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 primary patient peripheral blood mononuclear cells (PBMCs) from frozen stock in a 37 &#176;C water bath. Immediately after cells have thawed, transfer cells in their freezing media (90% FBS + 10% dimethyl sulfoxide [DMSO]) to a 15 mL conical tube with slow pipetting, avoiding air bubbles. Add 10 mL of prewarmed 37 &#176;C thawing media (25% fetal bovine serum [FBS] in Iscove&#8217;s modified Dulbecco&#8217;s medium [IMDM]) dropwise (approximately 2&#8722;3 s per mL) to the cells in the 15 mL conical tube. 
	NOTE: PBMCs were collected from patient blood samples at the time of diagnosis. The buffy coat was separated by density centrifugation and cells were washed 2x in RPMI 1640 + 10% FBS. Cells were counted and 107 cells were frozen per cryovial in 1 mL of freezing media, and stored at -80 &#176;C.</li>
	<li>Centrifuge cells at 100 x g for 10 min and aspirate media from the cell pellet. Repeat the addition of thaw media, centrifugation, and aspiration one additional time to remove any residual DMSO.</li>
	<li>Resuspend cells in 5 mL of phosphate-buffered saline (PBS) and remove 10 &#956;L for counting on an automated cell counter or hemocytometer. Add 10 &#956;L of trypan blue to 10 &#956;L of cells removed for counting. Count number of cells per mL and record to later calculate the volume to resuspend cells in for xenografting (see step 2.5). 
	NOTE: Typically, 500 cells are xenografted per larval zebrafish. For example, 5 x 105 cells are needed to inject 1,000 zebrafish. Viability should be &#62;85% to use for xenografting. In this experiment, cell viability was 96% after thawing, assessed by trypan blue staining.</li>
</ol>
2. Fluorescently Labeling Cells with DiI
<ol>
	<li>Centrifuge the desired cell number in 5 mL of PBS at 200 x g for 5 min and aspirate supernatant. Stain a minimum of 2 x 106 cells in case of clumping or problems loading the needle during injection.</li>
	<li>Make 1,1&#8217;-dioctadecyl-3,3,3&#8217;,3&#8217;-tetramethylindocarbocyanine perchlorate (DiI) staining solution (5 mL of PBS containing 4 &#181;L per mL of DiI stain, Table of Materials) and resuspend the cell pellet in the staining solution. 
	NOTE: Cell density should not exceed 2 x 106 cells/mL when resuspended in the staining solution.</li>
	<li>Incubate cells at 37 &#176;C protected from light for 20 min, vortex gently, then incubate cells on ice for 15 min protected from light.</li>
	<li>Centrifuge cells at 200 x g for 5 min and aspirate supernatant. Wash cells with 5 mL of PBS, centrifuge at 200 x g for 5 min and aspirate supernatant. Repeat wash, centrifugation, and aspiration one additional time.</li>
	<li>Resuspend cells in 1 &#181;L of PBS per 250,000 live cells and transfer to a 1.5 mL microcentrifuge tube. Keep resuspended cells on ice in the dark and immediately continue to microinjections. 
	NOTE: This ensures that 500 patient cells are injected into the zebrafish with each injection pump of 2 nL volume.</li>
</ol>
3. Microinjecting Zebrafish Larvae
NOTE: Microinjections should be completed within 1&#8722;3 h of staining to improve viability of cells.
<ol>
	<li>Prior to staining cells and injecting, make agarose plates for injecting by pouring 25 mL of 3% agarose in 1x Tris/borate/EDTA (TBE) into a Petri dish and allow it to solidify. Store plates at 4 &#176;C for up to 2 weeks.</li>
	<li>Also prior to staining cells and injecting, dechorionate 2 dpf zebrafish using forceps under a dissecting microscope. For manual dechorionation, pull from opposite ends of the protective chorion of the zebrafish with forceps until the chorion tears and the zebrafish becomes unenveloped10. 
	NOTE: Dechorionation can also be performed by using enzymatic treatment with pronase, as previously described10. Casper (roy-/-;nacre-/-) zebrafish were used for these experiments11. Any zebrafish larval strain can be used for xenografting. If pigment interferes with imaging or visualization, propylthiouracil (PTU) treatment can be used to block melanin synthesis if optically clear zebrafish strains are not available12.</li>
	<li>Prechill needles at 4 &#176;C or on ice to prevent clumping of cells during microinjection. Load 5 &#181;L of stained cells into a chilled nonfilamentous borosilicate glass needle using microloader pipette tips. 
	NOTE: Microinjector and needle setup methods have been previously published13.</li>
	<li>Load the needle into the microinjector arm. Bevel the needle tip using a sterile razor blade. Measure the droplet size in mineral oil using a stage micrometer, keeping droplet volume consistently at 2 nL (~0.15 mm diameter) throughout.</li>
	<li>Use 350 &#181;L of 4 mg/mL tricaine-S to anesthetize ~30 dechorionated 2 dpf zebrafish in a Petri dish containing 25 mL of E3 media. After ~1 min transfer anesthetized larvae to a flat-surface injection plate (3% agarose in a Petri dish) and inject larvae with one pump of stained cells at the desired injection site (e.g., the yolk or the pericardium; see Figure 2A,B). 
	NOTE: Injection site should be chosen based on the goal of the experiment. The most common injection site is the yolk. To get cells circulating in the bloodstream, the pericardium, duct of Cuvier, perivitelline space, or retro-orbital space can be used as injection sites. Orthotopic injection sites can also be used, such as the brain.</li>
	<li>Wash larvae off the injection plate into a 10 cm2 Petri dish (30 larvae per plate) containing E3 media14 without methylene blue and incubate at 28 &#176;C for a 1 h recovery period. Continue injecting until a desired number of larvae have been injected. 
	NOTE: Ideally, inject 2&#8722;2.5-fold the number of larvae needed for experiments. There will be some die-off of larvae due to stress from injection and the increased incubation temperature. Typically, after practice with the technique, 800&#8722;1,500 zebrafish larvae can be injected by a single person within the 1&#8722;3 h when stained cells should be injected.</li>
	<li>Move plates of injected larvae to a 34 &#176;C incubator. Do not place the Petri dishes of larvae directly on a metal shelf in the incubator to prevent overheating of the E3 water. For example, place an empty Petri dish between the shelf and Petri dish of larvae to act as a buffer. Remove dead zebrafish larvae after 24 and 48 hours post injection (hpi).</li>
</ol>
4. Setting Up Drug Screen with Xenografted Zebrafish
<ol>
	<li>At 48 hpi, screen zebrafish larvae for fluorescence/tumor engraftment and health (Figure 2C,D). Remove any dead or malformed zebrafish and select zebrafish with similar engraftment (Figure 2C,D, 1&#8722;3 and 1&#8217;&#8722;3&#8217;). Remove unengrafted zebrafish (Figure 2C,D, 5 and 5&#8217;).
	<ol>
		<li>For yolk injected fish, remove fish where borders of the yolk cannot be seen around engrafted cell mass (Figure 2C, 4) as it makes quantification difficult. For pericardium injected fish, remove fish where injected cell mass encroaches into the yolk sac (Figure 2D, 4&#8217;).</li>
	</ol>
	</li>
	<li>Add zebrafish to a 96-well plate. To do this, cut the tip off of a 200 &#181;L pipette tip, just large enough for a 4 dpf zebrafish to fit through. Aspirate 150 &#181;L of E3 media with one zebrafish from the plate using a P200 pipette, and add to an empty well of a flat-bottom 96-well plate.</li>
	<li>Dilute the drug to be tested in the required volume of E3 media, at 150 &#181;L per well. Prepare drug at 2-fold the desired concentration. For example, prepare 20 &#181;M of drug in E3 if the desired final concentration is 10 &#181;M, since half of the total volume in each well is comprised of the drug solution. For the DMSO control group, add DMSO at the same volume as the drug.</li>
	<li>Add 150 &#181;L of 2x diluted drug solution to each well containing zebrafish larvae in 150 &#181;L of E3, for a final volume of 300 &#181;L with 1x drug solution per well.</li>
	<li>Incubate the plate at 34 &#176;C. Check for dead zebrafish daily. After 2 days, if desired, refresh the drug by removing 200 &#181;L of liquid from each well of the 96-well plate and replacing with 200 &#181;L of 1x dilute drug solution or DMSO in E3 media. 
	NOTE: The best results were found after 3 days of drug treatment for this experiment; however, the length of drug treatment can vary between 2&#8722;4 days and may need to be optimized based on the experiment being done or drugs being used.</li>
</ol>
5. Imaging Xenografted Zebrafish Using a Fluorescence Microscope Equipped Imaging Unit and Automated Sampler Unit
<ol>
	<li>Prepare 1 L of fresh 4 mg/mL tricaine and 1.5 L of E3 media. Fill media bottle 1 with E3 media and media bottle 2 with tricaine.</li>
	<li>Remove all unwanted fluorescent channels in the imaging software and add the desired channel (DiI for this experiment). Check the desired fluorescent channel as an image will only be taken for the channels with a check mark. Also, select how the images will be taken (z-stacks, automation, loops in series, etc.). 
	NOTE: For this experiment, the focus was manually set for each fish imaged to obtain the highest number of images with optimal focus.</li>
	<li>Image the DMSO control fish before the drug-treated fish so that the appropriate exposure time can be set in the imaging software. Once the exposure is set, do not change the exposure time for the duration of the experiment. 
	NOTE: The focus can either be adjusted manually for each zebrafish to ensure there are no out of focus images, or for fully automated imaging, the same focus can be used between fish with out of focus images being discarded or fish reimaged prior to performing analysis. Furthermore, this experiment could be conducted by using any fluorescent imager followed by quantification of fluorescence using the ImageJ software.</li>
</ol>
6. Quantifying Fluorescence Using ImageJ
<ol>
	<li>Open ImageJ software.</li>
	<li>Go to File | Open and select the desired .czi file. The software will bring up an import options window.
	<ol>
		<li>For stack viewing select Hyperstacks, check Open Files Individually, check Autoscale, and check Split Channels. For the color option select Colorized.</li>
	</ol>
	</li>
	<li>Click Plugins | Macros | Record.</li>
	<li>Click Image | Adjust | Threshold. Select image type as red in the dropdown menu on the right side of the threshold window. Adjust the minimum threshold until the software is only highlighting areas with fluorescence (Figure 3A) and click Apply. The software will convert the photo to black and white, with the selected area in black. 
	NOTE: Use the same threshold for each image in the drug screen to keep results standardized and comparable.</li>
	<li>Click Analyze | Measure. The software will pull up a results window containing the fluorescent area for that image.</li>
	<li>Click Create on the Macro Recorder window. This will open up a new window with the code for the macro. Highlight all of the desired images for analysis and open as in step 6.2.</li>
	<li>Select Run on the window with the macro. The results window will now contain the area for each image. 
	NOTE: The image analysis can be done individually without recording and running a macro as well as by repeating the above steps for each image.</li>
	<li>Copy the measured data into a spreadsheet. Average the total fluorescence of all control (DMSO) samples. Calculate the percent difference using the following formula: &#8211;[(average DMSO area &#8211; experimental area)/average DMSO area] x 100% (Figure 3B).</li>
</ol>

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The authors have nothing to disclose.

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 ...

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

Research

JoVE Journal

Cancer Research

7.9K Views.  University of Kentucky. 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.

Video: Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

Flow Cytometry to Estimate Leukemia Stem Cells in Primary Acute Myeloid Leukemia and in Patient-derived-xenografts, at Diagnosis and Follow Up

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in adults. Initially, AML is a disease of hematopoietic stem cells (HSC) characterized by arrest of differentiation, subsequent accumulation of leukemia blast cells, and reduced production of functional hematopoietic elements. Heterogeneity extends to the presence of leukemia stem cells (LSC), with this dynamic cell compartment evolving to overcome various selection pressures imposed upon during leukemia progression and treatment. To further define the LSC population, the addition of CD90 and CD45RA allows the discrimination of normal HSCs and multipotent progenitors within the CD34+CD38- cell compartment. Here, we outline a protocol to detect simultaneous expression of several putative LSC markers (CD34, CD38, CD45RA, CD90) on primary blast cells of human AML by multiparametric flow cytometry. Furthermore, we show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts. This method of detection and quantification of putative LSC may be used for clinical follow-up of chemotherapy response (i.e., minimal residual disease), as residual LSC may cause AML relapse.

We outline a protocol to detect simultaneous expression of leukemia stem cell markers on primary acute myeloid leukemia cells by flow cytometry. We show how to quantify three progenitor populations and a putative LSC population with increasing degree of maturation. We confirmed the presence of these populations in corresponding patient-derived-xenografts.

Acute myeloid leukemia (AML) is a heterogeneous, and if not treated, fatal disease. It is the most common cause of leukemia-associated mortality in ...

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 ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 &#176;C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

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 zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

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 ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Patient-Derived Tumor Explants As a "Live" Preclinical Platform for Predicting Drug Resistance in Patients

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable preclinical models that can accurately predict patient responses. One of the disadvantages of existing preclinical models is the inability to contextually preserve the human tumor microenvironment (TME) and accurately represent intratumoral heterogeneity, thus limiting the clinical translation of data. By contrast, by representing the culture of live fragments of human tumors, the patient-derived explant (PDE) platform allows drug responses to be examined in a three-dimensional (3D) context that mirrors the pathological and architectural features of the original tumors as closely as possible. Previous reports with PDEs have documented the ability of the platform to distinguish chemosensitive from chemoresistant tumors, and it has been shown that this segregation is predictive of patient responses to the same chemotherapies. Simultaneously, PDEs allow the opportunity to interrogate molecular, genetic, and histological features of tumors that predict drug responses, thereby identifying biomarkers for patient stratification as well as novel interventional approaches to sensitize resistant tumors. This paper reports PDE methodology in detail, from collection of patient samples through to endpoint analysis. It provides a detailed description of explant derivation and culture methods, highlighting bespoke conditions for particular tumors, where appropriate. For endpoint analysis, there is a focus on multiplexed immunofluorescence and multispectral imaging for the spatial profiling of key biomarkers within both tumoral and stromal regions. By combining these methods, it is possible to generate quantitative and qualitative drug response data that can be related to various clinicopathological parameters and thus potentially be used for biomarker identification.

This paper describes methods for the generation, drug treatment, and analysis of patient-derived explants for assessing tumor drug responses in a live, patient-relevant, preclinical model system.

An understanding of drug resistance and the development of novel strategies to sensitize highly resistant cancers rely on the availability of suitable ...

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 visualizing the response to anti-cancer therapies (chemo, radiotherapy, and biologicals), angiogenesis and metastasis with single cell resolution, places the zebrafish xenograft model as a top choice to develop preclinical studies.

The zebrafish larval xenograft assay presents several experimental advantages compared to other models, but probably the most striking is the reduction of size scale and consequently time. This reduction of scale allows single cell imaging, the use of a relatively low number of human cells (compatible with biopsies), medium-high-throughput drug screenings, but most importantly enables a significant reduction of the time of the assay. All these advantages make the zebrafish xenograft assay extremely attractive for future personalized medicine applications.

Many zebrafish xenograft protocols have been developed with a wide diversity of human tumors; however, a general and standardized protocol to efficiently generate zebrafish larval xenografts is still lacking. Here we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Here, we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

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 ...