meer tumor knobbeltjes. Als de tumorcellen Toon specifieke orgel tropisme, moet metastase in die specifieke organen worden geobserveerd. De eerste stappen van de metastatische cascade (primaire tumorgroei en invasie) zijn niet inbegrepen in deze test, en zo de nadruk ligt op de interactie tussen het circulerende tumorcellen en de stromale cellen (endotheliale cellen, epitheliaale cellen en bloedcellen). In conventionele tumor cel injectie testen moeten onderzoekers wachten tot de tumor knobbeltjes groeien tot een tastbare grootte detecteren de metastase. In dit geval worden niet het exacte aantal tumorcellen in het bloedvat gekwantificeerd. Dit proces omvat bovendien een tumor hergroei fase, die aangeeft dat andere factoren in de tumorcellen van invloed kunnen zijn op het resultaat.
Tellen is met het label tumorcellen onder een fluorescentie stereomicroscoop een eenvoudig proces. De stereomicroscoop biedt een breed gezichtsveld zodat de hele Long kan worden gescand. Stroom cytometry is ook een nuttig hulpmiddel dat direct een nauwkeurige aantal tumorcellen in het weefsel geeft. Fluorescerende cellen in het weefsel onder een confocal microscoop tellen is ook mogelijk en geeft de meest nauwkeurige beelden van metastatische tumorcellen, maar het is tijdrovend en vooringenomen resultaten kan geven als alleen een geselecteerd gebied wordt geteld. Het uiteindelijke doel van deze test is om te observeren hoe gemakkelijk tumorcellen zich ophopen in de longen. Zoals gemeld eerder3,4, als de longen zijn in vooraf metastatische fase als gevolg van inflammatoire signalering van de primaire tumor, zijn de geïnjecteerde tumorcellen gevoelig voor zich ophopen in de longen, dus impliceert hogere kans op longkanker metastase. Andere aandoeningen (reumatoïde artritis, astma, enz.), en hun behandeling met anti-inflammatoire stoffen (zoals anti-TNFα) kan verzachten Long metastase. Dus, we concluderen dat deze bepaling een krachtig hulpmiddel voor het beoordelen van de mogelijkheid van longkanker metastase.

<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

Voor het onderzoek naar de moleculaire mechanismen bestuur tumor metastase, verschillende testen met behulp van de muis als een dier model voorgesteld. Hier tonen we een eenvoudige test om te evalueren van de tumor cel extravasation of micrometastasis. In deze test, tumorcellen waren geïnjecteerd door de ader van de staart, en na een korte periode, de longen werden ontleed en verteerd om te tellen de geaccumuleerde gelabelde tumorcellen. Deze test wordt overgeslagen de eerste stap van primaire tumor invasie in het bloedvat en vergemakkelijkt de studie van gebeurtenissen in het verre orgaan waar de uitzaaiing van de tumor zich voordoet. Het aantal cellen in het bloedvat geïnjecteerd kan worden geoptimaliseerd om te zien hoe een beperkt aantal metastasen. Er werd gemeld dat stromale cellen in het verre orgel aan metastase bijdragen. Dus zou deze test een nuttig instrument om te verkennen potentiële therapeutische drugs of apparaten voor preventie van uitzaaiing van de tumor.

De longen presenteren veel GFP-LLC cellen 2 h na injectie (figuur 1A). Opgemerkt moet worden dat de fluorescerende vlekken ook gedetecteerd in de rode filter moeten worden uitgesloten van de cel nummer tellen. De overgrote meerderheid van de LLC cellen verdwijnen uit de longen 24u na injectie (Figuur 1 c).
Om te bevestigen het nummer van het GFP-LLC in de longen, kan ee...

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

Lung Tumor Cell Recruitment Assay

Long Tumor cel aanwerving Assay

Dit manuscript beschrijft een methode om te kwantificeren tumor cel accumulatie in de longen in een diermodel van uitzaaiing van de tumor.

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

Deze methode kan helpen bij het beantwoorden van belangrijke vragen in de tumor biologie veld over de moleculaire mechanismen van metastase. Het belangrijkste voordeel van deze techniek is dat de rekrutering van tumorcellen binnen een afgelegen orgaan kan worden kwantificeren. Begin met het gebruik van een niet-enzymatische cel dissociatie reagens om fluorescerende eiwit-uitdrukkende Lewis longcarcinoom, of LLC, cellen uit hun cultuur container volgens standaard protocollen te verwijderen.Breng de resulterende celoplossing in een conische buis van 15 milliliter voor centrifugatie en was de pellets twee keer in vijf milliliter PBS per wasbeurt. Na de tweede wasbeurt, filter de cellen door een 40-micrometer porie cel zeef, en verdun de suspensie tot een keer 10 tot de zes cellen per milliliter concentratie. Laad vervolgens de cellen in een spuit van één milliliter uitgerust met een naald van 29 meter en injecteer 100 microliter cellen in de staartader van elke acht-tot-10-week-oude, C57-Black-6, mannelijke muis.Gebruik op het juiste experimentele eindpunt een schaar om de huid van het ventrale oppervlak van elke muis te verwijderen en snijd de ribben en het middenrif om de borstholte bloot te leggen. Gebruik een 25-gauge gevleugelde infuus naald en slang te spoelen 15 milliliter PBS door de rechter ventrikel, en verwijder het hart en de thymus. De luchtpijp vastpakken met tangen, optrekken, de bindweefsels rond de luchtpijp ontleden, om de longen te oogsten.Was vervolgens de longen in PBS, en los een kwab voor visualisatie van de fluorescerende cellen in situ onder een fluorescentie microscoop. Om het longweefsel te verteren, eerst de staartkwab met een schaar in blokjes gesneden en de longstukken in een spuit van 10 milliliter plaatsen. Sluit de spuit met de zuiger en zuig vijf milliliter spijsverteringsoplossing in de spuit, waarbij de zuiger in en uit de spuitschacht wordt verplaatst totdat het longweefsel door de hele oplossing wordt verspreid.Doseer de longdrijfmest in een buis van 50 milliliter en verteer het weefsel op een wederkerige shaker gedurende 15 minuten bij 37 graden Celsius en 150 rpm. Aan het einde van de incubatie trituraat het resterende weefsel totdat de longcellen volledig zijn verspreid, en de buis terug te keren naar de shaker voor een extra 30 minuten. Vervolgens filteren de cellen door een 40-micrometer porie zeef, en het verzamelen van de cellen door centrifugatie.Om de geïsoleerde longcellen te analyseren door flow cytometrie, resuspend de pellet in twee milliliter rode bloedcel lyse buffer voor een minuut bij kamertemperatuur, en het verzamelen van de cellen door centrifugatie. Centrifuge de pellet vervolgens twee keer in vijf milliliter verse PBS aangevuld met 1% runderserum albumine per wasbeurt. Na de tweede centrifugatie, resuspend de pellet in een milliliter verse PBS plus BSA, en filter de cellen door een nieuwe 40-micrometer porie zeef voor kwantificering van de tumorcellen door stroom cytometrie.De longen presenteren veel GFP-LLC cellen twee uur na injectie, met de overgrote meerderheid van de cellen verdwijnen uit de longen binnen 24 uur. Houd er rekening mee dat fluorescerende vlekken die in het rode filter worden gedetecteerd, moeten worden uitgesloten van het aantal cellen. Om het aantal GFP-LLC in de longen te bevestigen, kan een van de lobben worden gebruikt voor flow cytometrische analyse zoals aangetoond.Tijdens een poging tot deze procedure, is het belangrijk om te onthouden dat een bevroren sectie procedure is een meer nauwkeurige methode voor het observeren van de exacte locatie van tumorcellen.

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.

Voorwaardelijke Knockdown van genexpressie in kanker cellijnen te bestuderen van de aanwerving van monocyten/macrofagen naar de Tumor communicatie

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

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.

De oprichting van een Long kolonisatie Assay voor het circulerende Tumor visualisatie van de cel in de weefsels van de longen

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.

Acellulair en cellulaire Lung Model studie Tumor uitzaaiingen

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.

Live-cel Imaging tests studie Glioblastoma Brain Tumor stamcel migratie en invasie

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.

Digitale Polymerase Chain Reaction Assay voor de genetische variatie in een sporadische familiale adenomateuze Polyposis patiënt met behulp van de Chip-in-a-tube-indeling

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.

Mesenchymale stamcellen isolatie uit Pulp weefsel en co cultuur met kankercellen te bestuderen van hun interacties

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.

Real-time detectie van In Vitro Tumor cel Apoptosis geïnduceerd door CD8+ T cellen te bestuderen immuun onderdrukkende functies van Tumor-infiltreren myeloïde cellen

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.

Semi-automatische PD-L1 karakterisering en opsomming van circulerende tumor cellen van niet-kleincellige longkanker patiënten door immunofluorescentie

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 om tumor-macrofaag interactie te bestuderen

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.

Detectie van longtumorprogressie bij muizen door echografie

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