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>
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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. 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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. 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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., ...

The overall goal of this experiment is to quantitatively characterize angiogenic and non-angiogenic phenotypes of human osteosarcoma cells in a systematic manner combining analyses of cell morphology, proliferation, and motility. This method can help answer key questions with respect to human cancer establishment and progression. In particular, the escape from tumor dormancy and the endogenic switch.The main advantage of this technique is that multicellular parameters can be analyzed simultaneously by this integrated, high-spread method. This includes cell area, cell thickness, cell volume, proliferation rate, doubling time, motility speed, migration, and motility. To begin, prewarm the culture medium to 37 degrees Celsius in a water bath.Then remove cryogenic vials of angiogenic and non-angiogenic human osteosarcoma cells from the liquid nitrogen tank. Immerse the bottom of the vials into a 37 degree Celsius water bath and gently shake the cryogenic vials to accelerate the thawing process. As soon as cells are thawed, spray the vial with 70%ethanol and wipe the vial to sterilize it.Then immediately aspirate the cell suspension from the vial and gently suspend them in 10 milliliters of warmed culture medium. Next centrifuge the cell suspensions for five minutes to pellet the cells. Back in the hood, remove the supernatant and re-suspend the cell pellet with 10 milliliters of warmed culture medium.Then transfer the cell suspension into a T75 flask. Gently pipette the cell suspension several times to evenly disperse the cells. Place the flask into an incubator and allow the cells to attach for at least 12 hours.Change the culture medium every two to three days. When the cells reach 80 to 100 percent confluence, remove the culture media from the flask and wash the cells with five milliliters of sterile PBS without calcium and magnesium. Then add one milliliter of a 0.25%tripson EDTA solution into the flask and briefly swirl the flask.Incubate the flask for two to three minutes and then check the cells under a microscope to make sure the cells are rounded up and detached. Once the cells are detached, use a pipette to add 10 milliliters culture medium and gently pipette the medium up and down several times to break up any remaining cell aggregates. After counting the cells, seed two million of them into a new T75 flask.Place the flask in an incubator and grow the cells to between 80 and 100 percent confluence before seeding the cells for the quantitative phase imaging experiment. Harvest cells by tripson digestion and seed angiogenic and non-angiogenic human osteosarcoma cells in six well plates at the density of 50, 000 to 300, 000 cells per well. Cover the cells with a total of five milliliters of culture medium.Then incubate the plates for at least 12 hours to allow time for the cells to attach. Replace the medium with fresh media before imaging. Clean a cover slip by rinsing it several times with running deionized water.Then immerse it in a 75%ethanol solution for 15 minutes. Place the sterilized cover slip into a laminar flow hood to dry. Next carefully place the cover slip into a six well plate, avoiding obvious bubbles.Place the plate into the incubator to equilibrate for at least 15 minutes. If fog forms on the lid of the plate, use a sterile cotton swab to wipe it clean prior to imaging. Open the quantitative phase imaging software and initialize the system.Make sure that the values are acceptable in the self-calibration of exposure time, pattern contrast, and hologram noise. Next, place the six well plate onto the stage of the quantitative phase image microscope. Back in the software, click on live capture.Then go to the software focus menu and select manual. Next go to the microscope setting menu and coarsely focus the phase images of the cells by adjusting the work distance to obtain outlines for cells in phase images. When finished, go back to the software focus menu and change the focus setting to automatic.Now click on new experiment. Begin selecting image acquisition positions using the mouse or the arrow keys on the number pad. Click remember after each selection.Once the positions have all been selected, go to the time lapse section and set up the imaging intervals so that they are shorter than five minutes and the total time period is 48 hours. Start the experiment by clicking capture. This will automatically focus and acquire images at the selected positions at each interval time points.Following image acquisition, analyze the morphology of the cells by first clicking on identify cells. Adjust the numeric setting for the background threshold so that cell areas are well separated from the background noise. Next adjust the setting number for object size to make sure that each cell has only one nucleus.Manually modify any cell segments not matching this requirement by using manual changes. Then use the analyze data function in the software to analyze cells. Start a new data analysis by clicking new analysis.Drag selected images into the source frames tab and record cell morphology parameters, including cell area, optical thickness, and volume for each individual cell. When finished, choose the scatter plot or histogram mode and export the data as tables or figures. Select at least five images spaced 12 hours apart and record cell numbers that are provided by the software.Estimate the proliferation rate of the cells by normalizing with the initial cell numbers and then plotting the cell numbers. Finally, estimate the doubling time by fitting the data points using an exponential growth curve. To analyze cell motion use the track cells function in the software.Start by clicking on new analysis to start a new data analysis. Then drag a series of images from the time period of interest into the source frames tab. Next, under the add cells selection mode, randomly choose 10 to 30 cells by clicking on them.Be sure to review the tracking in the series of images. In the event that the system loses track of a cell or tracks the wrong cell, manually adjust the tracking cell by clicking on identify to identify cells or by going to the select mode and clicking on modify location to modify a cell's location. When finished go to plot movement and choose rows plot of cell trajectories or go to plot features and plot for the other motion related parameters, such as motility speed, motility, migration, and migration directness.Export the data as tables or figures. In this protocol, quantitative phase images are presented as holographs. The cells shown here depict the typical cell morphology for angiogenic and non-angiogenic human osteosarcoma cells.The cell thickness is calculated by the refractive index and optical path length and is shown here as a scatter plot. The two difference cell pheno types displayed different distribution patters and indicate that the angiogenic cells have smaller cell areas and a greater cell thickness than the non-angiogenic cells. The cell proliferation rate of the two cell types can be calculated from 2D images such as these, which have been segmented and span the course of two days.For these specific cell types, no significant differences were found. To determine cell motion characteristics for the cells, individual cells must be selected and tracked over time. Here the tracked cells are outlined with different colors.Each individual cell was tracked over the course of four hours with their total path trajectory described here where the zero, zero point is their starting location and their final location is indicated by the colored dot. After watching this video you should have a good understanding of how to use quantitative phase imaging to quantitatively compare a pan of cell morphological and behavior parameters. In this case, between angiogenic and non-angiogenic cell phenotypes.After this development, this technique paved the way for researchers in the field of cancer research to explore the escape from tumor dormancy and the role of angiogenecy in this process. In a continuous and non-labeling platform. Don't forget that working with human cell lines can be extremely hazardous and precautions such as wearing personal protective equipment should always be taken while performing this procedure.

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Cancer Research

7.3K vistas.  Boston Children's Hospital. The overall goal of this experiment is to quantitatively characterize angiogenic and non-angiogenic phenotypes of human osteosarcoma cells in a systematic manner combining analyses of cell morphology, proliferation, and motility. This method can help answer key questions with respect to human cancer establishment and progression. In particular, the escape from tumor dormancy and the endogenic switch.The main advantage of this technique is that multicellular parameters can be analyzed s...