Name: a time-lapse, label-free, quantitative phase imaging study of dormant and active human cancer cells
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Description: Watch this Scientific Journal Video about A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells at JoVE.com

De auteurs mijn dankbaarheid uitspreken voor de steun van de Breast Cancer Research Foundation en de Advanced Medical Research Foundation.

Alle methoden die hier worden beschreven zijn door de Boston Children's Hospital institutionele bioveiligheid Comité goedgekeurd.
1. cel voorbereiding
<ol><li>
Ontdooien KHOS-A en -N cellen
	<ol>
<li>Opwarmen kweekmedium, dat wil zeggen, de Dulbecco bewerkt Eagle medium aangevuld met 10% (vol/vol) foetaal kalfsserum (FBS) en 1% (vol/vol) penicilline/streptomycine.</li>
		<li>Nemen van de cryogene flesjes van cellen uit de vloeibare stikstof tank, de onderkant van d...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+regulation+of+tumor+angiogenesis:+mechanisms+and+therapeutic+implications.">Molecular regulation of tumor angiogenesis: mechanisms and therapeutic implications.</a> EXS. (96), 223-268 (2006).</li><li>Naumov, G. 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=A+model+of+human+tumor+dormancy:+an+angiogenic+switch+from+the+nonangiogenic+phenotype.">A model of human tumor dormancy: an angiogenic switch from the nonangiogenic phenotype.</a> Journal of the National Cancer Institute. 98 (5), 316-325 (2006).</li><li>Fang, 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=Matrix+metalloproteinase-2+is+required+for+the+switch+to+the+angiogenic+phenotype+in+a+tumor+model.">Matrix metalloproteinase-2 is required for the switch to the angiogenic phenotype in a tumor model.</a> Proceedings of the National Academy of Sciences of the United States of America. 97 (8), 3884-3889 (2000).</li><li>Almog, 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=Transcriptional+switch+of+dormant+tumors+to+fast-growing+angiogenic+phenotype.">Transcriptional switch of dormant tumors to fast-growing angiogenic phenotype.</a> Cancer Research. 69 (3), 836-844 (2009).</li><li>Hu, 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=Gene+expression+signature+for+angiogenic+and+nonangiogenic+non-small-cell+lung+cancer.">Gene expression signature for angiogenic and nonangiogenic non-small-cell lung cancer.</a> Oncogene. 24 (7), 1212-1219 (2005).</li><li>Harper, 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=Repression+of+vascular+endothelial+growth+factor+expression+by+the+zinc+finger+transcription+factor+ZNF24.">Repression of vascular endothelial growth factor expression by the zinc finger transcription factor ZNF24.</a> Cancer Research. 67 (18), 8736-8741 (2007).</li><li>Jia, D., 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=Transcriptional+repression+of+VEGF+by+ZNF24:+mechanistic+studies+and+vascular+consequences+in+vivo.">Transcriptional repression of VEGF by ZNF24: mechanistic studies and vascular consequences in vivo.</a> Blood. 121 (4), 707-715 (2013).</li><li>Jia, D., Huang, L., Bischoff, J., Moses, M. 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+endogenous+zinc+finger+transcription+factor,+ZNF24,+modulates+the+angiogenic+potential+of+human+microvascular+endothelial+cells.">The endogenous zinc finger transcription factor, ZNF24, modulates the angiogenic potential of human microvascular endothelial cells.</a> FASEB journal: official publication of the Federation of American Societies for Experimental Biology. 29 (4), 1371-1382 (2015).</li><li>Almog, 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=Prolonged+dormancy+of+human+liposarcoma+is+associated+with+impaired+tumor+angiogenesis.">Prolonged dormancy of human liposarcoma is associated with impaired tumor angiogenesis.</a> FASEB journal: official publication of the Federation of American Societies for Experimental Biology. 20 (7), 947-949 (2006).</li><li>Almog, 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=Consensus+micro+RNAs+governing+the+switch+of+dormant+tumors+to+the+fast-growing+angiogenic+phenotype.">Consensus micro RNAs governing the switch of dormant tumors to the fast-growing angiogenic phenotype.</a> PloS One. 7 (8), e44001 (2012).</li><li>Satchi-Fainaro, R., 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=Prospective+identification+of+glioblastoma+cells+generating+dormant+tumors.">Prospective identification of glioblastoma cells generating dormant tumors.</a> PloS One. 7 (9), e44395 (2012).</li><li>Almog, 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=Transcriptional+changes+induced+by+the+tumor+dormancy-associated+microRNA-190.">Transcriptional changes induced by the tumor dormancy-associated microRNA-190.</a> Transcription. 4 (4), 177-191 (2013).</li><li>Gao, D., Nolan, D. 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In deze studie beschrijven we een in vitro, niet-invasieve en label-vrije methode QPI met kwantitatief karakteriseren de fenotypen van het angiogenic en niet-angiogenic van menselijke Osteosarcoom cellen. Meerdere cellulaire parameters hebben gelijktijdig met deze geïntegreerde, hoge-doorvoer methode, met inbegrip van cel gebied, de dikte van de cel, cel volume, proliferatie tarief, verdubbeling van de tijd, migratie directheid, beweeglijkheid, snelheid, migratie en motiliteit is geanalyseerd.
<p class="jov...

Een van de vroegste controlepunten in de ontwikkeling en de vooruitgang van een solide tumor is de overname van het fenotype van de angiogenic, een kenmerk van kanker. Deze progressie omvat allerlei biochemische en moleculaire processen1,2,3. Een technische uitdaging in de studie van deze belangrijke stap in de tumor progressie is het gebrek aan tools om continu en kwantitatief karakteriseren en onderscheid maken tussen angiogenic en niet-angiogenic fenotypes van levende kankercellen op onpartijdige wijze. De traditionele testen wordt gebruikt om te onderzoeken het cellulaire gedrag van angiogenic en niet-angiogenic cellen meestal dure reagentia en instrumenten vereisen, bijvoorbeeld cel proliferatie/migratie testen4,5, 6,7,8,9,10,11,12,13,14 of aanvullende in-vivo evaluaties4,5,6,8,15,16, evenals vereisen aanzienlijke expertise en intensieve tijd en arbeid consumptie.
Onlangs, kwantitatieve fase imaging (QPI) heeft ontpopt als een nieuwe techniek waarmee time-lapse en labeling-vrije beoordeling van een verscheidenheid van cel morfologie en gedrag parameters17,18,19, 20 , 21 , 22. in tegenstelling tot conventionele optische microscopie, QPI kwantificeert variaties van faseverschuiving pixel voor pixel nadat licht een optische object passeert, en een holograaf reconstrueert met geconverteerde optische dikte en volume, waardoor de directe analyse van levende cellen en de volgende functies: (1) kwantitatieve beeldvorming, (2) niet-invasieve en time-lapse imaging, beeldvorming (3) label-vrij, en (4) gelijktijdige Multi-parameter imaging. Deze eigenschappen maken QPI een krachtig hulpmiddel om te beoordelen en begrijpen van pathologische processen op cellulair niveau.
In een recente studie, die we gebruikt QPI kwantitatief karakteriseren en onderscheid maken tussen angiogenic KHOS-A en niet-angiogenic KHOS-N fenotypes van menselijke Osteosarcoom cellen op een systematische en kwantitatieve wijze combineren van analyses van cel morfologie, proliferatie, en motiliteit23. Met behulp van de software van de analyse van de afbeelding, een panel van cel morfologische en gedrag parameters werden kwantitatief vergeleken tussen angiogenic en niet-angiogenic menselijke Osteosarcoom cellen en vijf kenmerkende verschillen tussen deze twee werden geïdentificeerd fenotypen. Deze nieuwe benadering biedt een geïntegreerde en kwantitatieve platform voor de beoordeling van een verscheidenheid aan biologisch relevante cellulaire kenmerken.

<table border="0" cellpadding="0" cellspacing="0" width="908">
	<tbody>
		<tr height="33">
			<td height="33" style="height:34px;width:227px;">T75 flask</td>
			<td style="width:227px;">Corning, NY, USA</td>
			<td style="width:227px;">353136</td>
			<td style="width:227px;"></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:34px;width:227px;">6-well plates&#160;</td>
			<td style="width:227px;">Corning, NY, USA</td>
			<td style="width:227px;">3506</td>
			<td></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:227px;">Dulbecco&#8217;s modified Eagle medium (DMEM)</td>
			<td style="width:227px;">Thermo Fisher Scientific, MA, USA</td>
			<td style="width:227px;">11965092</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:34px;width:227px;">Fetal bovine serum (FBS)&#160;</td>
			<td style="width:227px;">Atlanta Biologicals, GA, USA</td>
			<td style="width:227px;">S11550</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:34px;width:227px;">Penicillin Streptomycin</td>
			<td style="width:227px;">Thermo Fisher Scientific, MA, USA</td>
			<td style="width:227px;">15140122</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:34px;width:227px;">Phosphate buffered saline (PBS)</td>
			<td style="width:227px;">Thermo Fisher Scientific, MA, USA</td>
			<td style="width:227px;">10010023</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:34px;width:227px;">Beckman Z1 Coulter counter</td>
			<td style="width:227px;">Beckman Coulter, IN, USA</td>
			<td style="width:227px;">Z1&#160;</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:227px;">HoloMonitor M4</td>
			<td style="width:227px;">Phase Holographic Imaging Phi AB, Lund, Sweden</td>
			<td style="width:227px;">M4</td>
			<td>Microscope</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:227px;">Hololid</td>
			<td style="width:227px;">Phase Holographic Imaging Phi AB, Lund, Sweden</td>
			<td style="width:227px;">PHI 8020</td>
			<td></td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:227px;">HStudioM4</td>
			<td style="width:227px;">Phase Holographic Imaging Phi AB, Lund, Sweden</td>
			<td style="width:227px;">HStudioM4</td>
			<td>Software</td>
		</tr>
	</tbody>
</table>

a time-lapse, label-free, quantitative phase imaging study of dormant and active human cancer cells

De overname van het fenotype van de angiogenic is een essentieel onderdeel van de ontsnapping uit de tumor rustperiode. Hoewel verschillende klassieke in vitro testen (bijvoorbeeld, proliferatie, migratie en anderen) en in vivo modellen zijn ontwikkeld om te onderzoeken en karakteriseren van angiogenic en niet-angiogenic cel fenotypen, zijn deze methoden tijd en intensieve arbeid, en vereisen vaak dure reagentia en instrumenten, alsmede aanzienlijke deskundigheid. In een recente studie gebruikten we een nieuwe kwantitatieve fase imaging (QPI) techniek te voeren time-lapse en labeling-gratis karakterisaties voor angiogenic en niet-angiogenic menselijke Osteosarcoom KHOS cellen. Een panel van cellulaire parameters, met inbegrip van de morfologie van de cel proliferatie en motiliteit, werden kwantitatief gemeten en geanalyseerd met behulp van QPI. Deze roman en kwantitatieve benadering biedt de mogelijkheid om voortdurend en niet-gebeurt studie relevante cellulaire processen, gedrag, en kenmerken van kankercellen en andere celtypes in een eenvoudige en geïntegreerde wijze. Dit verslag beschrijft onze experimentele protocol, met inbegrip van de voorbereiding van de cel, QPI acquisitie en data-analyse.

Figuur 1 toont een typische cel morfologie karakterisering. Afbeeldingen worden gepresenteerd als holographs (figuur 1A-B) en 2D-afbeeldingen (Figuur 1 c-D). Optische cel diktes (berekend op basis van de brekingsindex en optische weglengte) zijn gekwantificeerd via lijn-profiel of een hele cel meting. Scatter percelen van het gebied en de dikte van KHOS-A en KHOS-N c...

Watch this Scientific Journal Video about A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells at JoVE.com

A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells

Slapende en actieve kanker cel fenotypen werden gekenmerkt met behulp van kwantitatieve fase imaging. Cel proliferatie, migratie en morfologie assays werden geïntegreerd en geanalyseerd in één eenvoudige methode.

Een Time-lapse, Label-vrije, kwantitatieve fase Imaging studie van slapende en actieve menselijke kankercellen

The acquisition of the angiogenic phenotype is an essential component of the escape from tumor dormancy. Although several classic in vitro assays (e.g., ...

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