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 procedurer udføres med mus blev godkendt af Animal forskning Udvalget af Tokyo Women's Medical University.
1. hale vene injektion af Lewis lunge karcinom (LLC) celler
<ol><li>Vedligeholde LLC celler i DMEM suppleret med 10% FCS, i en fugtig 5% CO2 inkubator ved 37 ° C. Celler bør indeholde en fluorescerende proteiner udtryk system (f.eks. normal god landbrugspraksis).</li>
	<li>Fjerne celler fra kultur parabol ved hjælp af en ikke-enzymatiske celle dissociation r...

<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>

Forbehandling af mus med antistoffer, narkotika, højt fedtindhold kost eller tumor-aircondition medium før tumor celle indsprøjtning er muligt. Det sværeste skridt i denne analyse er hale vene injektion. Ufuldstændig injektion resulterer i uafklarede data. Hale vene i C57BL/6 er særligt vanskeligt at identificere, hvilket resulterer i fejlbehæftede injektioner. Placere mus på en varmepude (37 ° C) spile hjælper hale vene, således at injektionen bliver lettere.
Denne analyse bruger e...

Tumor metastaser konti for høj dødelighed i cancer-relaterede sygdomme. Fra synspunkt af undersøgelser på det molekylære niveau, metastaser kan opdeles i flere trin: tumor indledningen på den primære tumor site, primær tumorvækst og invasion i omkringliggende væv, intravasation, omsætning gennem blodkarrene , ekstravasation på fjerntliggende organer, og tumor re-vækst. Hvert trin indebærer forskellige sæt af molekyler/signaler1.
Undersøgelser til dato har drejet sig om hændelser, som forekommer i fjerntliggende organer før tumorcellerne, cirkulerer i blodet, hvilket er også kendt som den præ metastatisk fase2,3,4,5, 6. Vores mus model undersøgelser afslørede, at fasen forud metastatisk letter lunge metastaser ved at skabe langvarig og low-level inflammatoriske respons i lungerne. Den pre-metastatisk fase er kendetegnet ved hyperpermeability af themicrovasculature, rekruttering af knoglemarven afledt celler (BMDC) og opregulering af cytokin/chemokine-lignende molekyler herunder S100A8 og SAA33,4 . Det er blevet rapporteret, at lysyl oxidase ændrer den ekstracellulære matrix i lungerne til at rekruttere BMDCs7. En anden betænkning har afsløret betydningen af Angpt2, MMP3 og MMP10 i pre metastatisk fase8. Med andre ord, det indviklede samspil mellem primære tumorer, knoglemarv og fjerntliggende organer skal være forstået og korrekt moduleret (for eksempel ved hjælp af narkotika, der er målrettet proteiner involveret i disse interaktioner) at forhindre fremtidige metastase9 . For at regulere den pre metastatisk fase, bør det først visualiseret og evalueres for diagnostiske eller terapeutiske indgreb. Her rapporterer vi en lunge tumor celle rekruttering assay, der giver mulighed for undersøgelse af pre metastatisk fase i lungerne.
Tumorceller direkte sprøjtet ind i blodkar af musen er lig cirkulerende tumorceller i kræftpatienter, selv om der kan være forskelle mellem kulturperler celler og cirkulerende tumorceller. Cirkulerende tumorceller undergå sig nogle karakteristiske ændringer, når de indtaster omsætning fra den primære tumor. Ikke desto mindre kan kulturperler celler danne tumorer hos immundefekte mus når de er indsprøjtet i halen vene. Derudover i mus modelsystem, kan fluorescens mærket tumorceller bruges som tillader sporing af disse celler i kroppen. Opførsel af cirkulerende tumorceller er kritisk, da de afgør de endelige konsekvenser i kræft patienter10. Blod er ikke et godt sted for overlevelse af tumor celler2. De har brug at flygte fra angreb af immunsystemet vært og behovet for anti-apoptotiske signaler at forhindre apoptose mangel af fysisk støtte. Tumorceller med højere evner af tumor indledning på metastatisk sites generere flere tumor knuder. Hvis tumorceller vise specifikke orgel tropisme, bemærkes metastaser i disse særlige organer. De første trin af den metastatisk kaskade (primær tumorvækst og invasion) er ikke inkluderet i denne analyse, og så fokus er på samspillet mellem cirkulerende tumorceller og stromale celler (endotelceller, epitelceller og blodlegemer). I konventionelle tumor celle indsprøjtning assays har forskere til at vente indtil tumor knuder vokser til en konkret størrelse at påvise metastaser. I dette tilfælde ikke kan det nøjagtige antal tumorceller i blodkar kvantificeres. Denne proces indeholder desuden en tumor genvækst fase, der angiver, at andre faktorer i tumorcellerne kan påvirke resultatet.
Tælle er mærket tumorceller under et fluorescens stereomikroskop en enkel proces. Stereomikroskopet giver et bredt synsfelt, så hele lungen kan scannes. Flowcytometri er også et nyttigt værktøj, der straks giver en præcis antal tumorceller i vævet. Tælle fluorescerende celler i vævet under en Konfokal mikroskop er også muligt og viser de mest præcise billeder af metastatisk tumorceller, men det er tidskrævende og kan give partisk resultater, hvis kun et markeret område er optalt. Det ultimative mål for denne analyse er at observere, hvordan let tumorceller ophobes i lungerne. Som rapporteret tidligere3,4, hvis lungerne er i pre metastatisk fase på grund af inflammatorisk signalering fra den primære tumor, er injiceres tumorcellerne tilbøjelige til at ophobe sig i lungerne, hvilket således indebærer større chancer for lunge metastaser. Andre betingelser (leddegigt, astma, etc.), og deres behandling med anti-inflammatoriske lægemidler (såsom anti-TNFα) kan dæmpe lunge metastaser. Dermed, vi konkludere, at denne analyse er et kraftfuldt værktøj til at vurdere muligheden for lunge metastaser.

<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

For at undersøge de molekylære mekanismer for tumor metastaser, er blevet foreslået forskellige assays ved hjælp af musen som en model dyr. Her viser vi en simpel analyse for at vurdere tumor celle ekstravasation eller micrometastasis. I denne analyse, tumorceller blev injiceret gennem halen vene, og efter en kort periode, lungerne blev dissekeret og fordøjet for at tælle de akkumulerede mærket tumorceller. Denne analyse springer det første trin af primær tumor invasion ind i blodkar og letter undersøgelsen af begivenhederne i fjernt organ hvor tumor metastaser opstår. Antallet af celler injiceres ind i blodkar kan optimeres for at observere et begrænset antal metastaser. Det er blevet rapporteret, at stromale celler i Fjern organ bidrage til metastaser. Således, denne analyse kunne være et nyttigt redskab til at udforske potentielle terapeutiske lægemidler eller anordninger til forebyggelse af tumor metastaser.

Lungerne præsentere mange normal god landbrugspraksis-LLC celler 2 h efter injektion (figur 1A). Det skal bemærkes, at de fluorescerende steder også opdaget i den røde filter bør udelukkes fra nummer celletal. Langt størstedelen af LLC celler forsvinder fra lungerne 24 timer efter injektion (figur 1 c).
For at bekræfte antallet af normal god landbrugspraksis-LLC ...

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

Lung Tumor Cell Recruitment Assay

Lunge Tumor celle rekruttering Assay

Dette manuskript beskriver en metode til at kvantificere tumor celle ophobning i lungerne i en dyremodel af tumor metastaser.

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

Denne metode kan hjælpe med at besvare centrale spørgsmål i tumor biologi felt om de molekylære mekanismer metastase. Den største fordel ved denne teknik er, at rekrutteringen af tumorceller i et fjerntliggende organ kan kvantgøre. Begynd med at bruge en ikke-enzymatisk celle dissociation reagens til at fjerne fluorescerende protein-udtryk Lewis lungekarcinom, eller LLC, celler fra deres kultur beholder i henhold til standard protokoller.Den resulterende celleopløsning overføres til et 15 milliliter konisk rør før centrifugering, og centrifugevask pellets to gange i fem milliliter PBS pr. vask. Efter den anden vask filtreres cellerne gennem en porecellest si på 40 mikrometer, og suspensionen fortyndes til en gang 10 til de seks celler pr. milliliterkoncentration. Derefter indlæses cellerne i en en milliliter sprøjte udstyret med en 29-gauge nål, og injicere 100 mikroliter af celler i hale venen i hver otte-til-10-ugergamle, C57-Black-6, mandlig mus.Ved det relevante eksperimentelle endepunkt skal du bruge en saks til at fjerne huden fra hver muss ventrale overflade og skære ribbenene og mellemgulvet for at afsløre brysthulen. Brug en 25-gauge vingede infusionsnål og slanger til at skylle 15 milliliter PBS gennem højre hjertekammer, og fjern hjertet og thymusen. Gribende luftrøret med stæcener, trække op, dissekere bindevæv omkring luftrøret, at høste lungerne.Vask derefter lungerne i PBS, og løsrive en lap til visualisering af fluorescerende celler in situ under et fluorescensmikroskop. At fordøje lungevæv, første terninger caudal lap med en saks, og læg lungestykkerne i en 10-milliliter sprøjte. Luk sprøjten med stemplet, og aspirere fem milliliter fordøjelsesopløsning ind i sprøjten, og flytte stemplet ind og ud af sprøjteskakten, indtil lungevævet spredes i hele opløsningen.Dispenseres lungesalgeren i et 50-milliliterrør, og væv på en gensidig shaker i 15 minutter ved 37 grader Celsius og 150 omdr./min. Ved slutningen af inkubationen tritureres det resterende væv, indtil lungecellerne er helt spredt, og røret returneres til shakeren i yderligere 30 minutter. Filtrer derefter cellerne gennem en 40-mikrometer pore si, og indsamle cellerne ved centrifugering.For at analysere de isolerede lungeceller ved flowcytometri skal pelleten resuspendere pellet i to milliliter lysisbuffer for røde blodlegemer i et minut ved stuetemperatur og indsamle cellerne ved centrifugering. Derefter centrifuge pellet to gange i fem milliliter frisk PBS suppleret med 1% kvæg serum albumin per vask. Efter den anden centrifugering, resuspend pellet i en milliliter frisk PBS plus BSA, og filtrere cellerne gennem en ny 40-mikrometer pore si til kvantificering af tumorcellerne ved flow cytometri.Lungerne præsentere mange GFP-LLC celler to timer efter injektion, med langt de fleste celler forsvinder fra lungerne inden for 24 timer. Bemærk, at eventuelle fluorescerende pletter, der registreres i det røde filter, bør udelukkes fra celletallet. For at bekræfte antallet af GFP-LLC i lungerne, en af lapperne kan bruges til flow cytometrisk analyse som påvist.Mens du forsøger denne procedure, Det er vigtigt at huske, at en frossen sektion procedure er en mere præcis metode til at observere den nøjagtige placering af tumorceller.

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.

Betinget Knockdown af genekspression i kræft cellelinjer til at studere ansættelse af monocytter/makrofager at Tumor mikromiljø

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.

Etablering af en lunge kolonisering Assay for Cirkulerende Tumor Cell visualisering i lungevæv

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.

Acellulær og cellulære Lung Model til at studere Tumor metastaser

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-celle Imaging Assays til undersøgelse glioblastom hjerne Tumor stamcelle Migration og 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 den genetiske Variation i en sporadisk familiær adenomatøs polypose Patient med formatet Chip i rør

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.

Mesenkymale stamceller isoleret fra Pulp væv og fælles kultur med kræftceller til at studere deres interaktioner

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.

Realtid påvisning af i Vitro Tumor celle apoptose induceret af CD8+ T-celler til at studere immun suppressiv funktioner af Tumor infiltrerer myeloide celler

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-automatisk PD-L1 karakterisering og optælling af cirkulerende tumor celler fra ikke-småcellet lunge cancer patienter ved immunofluorescens

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-analyse for at studere tumor-makrofag interaktion

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.

Påvisning af lungetumorprogression hos mus ved ultralydsscanning

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