Name: preclinical assessment of the bioactivity of the anticancer coumarin ot48 by spheroids, colony formation assays, and zebrafish xenografts
Uploaded: 2018-06-26T13:00:00
Description: Watch this Scientific Journal Video about Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts at JoVE.com

Research at SNU is supported by the National Research Foundation (NRF) by the MEST of Korea for Tumor Microenvironment Global Core Research Center (GCRC) grant, [grant number 2011-0030001]; by the Seoul National University Research Grant and by Brain Korea (BK21) PLUS program.

1. Colony Formation Assays
<ol>
	<li>Culture A549 cells at a concentration of 20,000 cells/cm2 in 15 mL of RPMI1640 cell culture medium supplemented with 10% FBS (Fetal Bovine Serum) and 1% antibiotics in a 75 cm2 flask in a CO2 incubator at 37&#160;&#176;C and 5% CO2.</li>
	<li>On the day of the experiment, determine the cell concentration by using a hemocytometer. Collect 50,000 cells by centrifugation at 400 x g for 7 min in a 1.5 mL microcentrifuge tube and re-su...

<ol>
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M., Stap, J., Haveman, J., van Bree, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonogenic+assay+of+cells+in+vitro.">Clonogenic assay of cells in vitro.</a> Nature Protocols. 1 (5), 2315-2319 (2006).</li><li>Katt, M. E., Placone, A. L., Wong, A. D., Xu, Z. S., Searson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+Vitro+Tumor+Models:+Advantages,+Disadvantages,+Variables,+and+Selecting+the+Right+Platform.">In Vitro Tumor Models: Advantages, Disadvantages, Variables, and Selecting the Right Platform.</a> Frontiers in Bioengineering and Biotechnology. 4, 12 (2016).</li><li>Lee, J. 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=Cytostatic+hydroxycoumarin+OT52+induces+ER/Golgi+stress+and+STAT3+inhibition+triggering+non-canonical+cell+death+and+synergy+with+BH3+mimetics+in+lung+cancer.">Cytostatic hydroxycoumarin OT52 induces ER/Golgi stress and STAT3 inhibition triggering non-canonical cell death and synergy with BH3 mimetics in lung cancer.</a> Cancer Letters. 416, 94-108 (2018).</li><li>Rotem, 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=Alternative+to+the+soft-agar+assay+that+permits+high-throughput+drug+and+genetic+screens+for+cellular+transformation.">Alternative to the soft-agar assay that permits high-throughput drug and genetic screens for cellular transformation.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (18), 5708-5713 (2015).</li><li>Spoerri, L., Beaumont, K. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+cell+culture+techniques+for+cancer+drug+discovery.">Advanced cell culture techniques for cancer drug discovery.</a> Biology (Basel). 3 (2), 345-367 (2014).</li><li>Ji, S., 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+dialkyl+resorcinol+stemphol+disrupts+calcium+homeostasis+to+trigger+programmed+immunogenic+necrosis+in+cancer.">The dialkyl resorcinol stemphol disrupts calcium homeostasis to trigger programmed immunogenic necrosis in cancer.</a> Cancer Letters. 416, 109-123 (2018).</li></ol>

The rates of colony formation obtained with MCBM depends on the cell type. Usually, for non-adherent cells, the number of colonies is much higher compared to adherent cells. We observed that A549 cells formed 30 to 40 colonies after 10 days. Previously we have reported for different leukemia cells that number of colonies is between 200 to 2509. Our results showed that OT48 alone did not produce a significant decrease of the number of colonies alone. However, in combination with BH3 mimetic A121047...

Cancer is caused by cellular mutations and as a consequence biochemical signaling pathways are disrupted triggering uncontrolled cell division and resistance to cell death. Tumors interfere with physiological functions of digestive, nervous, circulatory systems and subsequently neighboring tissues1. Despite extensive research efforts, cancer remains the most prevalent life-threatening disease in the world2. Precision medicine has been recognized as the foundation of future cancer therapeutics. New molecular entities are tested routinely in combination with existing drugs to improve clinical outcome.
However, one of the significant limitations associated with the development of new efficient targeted therapies is the failure of commonly used assays to simulate the exact biological outcome of drug exposure3. Cancer drug discovery still mostly relies on testing the efficacy of therapeutic agents in cancer cell lines cultured in 2D monolayer cultures, which are difficult to validate in clinical trials4. Therefore, there is a growing interest to develop better cancer models that better mimic the native features of tumors in vivo5. In recent years, an increasing interest in 3D culture models resulted in the development of improved methodologies to produce 3D tumor models5.
Here, we present an approach with three different 3D cell culture techniques allowing to improve the understanding of the potency of the hydroxycoumarin OT486 in combination with BH3 mimetics before more in depth in vivo assays. Our method consists of combining colony and SFAs with an in vivo tumor formation test based on a zebrafish model to further validate the efficacy of the combination of OT48 and BH3 mimetics in lung cancer cells and monitor cancer progression in a living organism.
Colony formation assays are routinely used to assess efficacy of anticancer agents for both solid as well as hematopoietic cancers. The assay determines the ability of a cell to proliferate indefinitely and form colonies7. The effect of an anticancer agent on the colony forming ability of cells is determined by decrease of the number and/or size of the colonies.
Spheroids represent in vitro tumor models and serve as a low-cost screening platform for anticancer agents. Spheroids are aggregates of cells growing in suspension or embedded in a 3D matrix. This approach is widely used for drug screening and studies of tumor growth, proliferation and immune interaction8.
To fully understand the properties of a new drug, it is essential to conduct in vivo experiments on rodents. However, this conventional method is expensive and time consuming. In recent years, zebrafish (Danio rerio) became a widely studied laboratory organism that is cheaper and faster to raise. Tumors developed in the zebrafish represent a 3D cell culture approach but within the in vivo setting of a vertebrate9.
Altogether, we use here three different 3D culture approaches including CFAs, SFAs and zebrafish in vivo tumor formation to demonstrate the anticancer capacity of hydroxycoumarin OT48 in a lung cancer A549 cell model in combination with BH3 mimetics.

<table>
	<tbody>
		<tr>
			<td>Materials required for colony formation assays</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A549</td>
			<td>ATCC</td>
			<td>CCL-185</td>
			<td>37 C&#176;</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Lonza</td>
			<td>30096</td>
			<td>4 C&#176;</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Biowest</td>
			<td>S1520-500</td>
			<td>-20 C&#176;</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Lonza</td>
			<td>17-602E</td>
			<td>-20 C&#176;</td>
		</tr>
		<tr>
			<td>Cell culture flask T75</td>
			<td>SPL</td>
			<td>70075</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>PBS solution</td>
			<td>Hyclone</td>
			<td>SH30256.02</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>1.5ml tube</td>
			<td>Extragene</td>
			<td>Tube-170-C</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>15 ml tube</td>
			<td>Hyundai Micro</td>
			<td>H20015</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>12 well plate</td>
			<td>SPL</td>
			<td>30012</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>MethoCult</td>
			<td>StemCell technologies</td>
			<td>4230</td>
			<td>-20 C&#176;</td>
		</tr>
		<tr>
			<td>Thiazolyl Blue Tetrazolium Bromide (MTT powder)</td>
			<td>Sigma</td>
			<td>M5622</td>
			<td>4 C&#176;</td>
		</tr>
		<tr>
			<td>LAS4000</td>
			<td>GE Healthcare Technologies</td>
			<td></td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Materials required for spheroid formation assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A549</td>
			<td>ATCC</td>
			<td>CCL-185</td>
			<td>37 C&#176;</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Lonza</td>
			<td>30096</td>
			<td>4 C&#176;</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Biowest</td>
			<td>S1520-500</td>
			<td>-20 C&#176;</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Lonza</td>
			<td>17-602E</td>
			<td>-20 C&#176;</td>
		</tr>
		<tr>
			<td>Cell culture flask T75</td>
			<td>SPL</td>
			<td>70075</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>PBS solution</td>
			<td>Hyclone</td>
			<td>SH30256.02</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25-300-054</td>
			<td>4 C&#176;</td>
		</tr>
		<tr>
			<td>Corning costar ultra low attachemnt 96 well plate</td>
			<td>Corning</td>
			<td>3474</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Microscopy</td>
			<td>Nikon</td>
			<td>Eclipse TS100</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Materials required for zebrafish xenografts</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A549</td>
			<td>ATCC</td>
			<td>CCL-185</td>
			<td>37 C&#176;</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Lonza</td>
			<td>30096</td>
			<td>4 C&#176;</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Biowest</td>
			<td>S1520-500</td>
			<td>-20 C&#176;</td>
		</tr>
		<tr>
			<td>Penicillin-streptomycin</td>
			<td>Lonza</td>
			<td>17-602E</td>
			<td>-20 C&#176;</td>
		</tr>
		<tr>
			<td>Cell culture flask T25</td>
			<td>SPL</td>
			<td>70025</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Cell culture flask T75</td>
			<td>SPL</td>
			<td>70075</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Cell culture flask T175</td>
			<td>SPL</td>
			<td>71175</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>1.5ml tube</td>
			<td>Extragene</td>
			<td>Tube-170-C</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>SPL</td>
			<td>30024</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Petridish</td>
			<td>SPL</td>
			<td>10100</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>PBS solution</td>
			<td>Hyclone</td>
			<td>SH30256.02</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25-300-054</td>
			<td>4 C&#176;</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>71382</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCL)</td>
			<td>Sigma-Aldrich</td>
			<td>P9541</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate (MgSO4.7H2O)</td>
			<td>Sigma-Aldrich</td>
			<td>M2773</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Calcium nitrate tetrahydrate (Ca(NO3)2)</td>
			<td>Sigma-Aldrich</td>
			<td>C1396</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>HEPES solution</td>
			<td>Sigma-Aldrich</td>
			<td>H0887</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Ethyl 3-aminobenzoate methanesulfonate (Tricaine)</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Phenol Red solution</td>
			<td>Sigma-Aldrich</td>
			<td>P0290</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>Sigma-Aldrich</td>
			<td>M0512</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>1-phenyl-2-thiourea (PTU)</td>
			<td>Sigma-Aldrich</td>
			<td>P7629</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>CM-Dil dye</td>
			<td>Invitrogen</td>
			<td>C7001</td>
			<td>-20 C&#176;</td>
		</tr>
		<tr>
			<td>Glass capillary</td>
			<td>World Precision Instruments</td>
			<td>TW 100F-4</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Shutter instrument, USA</td>
			<td>P-97</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Micro injector</td>
			<td>World Precision Instruments</td>
			<td>PV820</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>KOVAX</td>
			<td>1ml</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Micro loader</td>
			<td>Eppendorf</td>
			<td>5242956003</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>Marienfeld</td>
			<td>HSU-1000612</td>
			<td>RT</td>
		</tr>
		<tr>
			<td>Fluorescence microscopy</td>
			<td>Leica</td>
			<td>DE/DM 5000B</td>
			<td>RT</td>
		</tr>
	</tbody>
</table>

preclinical assessment of the bioactivity of the anticancer coumarin ot48 by spheroids, colony formation assays, and zebrafish xenografts

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines respond to therapeutic compounds in 2D (dimensional) monolayer cell cultures, 3D culture systems were developed to understand the efficacy of drugs in more physiologically relevant models. In recent years, a paradigm shift was observed in pre-clinical research to validate the potency of new molecules in 3D culture systems, more precisely mimicking the tumor microenvironment. These systems characterize the disease state in a more physiologically relevant manner and help to gain better mechanistic insight and understanding of the pharmacological potency of a given molecule. Moreover, with the current trend in improving in vivo cancer models, zebrafish has emerged as an important vertebrate model to assess in vivo tumor formation and study the effect of therapeutic agents. Here, we investigated the therapeutic efficacy of hydroxycoumarin OT48 alone or in combination with BH3 mimetics in lung cancer cell line A549 by using three different 3D culture systems including colony formation assays (CFA), spheroid formation assay (SFA) and in vivo zebrafish xenografts.

In Figure 2, lung cancer cell line A549 was seeded in MCBM to form colonies after treatment with OT48 alone or in combination with BH3 mimetic A1210477 at the indicated concentrations. The results showed that the combination significantly reduced number, size and total surface area of the colonies after 10 days of incubation.
In Figure 3, A549 cells were treated with OT...

Watch this Scientific Journal Video about Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts at JoVE.com

Preclinical Assessment of the Bioactivity of the Anticancer Coumarin OT48 by Spheroids, Colony Formation Assays, and Zebrafish Xenografts

Here, we present the preclinical screening of anticancer coumarins using 3D culture and zebrafish.

In vitro and in vivo pre-clinical screening of novel therapeutic agents are an essential tool in cancer drug discovery. Although human cancer cell lines ...

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