This work was supported in part by the Koch Institute Support (core) Grant P30-CA 14051 from the National Cancer Institute, by the National Institutes of Health Grant (CA206157-22 and GM118066) and Curt Marble Cancer Research Fund to Angelika Amon. Angelika Amon is also an investigator of the Howard Hughes Medical Institute and the Glenn Foundation for Biomedical Research. S. S. was supported by the American Italian Cancer Foundation (AICF), by a Fellowship in Cancer Research from Marie Curie Actions and the Italian Association for Cancer Research (AIRC), and by a KI Quinquennial Cancer Research Fellowship. E.M. was supported by a fellowship from the Howard Hughes Medical Institute.

Culture condition
RPE-1 hTERT cell line was cultured in Dulbecco&#39;s Modified Eagle Medium (Table of Materials) supplemented with 10% Fetal Bovine Serum, 2 mM L-glutamine, and 100 U/ml penicillin/streptomycin. Cells were incubated at 37 &#176;C with 5% CO2 in a humidified environment. The protocol described below has been developed by using 10-cm dishes and all technical details reported refer to those dishes. If using different dishes, scale up or down accordingly.
1. Synchronization of RPE-1 cells
<ol>
	<li>Use freshly thawed cells that have not undergone more than 15 passages for all experiments. On day 1, thaw and dispense ~2.5 - 5.0 x 105 RPE-1 hTERT cells into a 10-cm tissue culture dish. Add 8 mL of standard growth medium and incubate overnight. Determine cell number using a standard cell counter (such as a hemocytometer). 
	NOTE: Cell density refers to growth conditions of RPE-1 cells. Growth conditions might vary among different cell lines; consider adjusting them accordingly.</li>
	<li>On day 2, synchronize cells at the G1/S border by aspirating regular growth medium and adding 8 mL medium containing 5 mM thymidine. 
	NOTE: Thymidine concentration and length of treatment might vary across different cell lines. It is advisable to perform pilot experiments to determine the best conditions for the cell line(s) to be tested.</li>
	<li>On day 3, 24 h after adding thymidine medium (step 1.2), release from thymidine block by aspirating medium and washing cells three times with 1x PBS. Add 8 mL of regular growth medium and return plates to the incubator.</li>
</ol>
2. Generation of Aneuploid Cells by Interference with the Activity of the Mitotic Kinase Mps1
<ol>
	<li>On day 3, 6 h after release from thymidine block (step 1.3; this is about 3 - 6 h before cells enter mitosis), aspirate medium, wash once with 1x PBS, and replace with medium containing 500 nM reversine (the Mps1 inhibitor). 
	NOTE: RPE-1 cells enter mitosis 9 - 12 h after thymidine wash-out10,11. However, cell cycle kinetics vary among different cell lines. Therefore, it is crucial to determine those kinetics in pilot experiments by performing cell cycle analysis by established methods (e.g., by measuring DNA content via FACS analysis). Additionally, the optimal working concentration of Mps1 inhibitor may vary between cell lines. Previous experiments using RPE1 cells have been successful using reversine at a working concentration of 500 nM10,11,12. It is advisable to perform pilot experiments to determine the optimal concentration for the cell line to be tested.</li>
	<li>On day 4, 12 h after reversine treatment (about 6 h after cells were in mitosis), aspirate reversine medium and wash cells three times with 1x PBS. Add 8 mL regular growth medium to the plate and return cells to the incubator.</li>
</ol>
3. Removal of Aneuploid Cycling Cells and Enrichment of ArCK Population
<ol>
	<li>On day 6, about 72 h after cells enter the first mitosis (step 2.2; this is about 66 h after reversine wash-out, which corresponds to about 2 - 3 cell cycles), aspirate medium, wash once with 1x PBS and replace with medium containing 300 nM nocodazole. 
	NOTE: Recent work has shown that RPE-1 cells harboring aneuploid karyotypes are genomically unstable and arrest their proliferation about 2 - 3 cell cycles after the first faulty mitosis10. However, this kinetics might vary among different cell lines. Therefore, it is crucial to perform pilot experiments to determine the proper timing for the removal of aneuploid cycling cells.</li>
	<li>On day 7, 12 h after nocodazole treatment, aspirate medium and add 3 mL of 1x PBS to the plate. Shake off the mitotic cells by tapping the side of the plate. Rotate the plate between each tap to allow even removal of mitotic cells.</li>
	<li>To ensure complete removal of mitotic cells, repeat the shake-off process for multiple rounds (3 - 5 times recommended), aspirating and adding 3 mL of 1x PBS between each round.</li>
	<li>After shake-off, gently aspirate any liquid that adhered to the lid or the edge of the plate. Briefly, check the plate under a microscope at 10X magnification. Ensure that few mitotic cells are observed. If more than five mitotic cells are seen under 10X objective, repeat another round of shake-off. 
	NOTE: Mitotic cells would appear rounded and may be partially detached after shake-off. It is crucial to ensure the complete removal of mitotic cells before exposing the cells to the next round of nocodazole treatment. Failure to do so may lead to mitotic cell death and/or result in the generation of tetraploid cells.</li>
	<li>After final shake-off, wash cells once with 5 mL of 1x PBS. Add 8 mL medium containing 330 nM nocodazole into the plate and incubate for 12 h.</li>
	<li>Repeat nocodazole treatment/shake-off (steps 3.1 - 3.5) every 12 h until no mitotic cells are observed after incubation. Usually, 4-5 rounds of treatment/shake-off are recommended. 
	NOTE: The information provided here refer to RPE-1 cells. The number of rounds of treatment/shake-off might vary among different cell lines. To avoid prolonged and unnecessary exposure to nocodazole it is crucial to carefully determine, in a pilot experiment, the number of rounds required for efficient and complete removal of aneuploid cycling cells.</li>
	<li>After 4 - 5 rounds of nocodazole treatment/shake-off (steps 3.1 - 3.6), add 10 mL of regular growth medium and incubate. Allow cells to rest for 12 - 24 h before future manipulation or assay use. 
	NOTE: After final shake-off, cells remaining on the plate are cell cycle arrested and, as shown recently10, harbor complex karyotypes. These cells are referred to as ArCK (Arrested with Complex Karyotype) cells. It is advisable to perform assays on ArCK populations within a week of isolation.</li>
	<li>Optionally, to assess the percentage of arrested cells with complex karyotypes, collect and count cells from each shake-off using a standard cell counter.</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57215/57215fig1.jpg" /> 
Figure 1: Overall schematic of the method used to generate and isolate aneuploid cells with complex karyotype (ArCK) and representative images. (A) Schematic of ArCK cells generation and isolation protocol. (B) Representative images of RPE-1 hTERT cells at each stage of the treatment. The last panel (outlined in red) represents the isolated ArCK cells. Scale bar 100 &#181;m. <a href="//cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57215/57215fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Characterization of ArCK Population
<ol>
	<li>Confirm successful isolation of ArCK population by performing the following assays.
	<ol>
		<li>Asses beta-galactosidase staining (the marker of senescence) using a commercially available kit (see Table of Materials).</li>
		<li>Measure secretion of pro-inflammatory cytokines, such as CCL2, by employing commercially available kits (see Table of Materials).</li>
		<li>Confirm increased levels of the cell cycle inhibitors p53, p21, p16 by Western Blot. 
		NOTE: To properly control these assays, ArCK populations should be compared to both euploid and aneuploid cycling cells. The former is low passage, wild-type RPE-1 hTERT cells exponentially growing. The latter can be obtained by inducing chromosome mis-segregation in RPE-1 hTERT and harvesting cells 72 h after the first aberrant mitosis. To do so, follow the protocol described above from step 1.1 and harvest cells right before the first nocodazole treatment (step 3.1).</li>
	</ol>
	</li>
	<li>Perform single-cell sequencing to assess the karyotype of the arrested cells. 
	NOTE: Single-cell sequencing is a well-established method to determine chromosomal and sub-chromosomal changes across a cell population at the single cell level13. Detailed procedures for how to perform single-cell sequencing can be found elsewhere10.</li>
</ol>

<ol>
	<li>Santaguida, S., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-+and+long-term+effects+of+chromosome+mis-segregation+and+aneuploidy.">Short- and long-term effects of chromosome mis-segregation and aneuploidy.</a> Nature Reviews Molecular Cell Biology. 16 (8), 473-485 (2015).</li><li>Pfau, S. J., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+instability+and+aneuploidy+in+cancer:+from+yeast+to+man.">Chromosomal instability and aneuploidy in cancer: from yeast to man.</a> EMBO reports. 13 (6), 515-527 (2012).</li><li>Gordon, D. J., Resio, B., 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=Causes+and+consequences+of+aneuploidy+in+cancer.">Causes and consequences of aneuploidy in cancer.</a> Nature Reviews Genetics. 13 (3), 189-203 (2012).</li><li>Holland, A. J., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boveri+revisited:+chromosomal+instability,+aneuploidy+and+tumorigenesis.">Boveri revisited: chromosomal instability, aneuploidy and tumorigenesis.</a> Nature Reviews Molecular Cell Biology. 10 (7), 478-487 (2009).</li><li>Meena, J. K., Cerutti, 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=Telomerase+abrogates+aneuploidy-induced+telomere+replication+stress,+senescence+and+cell+depletion.">Telomerase abrogates aneuploidy-induced telomere replication stress, senescence and cell depletion.</a> The EMBO Journal. , 1-14 (2015).</li><li>Ohashi, A., Ohori, 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=Aneuploidy+generates+proteotoxic+stress+and+DNA+damage+concurrently+with+p53-mediated+post-mitotic+apoptosis+in+SAC-impaired+cells.">Aneuploidy generates proteotoxic stress and DNA damage concurrently with p53-mediated post-mitotic apoptosis in SAC-impaired cells.</a> Nature Communications. 6, 7668 (2015).</li><li>Passerini, V., Ozeri-Galai, 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=The+presence+of+extra+chromosomes+leads+to+genomic+instability.">The presence of extra chromosomes leads to genomic instability.</a> Nature Communications. 7, 10754 (2016).</li><li>Sheltzer, J. M., Blank, H. 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=Aneuploidy+drives+genomic+instability+in+yeast.">Aneuploidy drives genomic instability in yeast.</a> Science. 333 (6045), 1026-1030 (2011).</li><li>Zhu, J., Pavelka, N., Bradford, W. D., Rancati, G., Li, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Karyotypic+determinants+of+chromosome+instability+in+aneuploid+budding+yeast.">Karyotypic determinants of chromosome instability in aneuploid budding yeast.</a> PLoS Genetics. 8 (5), e1002719 (2012).</li><li>Santaguida, S., Richardson, 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=Chromosome+Mis-segregation+Generates+Cell-Cycle-Arrested+Cells+with+Complex+Karyotypes+that+Are+Eliminated+by+the+Immune+System.">Chromosome Mis-segregation Generates Cell-Cycle-Arrested Cells with Complex Karyotypes that Are Eliminated by the Immune System.</a> Developmental Cell. 41 (6), 638.e5-651.e5 (2017).</li><li>Santaguida, S., Vasile, E., White, E., Amon, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aneuploidy-induced+cellular+stresses+limit+autophagic+degradation.">Aneuploidy-induced cellular stresses limit autophagic degradation.</a> Genes &amp; Development. 29 (19), 2010-2021 (2015).</li><li>Santaguida, S., Tighe, A., D'Alise, A. M., Taylor, S. S., Musacchio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+role+of+MPS1+in+chromosome+biorientation+and+the+spindle+checkpoint+through+the+small+molecule+inhibitor+reversine.">Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine.</a> The Journal of Cell Biology. 190 (1), 73-87 (2010).</li><li>Gawad, C., Koh, W., Quake, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+genome+sequencing:+current+state+of+the+science.">Single-cell genome sequencing: current state of the science.</a> Nature Reviews Genetics. 17 (3), 175-188 (2016).</li><li>L&#243;pez-Soto, A., Gonzalez, S., L&#243;pez-Larrea, C., Kroemer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunosurveillance+of+Malignant+Cells+with+Complex+Karyotypes.">Immunosurveillance of Malignant Cells with Complex Karyotypes.</a> Trends in cell biology. , 1-4 (2017).</li><li>Watson, E. V., Elledge, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aneuploidy+Police+Detect+Chromosomal+Imbalance+Triggering+Immune+Crackdown!.">Aneuploidy Police Detect Chromosomal Imbalance Triggering Immune Crackdown!.</a> Trends in genetics: TIG. , 1-3 (2017).</li></ol>

The authors have nothing to disclose.

This novel method to generate and enrich for arrested cells with complex karyotypes (ArCK) allows for the study of cells that have multiple chromosome gains or losses and cease to divide. The method setup has been designed to facilitate the isolation of ArCK cells in a quick and reliable way.
The most critical step in this assay is rigorously controlling the timeline of drug treatment and, most importantly, the removal of aneuploid cycling cells. For optimal results, the timing of nocodazole treatment and shake-off is of particular importance to ensure that cycling cells are removed from the plate while they are still rounded and mitotic, preventing the possibility of mitotic cell death or slippage into G1, potentially creating a tetraploid population. It is not recommended that the timing of shake-off deviates more than two hours from the recommended 12-h nocodazole incubation.
Future studies on ArCK cells have the potential to facilitate a deeper understanding of how complex karyotypes affect cell physiology. In particular, a recent study demonstrated that cells of the immune system are able to interact with and trigger immune clearance of ArCK cells10. The method described here provides an excellent starting point for further characterization of ArCK cells, including the clarification of the molecular mechanisms underlying immune clearance in untransformed cells and the study of how oncogenic transformation may bypass this surveillance mechanism, a crucial question in the field14,15.

Errors in the process of chromosome segregation lead to aneuploidy, a condition characterized by a chromosome number that is not a multiple of the haploid complement1,2,3,4. Aneuploid karyotypes trigger replication stress that generates further genomic instability5,6,7,8,9,10, increases karyotype complexity, and ultimately leads to cell cycle arrest of a subpopulation of cells10. The purpose of the method presented here is to generate and separate such a subpopulation from cycling cells. By employing inexpensive tissue culture techniques, this protocol facilitates the isolation and characterization of cell cycle-arrested aneuploid cells with complex karyotypes. These cells are referred to as ArCK (Arrested with Complex Karyotype) cells and their euploid cycling counterparts as controls.
This protocol is the first one to be established for this purpose and allows for the isolation and further study of ArCK lines including, but not limited to, their induced senescence and the senescence-associated secretory phenotype (SASP), their pro-inflammatory features, and their ability to engage with immune cells. The method presented here has been developed in untransformed, immortalized human cells but has not yet been tested in cancer lines. Some transformed cells may be insensitive to cell cycle arrest due to suppression of one or multiple pathways; therefore, further validation should be performed in other cell lines.

<table border="0" cellpadding="0" cellspacing="0" width="953">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">DMEM (Dulbecco&#8217;s Modified Eagle Medium)&#160;</td>
			<td style="width:311px;">Thermofisher</td>
			<td style="width:319px;">11995-073</td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thymidine</td>
			<td>Sigma Aldrich</td>
			<td>T1859</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reversine</td>
			<td>Cayman Chemical Company</td>
			<td>10004412</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nocodazole</td>
			<td>Sigma Aldrich</td>
			<td>M1410</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Anti-p53 antibody</td>
			<td style="width:311px;">Santa Cruz</td>
			<td style="width:319px;">sc-126</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Anti-p21 antibody</td>
			<td style="width:311px;">Santa Cruz</td>
			<td style="width:319px;">sc-6246</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:263px;">Anti-p16 antibody</td>
			<td style="width:311px;">BD</td>
			<td style="width:319px;">554079</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:263px;">Senescence b-Galactosidase Staining Kit</td>
			<td style="width:311px;">Cell Signaling Technology&#160;</td>
			<td style="width:319px;">9860</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proteome Profiler Human Cytokine Array Kit&#160;</td>
			<td style="width:311px;">R&#38;D Systems</td>
			<td style="width:319px;">ARY005B</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation and isolation of cell cycle-arrested cells with complex karyotypes

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly indicate that aneuploidy triggers genome instability, ultimately generating cells with complex karyotypes that arrest their proliferation. Isolation and characterization of cells harboring complex karyotypes are crucial to study the impact of an imbalanced chromosome number on cell physiology. To date, no methods have been established to reliably isolate such aneuploid cells. This paper provides a protocol for the enrichment and analysis of aneuploid cells with complex karyotypes utilizing standard, inexpensive tissue culture techniques. This protocol can be used to analyze several features of aneuploid cells with complex karyotypes including their induced senescence-associated secretory phenotype, pro-inflammatory properties, and ability to interact with immune cells. Because cancer cells often harbor imbalances in chromosome number, it is crucial to decipher how aneuploidy impacts cell physiology in normal cells, with the ultimate goal of uncovering both its pro- and anti-tumorigenic effects.

This method utilizes an in vitro tissue culture system to isolate aneuploid cells with complex karyotypes that arrest their proliferation. This population is referred to as ArCK (Arrested with Complex Karyotypes) cells. Figure 1A shows the scheme of the experiment. Wild-type cycling RPE-1 hTERT cells are synchronized at the G1/S border with thymidine and treated with a Mps1 inhibitor to induce chromosome mis-segregation. The spindle poison nocodazole is added to the cells 72 h after induction of chromosome mis-segregation. 12 h after nocodazole treatment, the aneuploid cells that are still able to proliferate enter mitosis and are trapped in this cell cycle stage due to interference with microtubule polymerization. These trapped cells are then easily removed by gentle shake-off. The shake-off process is repeated 3 - 5 times to ensure complete removal of cycling cells. Figure 1B shows the representative images of RPE-1 cells at each stage of the treatment.
ArCK cells display increased levels of cell cycle inhibitors, such as p53, p21, and p16 compared to euploid and aneuploid cycling cells, as shown in Figure 2A. Additionally, Figure 2B shows positive &#946;-galactosidase staining in ArCK cells in comparison to euploid control cells, a well-established marker for cellular senescence.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57215/57215fig2.jpg" /> 
Figure 2: Representative results utilizing ArCK cells. (A) Western blot assessing cell cycle inhibitor levels in euploid, aneuploid cycling, and ArCK cells. The levels of p53, p21, and p16 were determined by western blot analysis. Actin served as a loading control. (B) &#946;-galactosidase staining assessing the level of senescence in ArCK cells. Senescence-associated &#946;-galactosidase (&#946;-Gal) activity was determined in euploid cells and ArCK cells. Scale bar 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57215/57215fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
This cell cycle arrest is a prominent feature of aneuploid cells with complex karyotypes, as shown by representative karyotype analysis from single-cell sequencing in Figure 3, in which ArCK cells display multiple, random chromosome gains and losses. This finding is in full agreement with previous reports10.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57215/57215fig3.jpg" /> 
Figure 3: Representative single-cell sequencing of ArCK cells. Segmentation plots showing the karyotype of six representative ArCK cells. Segmentation plots show the copy number of all chromosomes from 1 to X relative to a euploid reference on a log2 scale (wild type RPE-1 is a diploid cell line of female origin). Chromosome gains are highlighted in red, chromosome losses in green. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57215/57215fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes at JoVE.com

Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Aneuploidy leads to genome instability, which eventually produces cell cycle-arrested cells with complex karyotypes. This paper provides a simple and convenient method to isolate aneuploid cells with complex karyotypes that cease to divide.

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly ...

generation-isolation-cell-cycle-arrested-cells-with-complex

Nanyang Technological University

Research

JoVE Journal

Cancer Research

10.3K Views.  Massachusetts Institute of Technology. Aneuploidy leads to genome instability, which eventually produces cell cycle-arrested cells with complex karyotypes. This paper provides a simple and convenient method to isolate aneuploid cells with complex karyotypes that cease to divide.

Video: Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Generation of Prostate Cancer Cell Models of Resistance to the Anti-mitotic Agent Docetaxel

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a common mechanism of disease progression and a prognostic-determinant feature of malignant tumors. In prostate cancer (PC), resistance to MTAs such as the taxane Docetaxel dictates treatment failure as well as progression towards lethal stages of disease that are defined by a poor prognosis and high mortality rates. Though studied for decades, the array of mechanisms contributing to acquired resistance are not completely understood, and thus pose a significant limitation to the development of new therapeutic strategies that could benefit patients in these advanced stages of disease. In this protocol, we describe the generation of Docetaxel-resistant prostate cancer cell lines that mimic lethal features of late-stage prostate cancer, and therefore can be used to study the mechanisms by which acquired chemoresistance arises. Despite potential limitations intrinsic to a cell based model, such as the loss of resistance properties over time, the Docetaxel-resistant cell lines produced by this method have been successfully used in recent studies and offer&#160;the opportunity to advance our molecular understanding of acquired chemoresistance in lethal prostate cancer.

Resistance to cancer therapies contributes to disease progression and death. Determining the mechanistic underpinnings of resistance is crucial for improving therapeutic response. This manuscript details the protocol to generate taxane-resistant cell models of prostate cancer (PC) to help dissecting the pathways involved in progression to Docetaxel resistance in PC patients.

Microtubule targeting agents (MTAs) are a mainstay in the treatment of a wide range of tumors. However, acquired resistance to chemotherapeutic drugs is a ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their antigen presentation and T cell activation capacities. Since most vaccinations of experimental tumor models require millions of DC, they are widely isolated from the bone marrow or spleen. However, these DC significantly differ from blood and tumor DC in their responses to immune complexes (IC), and presumably to other Syk-coupled lectin receptors. Importantly, given the sensitivity of DC to danger-associated molecules, the presence of endotoxins or antibodies that crosslink activation receptors in one of the isolating steps could result in the priming of DC and thus affect the parameters, or at least the dosage, required to activate them. Therefore, here we describe a detailed protocol for isolating MoDC from blood and tumors while avoiding their premature activation. In addition, a protocol is provided for MoDC activation with tumor IC, and their subsequent analyses.

Isolation Protocol of Mouse Monocyte-derived Dendritic Cells and Their Subsequent In Vitro Activation with Tumor Immune Complexes

Monocyte-derived DC (MoDC) can sense minor amounts of danger-associated molecules and are therefore easily primed. We provide a detailed protocol for the isolation of MoDC from blood and tumors and their activation with immune complexes while highlighting key precautions that should be considered in order to avoid their premature activation.

Dendritic cells (DC) are heterogeneous cell populations that differ in their cell membrane markers, migration patterns and distribution, and in their ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of patients with advanced cancer. Adoptive T cell transfer (ACT) of highly enriched tumor antigen-specific T cells has been shown to cause durable regression of metastatic cancer in some patients. However, during expansion, these cells may become exhausted or senescent, limiting their effector function and persistence in vivo. Induced pluripotent stem cell (iPSC) technology may overcome these obstacles by leading to in vitro generation of large numbers of less differentiated tumor antigen-specific T cells. Human iPSC (hiPSC) have the capacity to differentiate into any type of somatic cell, including lymphocytes, which retain the original T cell receptor (TCR) genomic rearrangement when a T cell is used as a starting cell. Therefore, reprogramming of human tumor antigen-specific T cells to hiPSC followed by redifferentiation to T cell lineage has the potential to produce rejuvenated tumor antigen-specific T cells. Described here is a method for generating tumor antigen-specific CD8&#945;&#946;+ single positive (SP) T cells from hiPSC using OP9/DLL1 co-culture system. This method is a powerful tool for in vitro T cell lineage generation and will facilitate the development of in vitro derived T cells for use in regenerative medicine and cell-based therapies.

This article describes a method to generate functional tumor antigen-specific induced pluripotent stem cell-derived CD8&#945;&#946;+ single positive T cells using OP9/DLL1 co-culture system.

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Isolation and Expansion of Cytotoxic Cytokine-induced Killer T Cells for Cancer Treatment

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced killer (CIK) T cell therapy has been reported to exert significant cytotoxic effects against cancer cells and to reduce the adverse effects of surgery, radiation, and chemotherapy in cancer treatments. CIK can be derived from peripheral blood mononuclear cells (PBMCs), bone marrow, and umbilical cord blood. CIK cells are a heterogeneous subpopulation of T cells with CD3+CD56+ and natural killer (NK) phenotypic characteristics that include major histocompatibility complex (MHC)-unrestricted antitumor activity. This study describes a qualified, clinically applicable, flow cytometry-based method for the quantification of the cytolytic capability of PBMC-derived CIK cells against hematological and solid cancer cells. In the cytolytic assay, CIK cells are co-incubated at different ratios with prestained target tumor cells. After the incubation period, the number of target cells are determined by a nucleic acid-binding stain to detect dead cells. This method is applicable to both research and diagnostic applications. CIK cells possess potent cytotoxicity that could be explored as an alternative strategy for cancer treatment upon its preclinical evaluation by a cytometer setup and tracking (CS &#38; T)-based flow cytometry system.

Here, we present a protocol to perform the isolation and expansion of peripheral blood mononuclear cells-derived cytokine-induced CD3+CD56+ killer cells and illustrate their cytotoxicity effect against hematological and solid cancer cells by using an in vitro diagnosis flow cytometry system.

Adoptive cellular immunotherapy focuses on restoring cancer recognition via the immune system and improves effective tumor cell killing. Cytokine-induced ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach.
Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient&#8217;s circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient&#39;s genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques.
In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Decellularization of the Murine Cardiopulmonary Complex

We present here a decellularization protocol for mouse heart and lungs. It produces structural ECM scaffolds that can be used to analyze ECM topology and composition. It is based on a microsurgical procedure designed to catheterize the trachea and aorta of a euthanized mouse to perfuse the heart and lungs with decellularizing agents. The decellularized cardiopulmonary complex can subsequently be immunostained to reveal the location of structural ECM proteins. The whole procedure can be completed in 4 days.
The ECM scaffolds resulting from this protocol are free of dimensional distortions. The absence of cells enables structural examination of ECM structures down to submicron resolution in 3D. This protocol can be applied to healthy and diseased tissue from mice as young as 4-weeks old, including mouse models of fibrosis and cancer, opening the way to determine ECM remodeling associated with cardiopulmonary disease.

This protocol aims to decellularize the heart and lungs of mice. The resulting extracellular matrix (ECM) scaffolds can be immunostained and imaged to map the location and topology of their components.

We present here a decellularization protocol for mouse heart and lungs. It produces structural ECM scaffolds that can be used to analyze ECM topology and ...