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., ...

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.

a-time-lapse-label-free-quantitative-phase-imaging-study-dormant

Research

JoVE Journal

Cancer Research

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

Phosphopeptide Enrichment Coupled with Label-free Quantitative Mass Spectrometry to Investigate the Phosphoproteome in Prostate Cancer

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction pathways and is tightly regulated by kinases and phosphatases. Therefore, characterizing the phosphoproteome may provide insights into identifying novel targets and biomarkers for oncologic therapy. Mass spectrometry provides a way to globally detect and quantify thousands of unique phosphorylation events. However, phosphopeptides are much less abundant than non-phosphopeptides, making biochemical analysis more challenging. To overcome this limitation, methods to enrich phosphopeptides prior to the mass spectrometry analysis are required. We describe a procedure to extract and digest proteins from tissue to yield peptides, followed by an enrichment for phosphotyrosine (pY) and phosphoserine/threonine (pST) peptides using an antibody-based and/or titanium dioxide (TiO2)-based enrichment method. After the sample preparation and mass spectrometry, we subsequently identify and quantify phosphopeptides using liquid chromatography-mass spectrometry and analysis software.

This protocol describes a procedure to extract and enrich phosphopeptides from prostate cancer cell lines or tissues for an analysis of the phosphoproteome via mass spectrometry-based proteomics.

Phosphoproteomics involves the large-scale study of phosphorylated proteins. Protein phosphorylation is a critical step in many signal transduction ...

Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth factors and matrix-associated proteins in the microenvironment in vivo. Furthermore, readily available reagents for the modeling of microenvironments in vitro typically utilize complex mixtures of proteins that are incompletely defined and suffer from batch to batch variability. The microenvironment microarray (MEMA) platform allows for the assessment of thousands of simple combinations of microenvironment proteins for their impact on cellular phenotypes in a single assay. The MEMAs are prepared in well plates, which allows the addition of individual ligands to separate wells containing arrayed extracellular matrix (ECM) proteins. The combination of the soluble ligand with each printed ECM forms a unique combination. A typical MEMA assay contains greater than 2,500 unique combinatorial microenvironments that cells are exposed to in a single assay. As a test case, the breast cancer cell line MCF7 was plated on the MEMA platform. Analysis of this assay identified factors that both enhance and inhibit the growth and proliferation of these cells. The MEMA platform is highly flexible and can be extended for use with other biological questions beyond cancer research.

The purpose of the method presented here is to show how microenvironment microarrays (MEMA) can be fabricated and used to interrogate the impact of thousands of simple combinatorial microenvironments on the phenotype of cultured cells.

Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth ...

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

Time-Lapse 2D Imaging of Phagocytic Activity in M1 Macrophage-4T1 Mouse Mammary Carcinoma Cells in Co-cultures

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer therapies. However, tumor-associated macrophages can be harmful to the tumor depending on the tumor microenvironment and can reversibly alter their phenotypic characteristics by either antagonizing the cytotoxic activity of immune cells or enhancing anti-tumor response. The molecular actions of macrophages and their interactions with tumor cells (e.g., phagocytosis) have not been extensively studied. Therefore, the interaction between immune cells (M1/M2-subtype TAM) and cancer cells in the tumor microenvironment is now a focus of cancer immunotherapy research. In the present study, a live cell coculture model of induced M1 macrophages and mouse mammary 4T1 carcinoma cells was developed to assess the phagocytic activity of macrophages using a time-lapse video feature using phase-contrast, fluorescent, and differential interference contrast (DIC) microscopy. The present method can observe and document multipoint live-cell imaging of phagocytosis. Phagocytosis of 4T1 cells by M1 macrophages can be observed using fluorescent microscopy before staining 4T1 cells with carboxyfluorescein succinimidyl ester (CFSE). The current publication describes how to coculture macrophages and tumor cells in a single imaging dish, polarize M1 macrophages, and record multipoint events of macrophages engulfing 4T1 cells during 13 h of coculture.

Macrophage phagocytic activity against cancer cells, specifically 4T1 mouse mammary carcinoma cells, was imaged in this study. The live cell coculture model was established and observed using a combination of fluorescent and differential interference contrast microscopy. This assessment was imaged using imaging software to develop multipoint time-lapse video.

Tumor-associated macrophages (TAMs) have been identified as an important component for tumor growth, invasion, metastasis, and resistance to cancer ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many studies in the last decades. Therefore, it is important to develop methods to investigate the role of key genes involved in cancer cell stemness. Gastric cancer (GC) is one of the most common and mortal types of cancers. Gastric cancer stem cells (GCSCs) are thought to be the root of gastric cancer relapse, metastasis and drug resistance. Understanding GCSCs&#160;biology is needed to advance the development of targeted therapies and eventually to reduce mortality among patients. In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Immunofluorescence Imaging of DNA Damage and Repair Foci in Human Colon Cancer Cells

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage response induced by neutron-gamma mixed-beam used in boron neutron capture therapy (BNCT). Specifically, the proposed methodology is applied for the detection of repair proteins activation which can be visualized as foci using antibodies specific to DNA double-strand breaks (DNA-DSBs). DNA repair foci were assessed by immunofluorescence in colon cancer cells (HCT-116) after irradiation with the neutron-mixed beam. DNA-DSBs are the most genotoxic lesions and are repaired in mammalian cells by two major pathways: non-homologous end-joining pathway (NHEJ) and homologous recombination repair (HRR). The frequencies of foci, immunochemically stained, for commonly used markers in radiobiology like &#947;-H2AX, 53BP1 are associated with DNA-DSB number and are considered as efficient and sensitive markers for monitoring the induction and repair of DNA-DSBs. It was established that &#947;-H2AX foci attract repair proteins, leading to a higher concentration of repair factors near a DSB. To monitor DNA damage at the cellular level, immunofluorescence analysis for the presence of DNA-PKcs representative repair protein foci from the NHEJ pathway and Rad52 from the HRR pathway was planned. We have developed and introduced a reliable immunofluorescence staining protocol for the detection of radiation-induced DNA damage response with antibodies specific for repair factors from NHEJ and HRR pathways and observed radiation-induced foci (RIF). The proposed methodology can be used for investigating repair protein that is highly activated in the case of neutron-mixed beam radiation, thereby indicating the dominance of the repair pathway.

Radiation-induced DNA damage response activated by neutron mixed-beam used in boron neutron capture therapy (BNCT) has not been fully established. This protocol provides a step-by-step procedure to detect radiation-induced foci (RIF) of repair proteins by immunofluorescence staining in human colon cancer cell lines after irradiation with the neutron-mixed beam.

The purpose of the manuscript is to provide a step-by-step protocol for performing immunofluorescence microscopy to study the radiation-induced DNA damage ...

A Three-dimensional Model of Spheroids to Study Colon Cancer Stem Cells

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for tumor development, maintenance, and resistance to drugs. A better understanding of CSC properties for their specific targeting is, therefore, a pre-requisite for effective therapy. However, there is a paucity of suitable preclinical models for in-depth investigations. Although in vitro two-dimensional (2D) cancer cell lines provide valuable insights into tumor biology, they do not replicate the phenotypic and genetic tumor heterogeneity. In contrast, three-dimensional (3D) models address and reproduce near-physiological cancer complexity and cell heterogeneity. The aim of this work was to design a robust and reproducible 3D culture system to study CSC biology. The present methodology describes the development and optimization of conditions to generate 3D spheroids, which are homogenous in size, from Caco2 colon adenocarcinoma cells, a model that can be used for long-term culture. Importantly, within the spheroids, the cells which were organized around lumen-like structures, were characterized by differential cell proliferation patterns and by the presence of CSCs expressing a panel of markers. These results provide the first proof-of-concept for the appropriateness of this 3D approach to study cell heterogeneity and CSC biology, including the response to chemotherapy.

This protocol presents a novel, robust, and reproducible culture system to generate and grow three-dimensional spheroids from Caco2 colon adenocarcinoma cells. The results provide the first proof-of-concept for the appropriateness of this approach to study cancer stem cell biology, including the response to chemotherapy.

Colorectal cancers are characterized by heterogeneity and a hierarchical organization comprising a population of cancer stem cells (CSCs) responsible for ...