Name: a time-lapse, label-free, quantitative phase imaging study of dormant and active human cancer cells
Uploaded: 2018-02-16T13:00:00
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

The authors gratefully acknowledge the support of the Breast Cancer Research Foundation and the Advanced Medical Research Foundation.

All the methods described here have been approved by the Boston Children&#39;s Hospital Institutional Biosafety Committee.
1. Cell Preparation
<ol>
	<li>Thawing KHOS-A and -N cells
	<ol>
		<li>Warm up culture medium, i.e., Dulbecco&#39;s modified Eagle medium supplemented with 10% (vol/vol) fetal calf serum (FBS) and 1% (vol/vol) penicillin/streptomycin.</li>
		<li>Take the cryogenic vials of cells from the liquid nitrogen tank, immerse the bottom of the vials into ...

<ol>
	<li>Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Angiogenesis+in+cancer,+vascular,+rheumatoid+and+other+disease.">Angiogenesis in cancer, vascular, rheumatoid and other disease.</a> Nature Medicine. 1 (1), 27-31 (1995).</li><li>Hanahan, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+and+emerging+mechanisms+of+the+angiogenic+switch+during+tumorigenesis.">Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis.</a> Cell. 86 (3), 353-364 (1996).</li><li>Harper, 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=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. J., Mellick, A. S., Bambino, K., McDonnell, K., Mittal, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+progenitor+cells+control+the+angiogenic+switch+in+mouse+lung+metastasis.">Endothelial progenitor cells control the angiogenic switch in mouse lung metastasis.</a> Science. 319 (5860), 195-198 (2008).</li><li>Folkman, J., Watson, K., Ingber, D., Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+angiogenesis+during+the+transition+from+hyperplasia+to+neoplasia.">Induction of angiogenesis during the transition from hyperplasia to neoplasia.</a> Nature. 339 (6219), 58-61 (1989).</li><li>Popescu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Quantitative phase imaging of cells and tissues. , (2011).</li><li>Lee, 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=Quantitative+Phase+Imaging+Techniques+for+the+Study+of+Cell+Pathophysiology:+From+Principles+to+Applications.">Quantitative Phase Imaging Techniques for the Study of Cell Pathophysiology: From Principles to Applications.</a> Sensors. 13 (4), 4170-4191 (2013).</li><li>Mir, M., Bhaduri, B., Wang, R., Zhu, R., Popescu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Phase+Imaging.">Quantitative Phase Imaging.</a> Progress in Optics. 57, 133-217 (2012).</li><li>Marrison, J., R&#228;ty, L., Marriott, P., O'Toole, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ptychography--a+label+free,+high-contrast+imaging+technique+for+live+cells+using+quantitative+phase+information.">Ptychography--a label free, high-contrast imaging technique for live cells using quantitative phase information.</a> Scientific Reports. 3, 2369 (2013).</li><li>Falck Miniotis, M., Mukwaya, A., Gj&#246;rloff Wingren, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+holographic+microscopy+for+non-invasive+monitoring+of+cell+cycle+arrest+in+L929+cells.">Digital holographic microscopy for non-invasive monitoring of cell cycle arrest in L929 cells.</a> PloS One. 9 (9), e106546 (2014).</li><li>Popescu, G., 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=Optical+imaging+of+cell+mass+and+growth+dynamics.">Optical imaging of cell mass and growth dynamics.</a> AJP: Cell Physiology. 295 (2), C538-C544 (2008).</li><li>Guo, P., Huang, 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=Characterization+of+dormant+and+active+human+cancer+cells+by+quantitative+phase+imaging.">Characterization of dormant and active human cancer cells by quantitative phase imaging.</a> Cytometry. Part A: The Journal of the International Society for Advancement of Cytometry. 91 (5), 424-432 (2017).</li><li>Mir, M., Bergamaschi, A., Katzenellenbogen, B. S., Popescu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+quantitative+imaging+for+monitoring+single+cancer+cell+growth+kinetics+and+drug+response.">Highly sensitive quantitative imaging for monitoring single cancer cell growth kinetics and drug response.</a> PloS One. 9 (2), e89000 (2014).</li><li>Mir, M., Tangella, K., Popescu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+testing+at+the+single+cell+level+using+quantitative+phase+and+amplitude+microscopy.">Blood testing at the single cell level using quantitative phase and amplitude microscopy.</a> Biomedical Optics Express. 2 (12), 3259-3266 (2011).</li><li>Park, 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=Measurement+of+red+blood+cell+mechanics+during+morphological+changes.">Measurement of red blood cell mechanics during morphological changes.</a> Proceedings of the National Academy of Sciences of the United States of America. 107 (15), 6731-6736 (2010).</li><li>Pham, H. V., Bhaduri, B., Tangella, K., Best-Popescu, C., Popescu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real+time+blood+testing+using+quantitative+phase+imaging.">Real time blood testing using quantitative phase imaging.</a> PloS One. 8 (2), e55676 (2013).</li><li>Sridharan, S., Macias, V., Tangella, K., Kajdacsy-Balla, A., Popescu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prediction+of+prostate+cancer+recurrence+using+quantitative+phase+imaging.">Prediction of prostate cancer recurrence using quantitative phase imaging.</a> Scientific Reports. 5, 9976 (2015).</li><li>Park, H., 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=Measuring+cell+surface+area+and+deformability+of+individual+human+red+blood+cells+over+blood+storage+using+quantitative+phase+imaging.">Measuring cell surface area and deformability of individual human red blood cells over blood storage using quantitative phase imaging.</a> Scientific Reports. 6, 34257 (2016).</li><li>Bishitz, Y., Gabai, H., Girshovitz, P., Shaked, N. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical-mechanical+signatures+of+cancer+cells+based+on+fluctuation+profiles+measured+by+interferometry.">Optical-mechanical signatures of cancer cells based on fluctuation profiles measured by interferometry.</a> Journal of Biophotonics. 7 (8), 624-630 (2014).</li></ol>

In this study, we describe an in vitro, non-invasive, and label-free method using QPI to quantitatively characterize the angiogenic and non-angiogenic phenotypes of human osteosarcoma cells. Multiple cellular parameters have been analyzed simultaneously by this integrated, high-throughput method, including cell area, cell thickness, cell volume, proliferation rate, doubling time, migration directness, motility speed, migration, and motility.
Compared with the conventional assays that ...

One of the earliest checkpoints in the development and progression of a solid tumor is the acquisition of the angiogenic phenotype, a hallmark of cancer. This progression involves a variety of biochemical and molecular processes1,2,3. A technical challenge in the study of this key step in tumor progression is the lack of tools to continuously and quantitatively characterize and differentiate between angiogenic and non-angiogenic phenotypes of live cancer cells in an unbiased manner. The traditional assays being used to investigate the cellular behaviors of angiogenic and non-angiogenic cells usually require expensive reagents and instruments, for example, cell proliferation/migration assays4,5,6,7,8,9,10,11,12,13,14 or complementary in vivo evaluations4,5,6,8,15,16, as well as requiring significant expertise and intensive time and labor consumption.
Recently, quantitative phase imaging (QPI) has emerged as a novel technique that enables time-lapse and labeling-free assessment of a variety of cell morphology and behavior parameters17,18,19,20,21,22. Unlike conventional optical microscopy, QPI quantifies variations of phase shift pixel by pixel after light passes through an optical object, and reconstructs a holograph with converted optical thickness and volume, thus enabling the direct analysis of live cells and the following features: (1) quantitative imaging, (2) non-invasive and time-lapse imaging, (3) label-free imaging, and (4) simultaneous multi-parameter imaging. These features make QPI a powerful tool to assess and understand pathological processes at cellular level.
In a recent study, we utilized QPI to quantitatively characterize and differentiate between angiogenic KHOS-A and non-angiogenic KHOS-N phenotypes of human osteosarcoma cells in a systematic and quantitative manner, combining analyses of cell morphology, proliferation, and motility23. Using image analysis software, a panel of cell morphological and behavior parameters were quantitatively compared between angiogenic and non-angiogenic human osteosarcoma cells and five characteristic differences were identified between these two phenotypes. This novel approach provides an integrated and quantitative platform for assessing a variety of biologically relevant cellular characteristics.

<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

The acquisition of the angiogenic phenotype is an essential component of the escape from tumor dormancy. Although several classic in vitro assays (e.g., proliferation, migration, and others) and in vivo models have been developed to investigate and characterize angiogenic and non-angiogenic cell phenotypes, these methods are time and labor intensive, and often require expensive reagents and instruments, as well as significant expertise. In a recent study, we used a novel quantitative phase imaging (QPI) technique to conduct time-lapse and labeling-free characterizations of angiogenic and non-angiogenic human osteosarcoma KHOS cells. A panel of cellular parameters, including cell morphology, proliferation, and motility, were quantitatively measured and analyzed using QPI. This novel and quantitative approach provides the opportunity to continuously and non-invasively study relevant cellular processes, behaviors, and characteristics of cancer cells and other cell types in a simple and integrated manner. This report describes our experimental protocol, including cell preparation, QPI acquisition, and data analysis.

Figure 1 depicts a typical cell morphology characterization. Images are presented as holographs (Figure 1A-B) and 2D images (Figure 1C-D). Optical cell thicknesses (calculated from the refractive index and optical path length) are quantified via line profile or a whole cell measurement. Scatter plots of the area and thickness of KHOS-A and KHOS-N cells measured for a...

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

Dormant and active cancer cell phenotypes were characterized using quantitative phase imaging. Cell proliferation, migration, and morphology assays were integrated and analyzed in one simple method.

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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