Funding sources of this research: a joint fund of Tel Aviv University's technology transfer company, RAMOT and the Sheba-Medical Center (M. C-A. and S.I.), ICRF - Israeli Cancer research foundation (M. C-A.) and Israel science Foundation (S.I.).

1. Cell Culture Preparation
MDA-MB-231 cells were purchased from ATCC (American Type Culture Collection) and stored in liquid nitrogen.
<ol>
	<li>Seed 106 MDA-MB-231 cells in 92 mm diameter Petri dish in 10 ml complete medium containing Dulbeco Modified Eagle Medium (DMEM), 10% horse serum, 1% L-glutamine, and 1% Penstrep-Amphotericine B. Allow cells to proliferate to about 80-100% confluence.</li>
	<li>Remove culture medium from dish and discard.</li>
	<li>Wash the cell layer briefly with 0.25% (w/v) Trypsin-EDTA solution to remove all traces of serum.</li>
	<li>Add 2.0 ml of Trypsin-EDTA solution to dish and observe cells by inverted microscope until cell layer is dispersed (usually within 5 to 15 min).</li>
	<li>Add 18 ml complete growth medium and gently aspirate the cells by pipette. Transfer to a tube.</li>
	<li>Centrifuge the cell suspension at 1,200 rpm.</li>
	<li>Re-suspend the cell pellet in 24 ml of culture medium.</li>
	<li>Add 2 ml of cell suspension to 35 mm glass bottom dishes (about 25% confluence) and place in incubator (5% CO2 , 37 &deg;C).</li>
	<li>Solutions:
	<ol>
		<li>Complete Medium for cells proliferation: DMEM with 10%FBS, 1% Antibiotics (100 units/ml penicillin G, 100 &mu;g/ml streptomycin, Pen-Strep-Ampho solution) and 2 mM L-glutamine.</li>
		<li>Trypsin-EDTA solution, containing 0.25%Trypsin-EDTA.</li>
	</ol>
	</li>
	<li>Dishes: 
	92 mm diameter Petri dishes. 
	35 mm diameter poly-D-lysine coated glass bottom culture dishes.</li>
</ol>
2. Preparation of Cells for Live Confocal Imaging
<ol>
	<li>Seed 2 x 105 MDA-MB-231 cells in glass bottom culture dishes in 2 ml complete medium as mentioned in section 1. When cell culture reaches confluence of 60-70% (about 3-4 x 105 cells per dish), proceed with transfection.</li>
	<li>Transfect the cells with two plasmids encoding the fusion proteins &gamma;-Tubulin-GFP (for fluorescent detection of centrosomes) and Histone-RED (H2b-RED, for fluorescent detection of chromosomes) using the liposomal transfection reagent Jet-PI, following the manufacture protocol. Briefly, mix 2 &mu;g from each plasmid in a tube with 100 &mu;l NaCl (150 mM). Mix the transfection reagent (100 &mu;l) with 100 &mu;l NaCl (150 mM) in a second tube, and incubate 5 min at room temperature (RT). Then combine the two solutions, mix (using mild vortex) and spin-down. Incubate for 30 min at RT.</li>
	<li>During the incubation of the transfection mixture, wash the cells once with PBS and replace the cell medium with 2 ml of warm DMEM with no supplements (37 &deg;C).</li>
	<li>Gently add the transfection mixture to the cells in DMEM and then return the cells to the incubator (37 &deg;C, 5% CO2) for 8 hr.</li>
	<li>After 8 hr of incubation, replace the DMEM with 2 ml complete medium and incubate the cells in the incubator for 24 hr.</li>
	<li>24 hr after transfection, replace the medium of the cells with 2 ml complete medium containing 20 &mu;M PJ-34.</li>
	<li>Incubate the cells for additional 18 hr (37 &deg;C, 5% CO2).</li>
	<li>Subject the cells to live confocal imaging for at least 16 hr in imaging chamber keeping the cells at 5% CO2 and 37 &deg;C.</li>
	<li>In parallel, examine transfection efficacy 36 hr post transfection using fluorescent microscopy as follow:
	<ol>
		<li>Seed 2 x 105 MDA-MB-231 cells in 6-well plate containing 1 coverslip per well in 2 ml complete medium.</li>
		<li>Transfect the cells as mentioned in sections 2.2-2.5.</li>
		<li>36 hr post transfection fix the transfected cells mounted on a coverslip by incubation in cold methanol:acetone (1:1) solution, 7 min, -20 &deg;C.</li>
		<li>Aspirate the fixation solution and let the coverslip with the mounted cells to dry in a chemical hood.</li>
		<li>Apply ProLong Gold antifade reagent with DAPI and let the coverslip to dry in the dark for 6 hr.</li>
		<li>Examine the slide under fluorescent microscope and calculate the percentage of the transfected cells (red and green signals) from the total population of cells (DNA staining by DAPI). The desired transfection percentage is about 20-40% when 100-200 cells are counted.</li>
	</ol>
	</li>
</ol>
3. Technical Parameters of the Live Confocal Imaging Scanner Settings
<ol>
 <li>ScanMode XYZT; Pinhole [airy] 1.00; Zoom 3.5; Resolution 8 bits; Laser DPSS 561 nm; Argon, visible laser 488 nm; Laser He/Ne visible 633 nm; Objective HCX PL APO CS 63X 1.40 OIL UV; Numerical aperture 1.4; Scan speed 700 Hz; Refraction index 1.52.</li>
 <li> Image 3-D presentation were prepared by IMARIS imaging software 7.0.</li>
</ol>
4. Confocal Imaging of Mitosis in Fixed Cells
<ol>
	<li>Seed 2 x 105 MDA-MB-231 breast cancer cells (ATCC), normal mouse embryonic fibroblasts (MEF), or PARP1 deficient MEF (PARP-/-, prepared by Dr Francoise Dantzer) on glass coverslips in 6-well plate in 2 ml complete medium. Coverslips were washed with 96% ethanol, following by wash with sterile DD water, dried for 2 hr, and placed in each well of 6-well dish.</li>
	<li>Add PJ-34 (10-30 &mu;M) to the medium and incubate the cells for the required period (usually up to 96 hr).</li>
	<li>Wash the coverslips once with PBS (phosphate buffered saline), and fix the cells using incubation in ice-cold methanol:acetone (1:1) solution, 7 min, -20 &deg;C.</li>
	<li>Aspirate the fixation solution and let the coverslips to dry in chemical hood (at this stage, the coverslips can be kept in -20 &deg;C for several weeks).</li>
	<li>Wash the coverslips once with PBST (PBS supplemented with 0.1% Tween-20) to permeabilize the cell membranes and block the cells with 10% NDS (Normal Donkey Serum) in PBST ('blocking solution') for 1 hr at RT.</li>
	<li>Incubate the permeabilized fixed cells with primary antibodies for 2 hr at RT (for spindles and centrosomes staining). The antibodies are diluted in the blocking solution as follow: anti-&alpha; tubulin (1:250 dilution) and anti-&gamma; tubulin (1:200 dilution). Primary antibodies are applied as follows: apply 100 &mu;l (in a drop) of a mixture of the antibodies in blocking solution for each coverslip on 6-well plate cover (the cover is upside down). Gently put the coverslip on the antibodies drop, seeded cells facing the drop. Incubate the coverslips facing the antibodies for 2 hr at room temperature.</li>
	<li>Place the coverslips back in the wells and wash the cells 3 times with PBST. Then use the same procedure described in 4.6 for labeling cells on the coverslips with the fluorescent secondary antibodies. Incubate the cells on the coverslips with the secondary antibodies for 1 hr, RT, in the dark. The antibodies are diluted in the blocking solution as follow: Alexa Fluor 488 (1:1,000 dilution; green) and Alexa Fluor 568 (1:1,000 dilution; red).</li>
	<li>Mount the coverslips using ProLong Gold antifade reagent with DAPI (for chromosomes staining) and incubate overnight at RT in dark to dry.</li>
	<li>Examine the cover slips by confocal microscopy.</li>
</ol>
5. Cell Viability Measured by ATP Production
ATP production is measured by a luminescent ATP detection assay kit.
<ol>
	<li>Seed the cells in 96-well plate, approximately 20,000 cells in 800 &mu;l medium in each well. Three blank wells should be used for determination of the background luminescence of the medium.</li>
	<li>Prepare ATP standard dilution series from approximately 10 &mu;M to 100 &mu;M and keep on ice.</li>
	<li>Add 50 &mu;l of detergent to each well and shake the plate for 5 min in orbital shaker, 700 rpm.</li>
	<li>Reconstitute each vial of the' lyophilized substrate' with 5 ml of 'substrate buffer' in the kit.</li>
	<li>Add 50 &mu;l of the reconstituted substrate solution to the wells, and shake the plate for 5 min on orbital shaker, 700 rpm.</li>
	<li>Keep the plate in dark for 10 min.</li>
	<li>Measure luminiscence of each well by ELISA microplate reader.</li>
</ol>

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Care Med. 30, 1071-1082 (2002).</li><li>Castiel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+small+molecule+exclusively+eradicates+human+cancer+cells:+Extra-centrosomes+de-clustering+agent.">A small molecule exclusively eradicates human cancer cells: Extra-centrosomes de-clustering agent.</a> BMC Cancer. 11 (1), 412 (2011).</li><li>Inbar-Rozensal, 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=A+selective+eradication+of+human+nonhereditary+breast+cancer+cells+by+phenanthridine+-derived+polyADP-ribose+polymerase+inhibitors.">A selective eradication of human nonhereditary breast cancer cells by phenanthridine -derived polyADP-ribose polymerase inhibitors.</a> Breast Cancer Res. 11 (6), R78 (2009).</li><li>Gergely, F., Basto, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+centrosomes:+together+they+stand,+divided+they+fall.">Multiple centrosomes: together they stand, divided they fall.</a> Genes Dev. 22, 2291-2296 (2008).</li><li>Godinho, S. A., Kwon, M., Pellman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centrosomes+and+cancer:+how+cancer+cells+divide+with+too+many+centrosomes.">Centrosomes and cancer: how cancer cells divide with too many centrosomes.</a> Canc. Met. Rev. 28, 85-98 (2009).</li><li>Doxsey, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Re-evaluating+centrosome+function.">Re-evaluating centrosome function.</a> Nat. Rev. Mol. Cell Biol. 2, 688-698 (2001).</li><li>Walczak, C. E., Heald, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+mitotic+spindle+assembly+and+function.">Mechanisms of mitotic spindle assembly and function.</a> International Rev. of Cytology. 265, 111-158 (2008).</li><li>Ogden, A., Rida, P. C. G., Aneja, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Let's+huddle+to+prevent+a+muddle:+centrosome+declustering+as+an+attractive+anticancer+strategy.">Let's huddle to prevent a muddle: centrosome declustering as an attractive anticancer strategy.</a> Cell Death Differ. 19, 1255-1267 (2012).</li><li>Kramer, A., Anderhub, S., Maier, B., Schatten, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+Consequences+of+centrosomes+clustering+in+cancer+cells.">Mechanisms and Consequences of centrosomes clustering in cancer cells.</a> The Centrosome: Cell and Molecular mechanisms of functions and disfunctions in disease. , 255-277 (2012).</li><li>Galimberti, F., 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=Anaphase+Catastrophe+Is+a+Target+for+Cancer+Therapy.">Anaphase Catastrophe Is a Target for Cancer Therapy.</a> Clin. Cancer Res. 17, 1218-1222 (2011).</li><li>Kanai, M., 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=Haploinsufficiency+of+poly(ADP-ribose)+polymerase-1-mediated+poly(ADP-ribosyl)ation+for+centrosome+duplication.">Haploinsufficiency of poly(ADP-ribose) polymerase-1-mediated poly(ADP-ribosyl)ation for centrosome duplication.</a> Biochem. Biophys. Res. Commun. 359, 426-430 (2007).</li><li>Gartner, E. M., Burger, A. M., Lorusso, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Poly(ADP-ribose)+polymerase+inhibitors:+a+novel+drug+class+with+a+promising+future.">Poly(ADP-ribose) polymerase inhibitors: a novel drug class with a promising future.</a> Cancer J. 16, 83-90 (2010).</li><li>Wahlberg, E., 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=Family-wide+chemical+profiling+and+structural+analysis+of+PARP+and+tankyrase+inhibitors.">Family-wide chemical profiling and structural analysis of PARP and tankyrase inhibitors.</a> Nature Biotechnology. 30, 283-288 (2012).</li><li>Rouleau, M., Patel, A., Hendze, M. J., Kaufmann, S. H., Poirier, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PARP+inhibition:+PARP1+and+beyond.">PARP inhibition: PARP1 and beyond.</a> Nature Rev. Cancer. 10, 293-301 (2010).</li><li>Leber, B., 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=Proteins+Required+for+Centrosome+Clustering+in+Cancer+Cells.">Proteins Required for Centrosome Clustering in Cancer Cells.</a> Sci. Transl. Med. 2 (33), 33-38 (2010).</li><li>Kwon, M., 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=Mechanisms+to+suppress+multipolar+division+in+cancer+cells+with+extra+centrosomes.">Mechanisms to suppress multipolar division in cancer cells with extra centrosomes.</a> Gene Dev. 22, 2189-2203 (2008).</li></ol>

The authors declare that they have no competing financial interests.

Live confocal imaging provided a real-time documentation of the cytotoxic effect of PJ-34 in live multi-centrosomal cells during mitosis (Figure 3 and Supplementary information). This was the first live documentation attributing the cytotoxicity of PJ-34 in human cancer cells to extra centrosomes de-clustering and cell death, suggesting induction of Mitotic Catastrophe cell death by PJ-345-9. In contrast, bi-focal clustering of super-numerary centrosomes was observed in live untreated MDA-MB-2...

Phenanthrene derived PARP1 inhibitors, including PJ-34, were designed to protect quiescent cells from apoptotic cell death induced by the energy consuming PARP1 mediated DNA-repair under stress conditions (stroke or myocardial infarction)1. However, recently we discovered that PJ-34, at twice higher concentration than that inducing PARP1 inhibition, can exclusively cause cell death in human cancer cells2,3. The more rapid the proliferation of the cell was, the more efficient the eradication of the cells was. The cytotoxic activity of PJ-34 was attributed to extra-centrosomes de-clustering in mitosis2. Many human cancer cells harbor multicentrosomes4,5. Incubation of human breast cancer cells MDA-MB-231, which harbor supernumerary centrosomes, with 20 &mu;M PJ-34 efficiently eradicated these cells within 72-96 hr without impairing quiescent cells or some benign proliferating cells harboring two centrosomes in mitosis2,3. Benign cells included human mammary epithelial cells MCF-10, Human endothelial cells (huvec) and primary mesenchymal cells prepared from human thymus. These cells were resistant to the cytotoxic activity of PJ-34. PJ-34 did not interfere with their cell cycle during 96 hr incubation or affect their centrosomes and bi-focal spindle formation2,3.
Bipolar centrosome assembly is crucial for bipolar spindle formation in mitosis4,5. Therefore, cells with more than two centrosomes have developed a scarcely understood molecular mechanism, clustering their extra centrosomes at two poles 4-9. Failure of bipolar assembly of their centrosomes may cause multipolar distorted spindles and aberrant chromosomes segregation that arrests the cell-cycle in G2/M arrest, and leads to cell death attributed to mitotic failure4,5. The molecular mechanisms underlying extra-centrosomes de-clustering are intensively investigated10. Understanding this death mechanism will enable exclusive eradication of cancer cells while sparing healthy tissues 5,10.
Thus, compounds that activate mitotic catastrophe cell death offer a new mode of a selective cancer therapy, which may be efficient in a wide range of human solid cancers.Our results suggest that confocal imaging can be used to identify molecules affecting extra-centrosomes clustering in mitosis2,3, rendering these compounds cancer targeting drug candidates.
We have documented the cytotoxic activity of the phenanthridine PJ-34 by scanning fixed and live human cancer cells (with high occurrence of extra-centrosomes in mitosis) versus normal cells. A step-by step description of the imaging procedures used to identify the cytotoxic activity of PJ-34 in human cancer cells is included below.

<table border="1">
	<tbody>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>REAGENTS </td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen (GIBCO)</td>
			<td>41965</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>FBS (Fetal bovine Serum)</td>
			<td>Invitrogen (GIBCO)</td>
			<td>12657</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pen-Strep-Ampho solution</td>
			<td>Biological Industries, Israel</td>
			<td>03-033-1B</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Invitrogen (GIBCO)</td>
			<td>25030-024</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>0.25% Tripsin-EDTA</td>
			<td>Invitrogen (GIBCO)</td>
			<td>25200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>92 mm Petri dishes</td>
			<td>Nunc,Thermo scientific</td>
			<td>150350</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>35 mm poly-D-lysine coated glass bottom culture dishes</td>
			<td>MatTek Corporation, USA</td>
			<td>P35GC-0-14-C</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Luminescent ATP detection assay kit</td>
			<td>Abcam</td>
			<td>ab113849</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NDS (Normal Donkey Serum)</td>
			<td>Jackson ImmunoResearch</td>
			<td>017-000-121</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti &alpha;-tubulin antibody</td>
			<td>Sigma</td>
			<td>T9026</td>
			<td>1:250 dilution (IF)</td>
		</tr>
		<tr>
			<td>Anti &gamma;-tubulin antibody</td>
			<td>Sigma</td>
			<td>T5192</td>
			<td>1:200 dilution (IF)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat Anti-Mouse IgG</td>
			<td>Invitrogen</td>
			<td>A-11017</td>
			<td>1:1,000 dilution (IF)</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Donkey Anti-Rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A-10042</td>
			<td>1:1,000 dilution (IF)</td>
		</tr>
		<tr>
			<td>ProLong Gold antifade reagent with DAPI (mounting)</td>
			<td>Invitrogen</td>
			<td>P36935</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>JetPEI (liposomal transfection reagent )</td>
			<td>Polyplus</td>
			<td>101-10</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>EQUIPMENT</td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica (Mannheim, Germany)</td>
			<td>TCS SP5II</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

cell death associated with abnormal mitosis observed by confocal imaging in live cancer cells

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human cancer cells in mitosis. The phenanthridine PJ-34 was the most potent molecule. Declustering of extra-centrosomes causes mitotic failure and cell death in multi-centrosomal cells. Most solid human cancers have high occurrence of extra-centrosomes. The activity of PJ-34 was documented in real-time by confocal imaging of live human breast cancer MDA-MB-231 cells transfected with vectors encoding for fluorescent &gamma;-tubulin, which is highly abundant in the centrosomes and for fluorescent histone H2b present in the chromosomes. Aberrant chromosomes arrangements and de-clustered &gamma;-tubulin foci representing declustered centrosomes were detected in the transfected MDA-MB-231 cells after treatment with PJ-34. Un-clustered extra-centrosomes in the two spindle poles preceded their cell death. These results linked for the first time the recently detected exclusive cytotoxic activity of PJ-34 in human cancer cells with extra-centrosomes de-clustering in mitosis, and mitotic failure leading to cell death. According to previous findings observed by confocal imaging of fixed cells, PJ-34 exclusively eradicated cancer cells with multi-centrosomes without impairing normal cells undergoing mitosis with two centrosomes and bi-focal spindles. This cytotoxic activity of PJ-34 was not shared by other potent PARP1 inhibitors, and was observed in PARP1 deficient MEF harboring extracentrosomes, suggesting its independency of PARP1 inhibition. Live confocal imaging offered a useful tool for identifying new molecules eradicating cells during mitosis.

PJ-34 is a stable water soluble phenanthridine1 (Figure 1). Our previous results revealed cell death and de-clustered extra-centrosomes in several types of fixed multi-centrosomal cancer cells that were treated with PJ-34. In contrast, normal proliferating cells were not impaired2,3. Centrosomes were identified by double labeling with antibodies directed against centrine1 and &gamma;-tubulin in the fixed extra-centrosomal cells2.
Here, the cyto...

Watch this Scientific Journal Video about Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells at JoVE.com

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

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14.8K Views.  Sheba Medical Center. The cytotoxic activity of the phenanthridine PJ-34 in cancer cells undergoing mitosis was documented in real time by live confocal imaging. PJ-34 eradicated human breast cancer MDA-MB-231 cells harboring extra-centrosomes in mitosis. Unlike normal bi-focal mitosis, the extra-centrosomes were not clustered in the two spindle poles in the presence of PJ-34.

Video: Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

High-Efficiency Transduction of Liver Cancer Cells by Recombinant Adeno-Associated Virus Serotype 3 Vectors

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a number of gene therapy clinical trials. More recently, a number of additional AAV serotypes have also been isolated, which have been shown to exhibit selective tissue-tropism in various small and large animal models1. Of the 10 most commonly used AAV serotypes, AAV3 is by far the least efficient in transducing cells and tissues in vitro as well as in vivo.
However, in our recently published studies, we have documented that AAV3 vectors transduce human liver cancer - hepatoblastoma (HB) and hepatocellular carcinoma (HCC) - cell lines extremely efficiently because AAV3 utilizes human hepatocyte growth factor receptor as a cellular co-receptor for binding and entry in these cells2,3.
In this article, we describe the steps required to achieve high-efficiency transduction of human liver cancer cells by recombinant AAV3 vectors carrying a reporter gene. The use of recombinant AAV3 vectors carrying a therapeutic gene may eventually lead to the potential gene therapy of liver cancers in humans.

In this article, we describe the identification of the adeno-associated virus serotype 3 (AAV3) as the most efficient vector for targeting human liver cancer cells.

Recombinant vectors based on a non-pathogenic human parvovirus, the adeno-associated virus 2 (AAV2) have been developed, and are currently in use in a ...

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Evaluation of Cancer Stem Cell Migration Using Compartmentalizing Microfluidic Devices and Live Cell Imaging

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major obstacle for improving patient outcomes is the poor understanding of mechanisms underlying cellular migration associated with aggressive cancer cell invasion, metastasis and therapeutic resistance1. Glioblastoma Multiforme (GBM), the most prevalent primary malignant adult brain tumor2, exemplifies this difficulty. Despite standard surgery, radiation and chemotherapies, patient median survival is only fifteen months, due to aggressive GBM infiltration into adjacent brain and rapid cancer recurrence2. The interactions of aberrant cell migratory mechanisms and the tumor microenvironment likely differentiate cancer from normal cells3. Therefore, improving therapeutic approaches for GBM require a better understanding of cancer cell migration mechanisms. Recent work suggests that a small subpopulation of cells within GBM, the brain tumor stem cell (BTSC), may be responsible for therapeutic resistance and recurrence. Mechanisms underlying BTSC migratory capacity are only starting to be characterized1,4.
Due to a limitation in visual inspection and geometrical manipulation, conventional migration assays5 are restricted to quantifying overall cell populations. In contrast, microfluidic devices permit single cell analysis because of compatibility with modern microscopy and control over micro-environment6-9.
We present a method for detailed characterization of BTSC migration using compartmentalizing microfluidic devices. These PDMS-made devices cast the tissue culture environment into three connected compartments: seeding chamber, receiving chamber and bridging microchannels. We tailored the device such that both chambers hold sufficient media to support viable BTSC for 4-5 days without media exchange. Highly mobile BTSCs initially introduced into the seeding chamber are isolated after migration though bridging microchannels to the parallel receiving chamber. This migration simulates cancer cellular spread through the interstitial spaces of the brain. The phase live images of cell morphology during migration are recorded over several days. Highly migratory BTSC can therefore be isolated, recultured, and analyzed further. 
Compartmentalizing microfluidics can be a versatile platform to study the migratory behavior of BTSCs and other cancer stem cells. By combining gradient generators, fluid handling, micro-electrodes and other microfluidic modules, these devices can also be used for drug screening and disease diagnosis6. Isolation of an aggressive subpopulation of migratory cells will enable studies of underlying molecular mechanisms.

A compartmentalizing microfluidic device for investigating cancer stem cell migration is described. This novel platform creates a viable cellular microenvironment and enables microscopic visualization of live cell locomotion. Highly motile cancer cells are isolated to study molecular mechanisms of aggressive infiltration, potentially leading to more effective future therapies.

In the last 40 years, the United States invested over 200 billion dollars on cancer research, resulting in only a 5% decrease in death rate. A major ...

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

Assessing Phagocytic Clearance of Cell Death in Experimental Stroke by Ligatable Fluorescent Probes

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination of dead cells in stroke by waste-management phagocytes of any cellular lineage. Although numerous cells of different origins that are capable of phagocytosis are present in ischemic brain, only part of them actively engulf and digest cell corpses. The selective visualization, quantification and analysis of such active phagocytic waste-management are helpful in assessing brain response to ischemia. Efficient cell death clearance is important for brain recovery from ischemic injury, as it opens the way for the subsequent regenerative processes. The failure to clean the corpses would result in a toxic reaction caused by non-degraded DNA and proteins. The described procedure uses fluorescent probes selectively ligated by a viral topoisomerase to characteristic DNA breaks produced in all phagocytes during engulfment and digestion of cells irreversibly damaged by ischemia. The method is a new tool for the investigation of brain reaction to ischemic injury.

We present a new fluorescence technique for selective in situ labeling of active phagocytic cells, which clear off cell corpses in stroke. The approach is important for assessing brain reaction to ischemia because only a small proportion of phagocytes present in ischemic brain participate in clearance of cell death.

We describe a new histochemical approach for visualization of phagocytic clearance in focal brain ischemia. The approach permits the study of elimination ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...