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
<ol>
	<li>Thaw pr...

<ol>
	<li>Lee, L. M., Seftor, E. A., Bonde, G., Cornell, R. A., Hendrix, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fate+of+human+malignant+melanoma+cells+transplanted+into+zebrafish+embryos:+assessment+of+migration+and+cell+division+in+the+absence+of+tumor+formation.">The fate of human malignant melanoma cells transplanted into zebrafish embryos: assessment of migration and cell division in the absence of tumor formation.</a> Developmental Dynamics. 233 (4), 1560-1570 (2005).</li><li>Topczewska, J. 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. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Leukaemia+xenotransplantation+in+zebrafish--chemotherapy+response+assay+in+vivo.">Leukaemia xenotransplantation in zebrafish--chemotherapy response assay in vivo.</a> British Journal of Haematology. 153 (6), 786-789 (2011).</li><li>Rennekamp, A. J., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=15+years+of+zebrafish+chemical+screening.">15 years of zebrafish chemical screening.</a> Current Opinion in Chemical Biology. 24, 58-70 (2015).</li><li>Ghotra, V. P., 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=Automated+whole+animal+bio-imaging+assay+for+human+cancer+dissemination.">Automated whole animal bio-imaging assay for human cancer dissemination.</a> PLoS One. 7 (2), 31281 (2012).</li><li>Chang, T. -. Y. Y., Pardo-Martin, C., Allalou, A., W&#228;hlby, C., Yanik, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+automated+cellular-resolution+vertebrate+screening+platform+with+parallel+animal+processing.">Fully automated cellular-resolution vertebrate screening platform with parallel animal processing.</a> Lab On a Chip. 12 (4), 711-716 (2012).</li><li>Pardo-Martin, C., 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=High-throughput+in+vivo+vertebrate+screening.">High-throughput in vivo vertebrate screening.</a> Nature Methods. 7 (8), 634-636 (2010).</li><li>Pulak, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tools+for+automating+the+imaging+of+zebrafish+larvae.">Tools for automating the imaging of zebrafish larvae.</a> Methods. 96, 118-126 (2016).</li><li>Henn, K., Braunbeck, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dechorionation+as+a+tool+to+improve+the+fish+embryo+toxicity+test+(FET)+with+the+zebrafish+(Danio+rerio).">Dechorionation as a tool to improve the fish embryo toxicity test (FET) with the zebrafish (Danio rerio).</a> Comparative Biochemistry and Physiology Part C: Toxicology &amp; Pharmacology. 153 (1), 91-98 (2011).</li><li>White, R. 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=Transparent+adult+zebrafish+as+a+tool+for+in+vivo+transplantation+analysis.">Transparent adult zebrafish as a tool for in vivo transplantation analysis.</a> Cell Stem Cell. 2 (2), 183-189 (2008).</li><li>Paatero, I., Alve, S., Gramolelli, S., Ivaska, J., Ojala, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+Embryo+Xenograft+and+Metastasis+Assay.">Zebrafish Embryo Xenograft and Metastasis Assay.</a> Bio-Protocol. 8 (18), (2018).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> Journal of Visualized Experiments. (25), e1115 (2009).</li><li>Cold Spring Harbor Laboratory Press. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E3+medium+(for+zebrafish+embryos).">E3 medium (for zebrafish embryos).</a> Cold Spring Harbor Protocols. 2011 (10), (2011).</li><li>Wertman, J., Veinotte, C. J., Dellaire, G., Berman, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zebrafish+Xenograft+Platform:+Evolution+of+a+Novel+Cancer+Model+and+Preclinical+Screening+Tool.">The Zebrafish Xenograft Platform: Evolution of a Novel Cancer Model and Preclinical Screening Tool.</a> Advances in Experimental Medicine and Biology. 916, 289-314 (2016).</li><li>Tang, Q., 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=Optimized+cell+transplantation+using+adult+rag2+mutant+zebrafish.">Optimized cell transplantation using adult rag2 mutant zebrafish.</a> Nature Methods. 11 (8), 821-824 (2014).</li><li>Moore, J. C., 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=Single-cell+imaging+of+normal+and+malignant+cell+engraftment+into+optically+clear+prkdc-null+SCID+zebrafish.">Single-cell imaging of normal and malignant cell engraftment into optically clear prkdc-null SCID zebrafish.</a> The Journal of Experimental Medicine. 213 (12), 2575-2589 (2016).</li><li>Yan, C., 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=Visualizing+Engrafted+Human+Cancer+and+Therapy+Responses+in+Immunodeficient+Zebrafish.">Visualizing Engrafted Human Cancer and Therapy Responses in Immunodeficient Zebrafish.</a> Cell. 177 (7), 1903-1914 (2019).</li><li>Kawahara, G., 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=Drug+screening+in+a+zebrafish+model+of+Duchenne+muscular+dystrophy.">Drug screening in a zebrafish model of Duchenne muscular dystrophy.</a> Proceedings of the National Academy of Sciences of United States of America. 108 (13), 5331-5336 (2011).</li></ol>

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">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<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 ...

Our zebrafish xenograft drug screening workflow allows for automated imaging and quantification of human cancer cells and their response to drug treatment. It can be used to rapidly test the response of a patience's cancer cells to known drugs or to identify new anticancer compounds. Using zebrafish xenograph models and drug screening allows for Inviva replicant numbers that were previously only obtainable with in vitro systems.It is also much more cost effective than mouse models at screening through large libraries of compounds. This workflow has several steps from interjection of cancer cells in zebrafish to imaging of drug response that are learned best by visual demonstration. To begin, centrifuge a minimum of two times 10 to the sixth cells in five millimeters of PBS at 200 times g for five minutes, and aspirate the supernatant.Resuspend the cell palette in five millimeters of dii staining solution. Incubate the cells at 37 degrees Celsius protected from light for 20 minutes. Then vortex gently and incubate the cells on ice for 15 minutes protected from light.Then centrifuge the cells at 200 times g for five minutes and aspirate the supernatant. Wash the cells by adding five milliliters of PBS, centrifuging at 200 times g for five minutes, and aspirating the supernatant. Repeat the wash one additional time.Resuspend the cells in one microliter of PBS per 250 thousand live cells and transfer them to a 1.5 millimeter micro centrifuge tube. Keep the resuspended cells on ice in the dark. Prior to staining cells and injecting, make agarose plates for injecting by pouring 25 millileiers of three percent agarose in 1X TBE into a petri dish and allow it to solidify.Store the plates at four degrees Celsius for up to two weeks. Also prior to staining cells and injecting, dechlorinate two day post fertilization zebra fish using forceps under a dissecting microscope. Pull from the opposite ends of the protective chorion of the zebra fish with forceps until the chorion tears and the zebra fish becomes unenveloped.To prevent clumping of cells during microinjection, pre chill non filamentous borosilicate glass needles at four degrees Celsius or on ice. Immediately after staining cells, use a microloader pipette tip to load five microliters of stained cells into the chilled needle. Load the needle into the microinjector arm.Level the needle tip using a sterile razor blade. Using a stage micrometer, measure the droplet size in mineral oil. Keep the droplet volume consistently at two nanoliters, which is around 1.5 millimeters in diameter.One minute after anesthesia, transfer the anesthetized larvae to a prepared flat surface injection plate with three percent agarose, and inject the larvae with one pump of stained cells at the desired injection site. All injections should be completed within one to three hours after staining the cells to prevent clumping of the needle. Wash the larvae off the injection plate into a 10 centimeter petri dish containing E3 media without methylene blue and incubate at 28 degrees Celsius for a one hour recovery period.Move the dish of the injected larvae into a 34 degree Celsius incubator. With an empty petri dish placed between the shelf and the petri dish of larvae to act as a buffer. At 48 hour post injection, screen zebra fish larvae for fluorescent tumor cell engraftment and health.Remove any dead or malformed zebra fish, and select zebra fish with similar engraftment. Remove unengrafted zebra fish. For yolk injected fish, remove fish where borders of the yoke cannot be seen around the engrafted cell mass.For pericardium injected fish, remove fish where injected cell mass encroaches into the yolk sack. To transfer the selected fish to a 96-well plate, first cut the tip off a 200 microliter pipette tip, just large enough for a four day post fertilization zebra fish to fit through. Aspirate 150 microliters of E3 media with one zebra fish from the plate using the P200 pipette and add it to an empty well of a flat bottom 96-well plate.Add 150 microliters of 2X diluted drug solution to each well containing zebra fish larvae to reach a final volume of 300 microliters with 1X drug solution per well. Incubate the plate at 34 degrees Celsius. Check for dead zebra fish daily.After two days, if desired, refresh the drug by replacing 200 microliters of liquid from each well with 1X drug solution or DMSO in E3 media. In the Zen blue software, remove all unwanted fluorescent channels in the imaging software and add the Dii channel. Check the desired fluorescent channel.In the same window, select how the images will be taken, Z stacks, automation, loops and series, et cetera. Image the DMSO control fish before the drug treated fish. And set the appropriate exposure time in the imaging software.To quantify the fluorescence, open imagej software. Go to file, open, and select the desired CZI file. The software brings up an import options window.For stack viewing, select hyperstacks, check open files individually, check auto scale, and check split channels. For the color option, select colorized. Then click plugins, macros, record.Click image, adjust, threshold. Select image type as red in the drop down menu on the right side of the threshold window. Adjust the minimum threshold until the software is only highlighting areas with fluorescence, and then click apply.The software converts the photo to black and white with the selected area in black. Click analyze and measure. The software pulls up a results window containing the fluorescent area for that image.Click create on the macro recorder window. This opens up a new window with the code for the macro. Highlight all of the desired images for analysis and open according to stacking viewing or color option.Select run on the window with the macro. The results window now contains the area for each image. In this protocol, at 48 hour post injection, xenografted fish were screened for fluorescently labeled tumor cells and treated with chemotherapy or DMSO.Overall, xenografted fish treated with Vincristine showed the largest and most consistent decrease in xenografted cell mass compared to DMSO treated fish. Dexamethasone treated fish showed about half the reduction in tumor area compared to Vincristine. But still showed a reduction in tumor area compared to DMSO.This procedure can be used to screen any cancer cell line or patient sample for response to different drugs or specific targeted therapies giving insight into each patient's tumor.

drug-screening-primary-patient-derived-tumor-xenografts

Research

JoVE Journal

Cancer Research

7.8K vistas.  University of Kentucky. Nuestro flujo de trabajo de detección de fármacos de xenoinjerto de peces cebra permite obtener imágenes automatizadas y cuantificar las células cancerosas humanas y su respuesta al tratamiento farmacológico. Se puede utilizar para probar rápidamente la respuesta de las células cancerosas de una paciencia a fármacos conocidos o para identificar nuevos compuestos contra el cáncer. El uso de modelos de xenografo de peces cebra y cribado de fármacos permite números replicantes de Invi...