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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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 ...

This method can help answer key questions in the field of drug discovery to identify anti-cancer effect and gain better mechanistic insight into novel molecules using in vitro and in vivo 3D culture systems. The main advantage of this technique is the varieties of pharmacological effect of compounds in a more physiologically relevant in vivo model. To begin, plate 20, 000 A549 cells per square centimeter in 15 milliliters of RPMI 1640 cell culture medium, supplemented with 10%FBS and 1%antibiotics, in a 75 square centimeter flask.Incubate the culture at 37 degrees Celsius and 5%CO2. On the day of the experiment, use a hemocytometer to determine the cell concentration. Collect 50, 000 cells in a 1.5 milliliter microcentrifuge tube by centrifugation at 400g for seven minutes and resuspend the cells in 100 microliters of sterile 1x PBS.Using a multi-pipet, dispense 1.1 milliliters of semi-solid methylcellulose-based medium, or MCBM, into tubes, preparing one tube for each condition. Then, add 110 microliters of FBS to each tube. Next, save 1000 cells per milliliter.Then, pipet 2.4 microliters of the stock cell solution into the tubes of MCBM and FBS. After adding the cells, hold the tubes vertically and vortex them at the highest speed for one minute to thoroughly mix the MCBM and cells. Now, add test compound OT48 alone or in combination with A1210477 to the samples.Prepare a separate tube as a control for the solvent used, such as DMSO. Then, vortex the samples for one minute. For each assay, pipet one milliliter of the mixture into the center well of a 12 well cell culture plate.Then, use one milliliter of sterile water or PBS to fill the empty wells. Incubate the plates at 37 degrees Celsius and 5%CO2 for 10 days. Then, add 200 microliters of a five milligram per milliliter MTT stock solution to each well.After incubating the plates for ten minutes, check for viable colonies which will turn violet. Using a white background plate, capture images using the following settings:For method, choose digitize. For exposure type, choose precision.For exposure time, choose manual and one second. For sensitivity and resolution, choose high resolution. Then, save the captured images in TIFF format.With ImageJ software, quantify viable colonies by selecting menu image, type, 8-bit. Select menu adjust and threshold. Set the threshold to control and keep it constant for all conditions.Then, select menu analyze, analyze particles. Save the data and analyze it with a statistic software for graphical representation. After preparing stock reagents according to the text protocol, separate one female and one male zebrafish in a partition tank filled with UV sterilized and filtered tap water at 28.5 degrees Celsius and place them in the dark.After 24 hours of separation, remove the partition to initiate mating. Transfer fertilized eggs from the mating tank to a Petri dish containing five milliliters of fresh Danios solution. Use five milliliters of Danios solution to wash the eggs three times by using a pipet to carefully aspirate the eggs and transfer them to five milliliters of fresh solution.Then, incubate the eggs at 28.5 degrees Celsius for eight hours. Change to Danios solution containing 0.03%PTU to inhibit pigmentation and sort out opaque unfertilized eggs to prevent contamination. 24 hours post-fertilization, use sharp forceps to dechorionate the embryos.Then, incubate the embryos in Danios solution with PTU up to 48 hours post-fertilization. After seeding A549 cells and incubating them with compounds according to the text protocol, 24 hours before the end of treatment, add four micromolar of the fluorescent cell tracker dye, CM-Dil, to stain the cells and incubate them for two hours. Following the incubation, use 0.05%Trypsin-EDTA to trypsinize the cells.Then, dilute ten to the sixth cells in 50 microliters of phenol red PBS solution prior to injection. To carry out microinjection of cancer cells, pull a 1.0 millimeter glass capillary using the following program settings:Use a syringe to cut the capillary to produce a sharp edge. Then, load the microcapillary with 20 microliters of the cell phenol red solution.After anesthetizing zebrafish according to the text protocol, inject 100 to 200 cells into the yolk sack by injecting six to ten nanoliters via three to five injections. Microinjections should be done very carefully and precisely to ensure that the correct volume of the cells is injected and injection injury is minimal and the carbon dioxide pressure should be optimal to avoid a fissure laterally. After injection, allow the fish to recover for ten to 30 minutes in five milliliters of fresh Danios solution containing PTU.Transfer the fish into 24 well plates with one milliliter of Danios PTU solution, and incubate them at 28.5 degrees Celsius for 72 hours. Following the incubation, after anesthetizing the fish according to the text protocol, immobilize them on a glass slide with a drop of 3%methyl cellulose. Take 5x fluorescent images at a wavelength of 620 to 750 nanometers.Use ImageJ software to quantify the size of fluorescence tumors by selecting analyze and measure. Then, save the fluorescence values and analyze them with a statistics software for graphical representation. In this experiment, 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 concentration seen here.The results showed that the combination significantly reduced the number, size and total surface area of the colonies after ten days of incubation. Here, after treatment with OT48 alone or in combination with BH3 mimetic, A1210477, the cells were allowed to form spheroids using the U-bottom plate technique. Following a three day incubation, the spheroids were imaged and quantified, and 3D images were generated.This figure illustrates the inhibitory potential of OT48 and/or A1210477 on tumor formation from the quantification of fluorescent tumors generated when treated cells were injected into the zebrafish yolk sack. While attempting this procedure, it is important to remember to keep the number of fish for each condition around ten or more to avoid the important variations affecting the statistical evaluation of the data. Following this procedure, other methods like histological analysis can be performed in order to asses target protein expression in zebrafish xenograft.This technique paves the way for researchers in the field of drug discovery to explore the efficacy of compounds in an in vivo micro-environment in zebrafish xenograft. After watching this video, you should have a good understanding of how to validate drug efficacy in a 3D culture environment.

Research

JoVE Journal

Cancer Research

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

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

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

Immunofluorescence Microscopy of &#947;H2AX and 53BP1 for Analyzing the Formation and Repair of DNA Double-strand Breaks

DNA double-strand breaks (DSB) are serious DNA lesions. Analysis of the formation and repair of DSB is relevant in a broad spectrum of research areas ...

Two-dimensional Gel Electrophoresis Coupled with Mass Spectrometry Methods for an Analysis of Human Pituitary Adenoma Tissue Proteome

Human pituitary adenoma (PA) is a common tumor that occurs in the human pituitary gland in the hypothalamus-pituitary-targeted organ axis systems, and may ...

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

Potentiation of Anticancer Antibody Efficacy by Antineoplastic Drugs: Detection of Antibody-drug Synergism Using the Combination Index Equation

Potentiation of hostile monoclonal antibodies (mAb) by chemotherapeutic agents constitutes a valuable strategy for designing effective and safer therapy ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly ...