Name: drug screening of primary patient derived tumor xenografts in zebrafish
Uploaded: 2020-04-10T14:00:00
Description: Watch this Scientific Journal Video about Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish at JoVE.com

This research was supported by a V Foundation V Scholar Award and NIH Grants DP2CA228043, R01CA227656 (to J.S. Blackburn) and NIH Training Grant T32CA165990 (to M.G. Haney).

All procedures described in this protocol have been approved by the University of Kentucky&#8217;s Institutional Animal Care and Use Committee (protocol 2015-2225). Patient samples were collected under University of Kentucky&#8217;s Institutional Review Board (protocol 44672). All animal experiments performed following this protocol must be approved by the user&#8217;s Institutional Animal Care and Use Committee.
1. Thawing Primary Patient Acute Lymphoblastic Leukemia Cells
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	<li>Thaw pr...

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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=Embryonic+and+tumorigenic+pathways+converge+via+Nodal+signaling:+role+in+melanoma+aggressiveness.">Embryonic and tumorigenic pathways converge via Nodal signaling: role in melanoma aggressiveness.</a> Nature Medicine. 12 (8), 925-932 (2006).</li><li>Haldi, M., Ton, C., Seng, W. L., McGrath, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+melanoma+cells+transplanted+into+zebrafish+proliferate,+migrate,+produce+melanin,+form+masses+and+stimulate+angiogenesis+in+zebrafish.">Human melanoma cells transplanted into zebrafish proliferate, migrate, produce melanin, form masses and stimulate angiogenesis in zebrafish.</a> Angiogenesis. 9 (3), 139-151 (2006).</li><li>Corkery, D. P., Dellaire, G., Berman, J. 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In this study, we demonstrated a standardized method for thawing and injection of primary patient leukemia cells into zebrafish as a xenograft model. We also established a protocol for high-throughput drug screening of xenografted zebrafish using a fluorescence microscope equipped imaging unit and automated sampler unit. Previously, xenografts have been reported with human cell lines, and quantification of xenografted tumors in a high-throughput manner has been a challenge in the field. This method serves as a basis for ...

Xenografting of primary patient cancers or human cancer cell lines into model organisms is a widely used technique to study tumor progression and behavior in vivo, tumor response to drug treatment, and cancer cell interaction with the microenvironment, among others. Traditionally, cells are xenografted into immune-compromised mice, and this remains the standard in the field. However, this model system has several limitations, such as high cost, low replicate numbers, difficulties in accurately quantifying tumor burden in vivo, and the extended time that it takes for tumors to engraft and drug testing to be completed. In recent years, zebrafish have emerged as an alternate xenograft model, with the first being reported in 2005, with green fluorescent protein (GFP)-labeled human melanoma cell lines transplanted into blastula-stage embryos1,2. More recently, 2 day post-fertilization (dpf) zebrafish larvae have been used as xenograft recipients to allow for control of anatomic location of injection and for use in high resolution in vivo imaging of tumor interaction with the surrounding microenvironment3,4.
Zebrafish offer many advantages as a xenograft model. First, adult zebrafish can be housed and rapidly bred in large quantities at a relatively low cost. Each mating pair of adult zebrafish can produce hundreds of larval fish per week. Due to their small size, these larval zebrafish can be maintained in 96-well plates for high-throughput drug screening. Larvae do not have to be fed during the course of a typical xenograft experiment, as their yolk-sac provides the nutrients to sustain them for their first week of life. Furthermore, zebrafish do not have a fully functional immune system until 7 dpf, meaning that they do not require irradiation or immunosuppressive regimens prior to xenograft injection. Finally, optically clear zebrafish lines allow for high-resolution imaging of tumor-microenvironment interactions.
Perhaps the most promising application of zebrafish as a xenograft model is the ability to perform high-throughput drug screening on human cancer samples in a way that is not possible using any other model organism. Larvae absorb drugs from the water through the skin, enhancing the ease of drug administration5. Because animals are maintained in 96-well plates, typically in 100&#8722;300 &#956;L of water, screens require smaller drug quantities compared to mice. Currently, there are several different methods for standardization and quantification of the effect of drugs on human tumor burden in zebrafish, some of which are more practical than others for scaling-up single drug testing to high-throughput screening. For example, some groups dissociate fish into single cell suspensions, and quantify fluorescently labeled or stained tumor cells by imaging individual droplets of the suspension and quantifying fluorescence using a semi-automated ImageJ macro4. A semi-automated whole-larvae imaging method was developed in which larval fish were fixed in 96-well plates and imaged using an inverted fluorescent microscope before realignment of composite images and quantification of tumor cell foci6. Both of these assays are fairly labor-intensive methods for quantification, which has made truly high-throughput drug screening in zebrafish xenograft models impractical.
This issue has been addressed by the development of the Vertebrate Automated Screening Technology (VAST) Bioimager and Large Particle (LP) Sampler, a fluorescence microscope equipped imaging unit and automated sampler unit (Figure 1 and Table of Materials), which is a truly automated method for high-throughput imaging of zebrafish larvae7,8,9. With this unit, fish are anesthetized, sampled automatically from a 96-well plate, positioned in a capillary and rotated into the set orientation based on a preset user preference, imaged, and then either placed back into the same well of a new 96-well plate for further studies or discarded. Combining this imaging technology with zebrafish xenografts may allow for the possibility of personalized medicine that uses high-throughput drug screening of large drug compound libraries against individual patient tumors. Zebrafish xenografts also offer a large-scale and low-cost method for testing both toxicity and efficacy of novel compounds in vivo. Zebrafish can be used as a preliminary screening step before proceeding to mouse xenograft models.
We have developed a streamlined workflow for xenografting primary patient leukemia cells into zebrafish and performing high-throughput drug screens with automated imaging and quantification, which can be applied to any other primary patient tumor cells or cancer cell line. This workflow utilized a fluorescence microscope equipped imaging unit and automated sampler unit to improve upon current standardization and quantification methods and offers an automated alternative to previous, more labor-intensive methods of quantifying tumor mass in vivo.&#160;

<table border="0" cellpadding="0" cellspacing="0" style="width:901px;" width="901">
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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">10x TBE Liquid Concentrate</td>
			<td style="width:139px;">VWR</td>
			<td style="width:115px;">0658-5L</td>
			<td style="width:360px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">96-well plate, flat bottom</td>
			<td>CELLTREAT</td>
			<td>229195</td>
			<td style="width:360px;">VAST is compatible with a variety of standard or deep well 24, 48, or 96 well plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Agarose</td>
			<td style="width:139px;">Fisher Scientific</td>
			<td style="width:115px;">BP160-500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:288px;">Borosilicate Glass Capillary without Filament</td>
			<td style="width:139px;">Sutter Instrument Company</td>
			<td style="width:115px;">B100-50-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dexamethasone</td>
			<td>Enzo Life Sciences</td>
			<td>BML-EI126-0001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D2438-5X10ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E3 media</td>
			<td></td>
			<td>N/A</td>
			<td>5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Femtotips Microloader Tips</td>
			<td style="width:139px;">Eppendorf</td>
			<td style="width:115px;">930001007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal Bovine Serum (Premium Heat Inactivated)</td>
			<td>Atlanta Biologicals</td>
			<td>S11150H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ</td>
			<td>FIJI</td>
			<td>N/A</td>
			<td>https://imagej.net/Fiji</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Iscove&#39;s Modified Dulbecco&#39;s Medium</td>
			<td>STEMCELL Technologies</td>
			<td>36150</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Large Particle (LP) Sampler</td>
			<td>Union Biometrica</td>
			<td>N/A</td>
			<td style="width:360px;">automated sampler unit http://www.unionbio.com/copas/features.aspx?id=8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methotrexate</td>
			<td>Sigma-Aldrich</td>
			<td>A6770-10MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mineral Oil</td>
			<td>Fisher Scientific</td>
			<td>BP26291</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline (1x)</td>
			<td>Caisson labs</td>
			<td>PBL06-6X500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stage Micrometer (400-Stage)</td>
			<td>Hausser Scientific</td>
			<td>400-S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:288px;">Tricaine-S</td>
			<td style="width:139px;">Pentair Aquatic</td>
			<td style="width:115px;">TRS1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypan Blue</td>
			<td>Thermo Fisher</td>
			<td>T10282</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">VAST Bioimager</td>
			<td>Union Biometrica</td>
			<td>N/A</td>
			<td style="width:360px;">fluorescent equipped microscope imaging unit https://www.unionbio.com/vast/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vincristine Sulfate</td>
			<td>Enzo Life Sciences</td>
			<td>BML-T117-0005</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vybrant DiI Stain</td>
			<td>Thermo Fisher</td>
			<td>V22885</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 standard in the field, but zebrafish have emerged as an alternative model with several advantages, including the ability for high-throughput and low-cost drug screening. Zebrafish also allow for in vivo drug screening with large replicate numbers that were previously only obtainable with in vitro systems. The ability to rapidly perform large scale drug screens may open up the possibility for personalized medicine with rapid translation of results back to clinic. Zebrafish xenograft models could also be used to rapidly screen for actionable mutations based on tumor response to targeted therapies or to identify new anti-cancer compounds from large libraries. The current major limitation in the field has been quantifying and automating the process so that drug screens can be done on a larger scale and be less labor-intensive. We have developed a workflow for xenografting primary patient samples into zebrafish larvae and performing large scale drug screens using a fluorescence microscope equipped imaging unit and automated sampler unit. This method allows for standardization and quantification of engrafted tumor area and response to drug treatment across large numbers of zebrafish larvae. Overall, this method is advantageous over traditional cell culture drug screening as it allows for growth of tumor cells in an in vivo environment throughout drug treatment, and is more practical and cost-effective than mice for large scale in vivo drug screens.

Following the protocol described above, zebrafish were xenografted in the yolk and pericardium with primary patient PBMCs that were originally isolated from a T-cell acute lymphoblastic leukemia (T-ALL) patient at diagnosis and banked as a viable, frozen sample. At 48 hpi, xenografted fish were screened for fluorescently labeled tumor cells (Figure 2C,D) and treated with chemotherapy (dexamethasone or vincristine) or DMSO. Fish were imaged at 7 dpi, after 3 days on drug trea...

Watch this Scientific Journal Video about Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish at JoVE.com

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Zebrafish xenograft models allow for high-throughput drug screening and fluorescent imaging of human cancer cells in an in vivo microenvironment. We developed a workflow for large scale, automated drug screening on patient-derived leukemia samples in zebrafish using an automated fluorescence microscope equipped imaging unit.

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

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