The authors thank Dr. Darrel Kotton at Boston University for providing the pHAGE-EF1aL-dsRed-UBC-GFP-W vector and protocols for high titer lentiviral production used in these studies. This work was funded by DOD BCRP W81XWH-11-1-0101 (DMC), ACS IRG # 57-001-53 (DMC), NCI K22CA181250 (DMC) and R01 CA140985 (CAS).NCI P30CA046934 Center grant supported in vivo imaging and tissue culture cores at University of Colorado AMC.

All steps requiring the use of animals in this protocol follows the guidelines of University of Colorado animal research ethics committee (IACUC).
1. Preparation of Instruments, Culture Media and Other Reagents
<ol>
	<li>Prepare 100 ml mammosphere media containing Dulbecco&#39;s Modified Eagle Medium and Han&#39;s F-12 medium (DMEM/F12) (1:1), basic Fibroblast Growth Factor (bFGF, 20 ng/ml), epidermal growth factor (EGF, 10 ng/ml), Heparin (4 &#181;g/ml), 1x B27, Penicillin (100 U/ml), streptomycin (100 &#181;g/ml). Make media in sterile conditions and store at 4 &#176;C for up to 3 months.</li>
	<li>Prepare epithelial enrichment buffer (EEB) containing PBS pH 7.2, 0.5% Bovine Serum Albumin (BSA), 2 mM EDTA. Filter sterilize and store at 4 &#176;C for up to 6 months.</li>
	<li>Make 5 ml aliquots of digestion buffer in sterile 15 ml conical tubes and store at -20 &#176;C for months. The night before tumor digestion, thaw digestion buffer (5 ml per 500 mg tumor) on ice, at 4 &#176;C. Add 1x antibiotic-antimycotic mix before use.</li>
	<li>Autoclave (121 &#176;C for 30 min) at least two forceps, scalpel and scissors for dissection of PDXs.</li>
	<li>Make 500 ml wash buffer containing DMEM:F12 and 5% Fetal Bovine Serum (FBS). Store at 4 &#176;C for months. Aliquot 10 ml per tumor the day of tumor dissection.</li>
	<li>Prepare a polybrene stock at 4 &#181;g/&#181;l with sterile water. Filter and aliquot into 1.5 ml microcentrifuge tubes containing 100 &#181;l each, store at -20 &#176;C.</li>
</ol>
2. Generation of High Titer Lentiviral Particles Carrying Traceable Markers
<ol>
	<li>Purchase or generate high titer lentiviral particles carrying a reporter gene (i.e., GFP and Luc for in vivo tracking of tumor growth) 18,19. 
	NOTE: A lentiviral titer of &#62; 108 TU/ml (transduction units per milliliter) is recommended for successful transduction of PDXs. The lentiviral particles should be pseudotyped with an envelope G glycoprotein from Vesicular Stomatitis Virus (VSV-G), allowing transduction of a wide variety of mammalian cells. 
	NOTE: The protocol used here for the generation and titration of high titer lentiviral particles is available at <a href="http://www.kottonlab.com">www.kottonlab.com</a>. Vectors used in this protocol are pSIH1-H1-copGFP-T2A-puro and the dual promoter pHAGE-EF1aL-luciferase-UBC-GFP-W. This vector was generated by replacing dsRed with the luciferase gene downstream of EF1aL promoter in the pHAGE-EF1aL-dsRed-UBC-GFP-W vector, a kind gift of Darrell Kotton, Boston University.</li>
</ol>
3. Generation of Patient-derived Xenografts
<ol>
	<li>Generate patient derived xenografts (PDX) from breast cancer by implanting primary or metastatic breast tumors in the mammary fat pad of immunocompromised mice3,4. Generate tumors at a maximum recommended size of 1 cm diameter as larger tumors are likely to contain necrotic cores. 
	NOTE: Detailed methods for establishing and transplanting xenografts are described in DeRose et al. 18. Growth of established-transplantable PDXs into &#62; 1 cm-diameter tumors takes between 4 and 24 weeks after implantation in immunocompromised NOD/SCID/ILIIrg&#8722;/&#8722; (NSG), depending on intrinsic tumor growth rates. While this protocol describes transduction of breast cancer PDXs, it can be used for viral transduction of any tumor suitable to short-term in vitro passage. The sensitivity of PDXs to short-term (24 - 96 hr) in vitro survival is intrinsic to each tumor and must be determined experimentally.</li>
</ol>
4. Tumor Dissection and Dissociation of PDX-derived Tumor Cells
<ol>
	<li>Euthanize mice carrying PDX using CO2 inhalation followed by cervical dislocation (follow institutional guidelines of animal research ethics committee). Submerge mice in a 0.2% Chlorhexidine solution for 1 min. Set mouse on a supine position on top of a dissection board, and use pins to fix the extended upper and lower limbs to the board.</li>
	<li>Using aseptic technique, use a scalpel to cut the skin around the tumor. Pull the skin with a set of forceps and separate it from the tumor using a clean scalpel until the tumor is completely exposed. Using a clean set of forceps and scalpel, remove the tumor and place it in 5 ml wash media on ice.</li>
	<li>Take dissected tumor into a BLS2 cabinet for further processing. Remove media and rinse tumor with 10 ml of Hank&#39;s Balanced Salt Sodium Modified with 10 mM Hepes (HBSS/Hepes), twice.</li>
	<li>Drop the tumor in a sterile pre-weighed 60 cm tissue culture plate. Weigh the plate containing the tumor to estimate tumor mass.</li>
	<li>Using a scalpel, cut the tumor in half, then cut a 3 mm disc from one half and place it in a container with 10% formalin for 24 hr. Use this tissue to verify the heterogeneity of the tumor sample (parental PDX). Add 200 &#181;l of HBSS/Hepes (enough to avoid the tissue to dry out) and mince the remaining tumor in the smallest possible pieces using forceps and a clean scalpel.</li>
	<li>Transfer the minced tumor into a sterile 50 ml conical tube. Add at least 1 ml of digestion buffer containing 1x antimycotic-antibiotic per 100 mg of tumor. 
	NOTE: Addition of antibiotics/antimycotic prevents contamination of tumor cells in vitro.</li>
	<li>Digest tumor for 3 hr at 30 &#176;C, shaking at 125 - 200 rpm. 
	NOTE: Increasing temperature up to 37 &#176;C increases the efficiency of the digestion (more single cells over time) but it is usually accompanied by increased cell death. For most tumors, digestion at 30 &#176;C for 3 hr results in sufficient numbers of viable dissociated cells to proceed to the labeling step.</li>
	<li>Stop the digestion adding 35 ml wash media (DMEM/F12 with 5% FBS). Filter through a 70 &#181;m nylon mesh into a clean 50 ml conical tube to remove undigested tissue. Centrifuge the filtered media containing digested cells at 400 x g for 5 min.</li>
	<li>Aspirate supernatant and resuspend pellet in 1 ml Red blood cell lysis buffer. Incubate at room temperature for 5 min.</li>
	<li>Add 10 ml Wash buffer (DMEM:F12, 5% FBS) to stop lysis. Pass cells through a 40 &#181;M nylon Mesh to remove clumps. Centrifuge at 400 x g for 5 min and remove supernatant, place on ice.</li>
	<li>Resuspend digested cells in 5 ml wash buffer and count both viable and non-viable cells using trypan blue exclusion. Briefly, mix 50 &#181;l of cells and 50 &#181;l of trypan blue and count trypan-blue excluding cells using a hemocytometer. 
	NOTE: This crude digestion product will contain human tumor cells derived from the PDX, as well as mouse stromal cells (fibroblasts, blood cells, etc.). Digested cells can now be processed for labeling (step 6), or human cancer cells can be enriched using one of the procedures described in Ref 5.</li>
</ol>
5. Enrichment of Human Epithelial Cancer Cells
<ol>
	<li>Deplete the mouse stromal cells using a lineage (Lin+) cell depletion kit20. 
	NOTE: Recommended for highly vascularized tumors and/or tumors with high stromal content in which EpCAM expression is unknown or lost).
	<ol>
		<li>Take up to 107 viable cells in a clean 5 ml polypropylene tube, add 2 ml EEB and centrifuge at 300 x g for 5 min. Pipette off supernatant completely.</li>
		<li>Resuspend cell pellet in 40 &#181;l of ice-cold EEB per 107 cells.</li>
		<li>Add 10 &#181;l of biotin-antibody cocktail per 107 cells, mix by gently pipetting and incubate at 4 &#176;C for 10 min (cocktail contains antibodies to Lin+ mouse cells).</li>
		<li>Add 30 &#181;l of cold EEB per 107 cells, mix by gently pipetting.</li>
		<li>Add 20 &#181;l of anti-biotin microbeads per 107 cells mix by gently pipetting and incubate at 4 &#176;C for 15 min.</li>
		<li>Wash cells with 3 ml cold EEB, centrifuge at 300 x g for 5 min at 4 &#176;C. Carefully aspirate supernatant. Resuspend pellet in 500 &#181;l of EEB and place on ice.</li>
		<li>Place column in the magnetic field of a magnetic separator. Rinse column with 0.5 ml epithelial enrichment buffer and allow it to drip through. Do not allow the column to dry out.</li>
		<li>Place a new polypropylene collection tube under the column. Slowly, add the 500 &#181;l containing labeled cells over the column (Lin+ labeled cells will be retained in the magnetic column). Collect effluent as a fraction with unlabeled cells, representing the enriched lineage negative (tumor) cell fraction.</li>
		<li>Rinse the column 3 times with 500 &#181;l of EBB and collect the effluent in the same tube as effluent of step 5.1.8. Keep effluent on ice and proceed to labeling step (6).</li>
		<li>Optional: Take the column outside of the magnetic field. Place a new polypropylene tube underneath and elute the LIN+ fraction by adding 500 &#181;l of EEB, three times and using the provided plunger.</li>
	</ol>
	</li>
	<li>Enrichment of human EpCAM+ epithelial cancer cells (recommended for tumors known to express CD326+ and contain high mouse stromal content).
	<ol>
		<li>Take up to 107 viable cells in a clean 5 ml polypropylene tube, add 2 ml cold EEB and centrifuge at 300 x g for 5 min. Pipette off supernatant completely.</li>
		<li>Resuspend cell pellet in 60 &#181;l of ice-cold EBB per 107 cells.</li>
		<li>Add 20 &#181;l of EpCAM microbeads per 107 cells, mix by gently pipetting and incubate at 4 &#176;C for 30 min.</li>
		<li>Wash cells with 3 ml cold EEB, centrifuge at 300 x g for 5 min at 4 &#176;C. Carefully aspirate supernatant. Resuspend pellet in 500 &#181;l of EEB and put on ice.</li>
		<li>Place column in the magnetic field of a mini separator. Rinse column with 0.5 ml EEB and allow it to drip through. Do not allow the column to dry out.</li>
		<li>Place a new polypropylene collection tube under the column. Slowly, add the 500 &#181;l containing labeled cells over the column (EpCAM+ labeled cells will be retained in the magnetic column). Collect effluent as a fraction with unlabeled cells, representing the mouse stromal cells.</li>
		<li>Rinse the column 4 times with 500 &#181;l of ME Buffer and collect the effluent in the same tube as effluent of step 5.1.8.</li>
		<li>Take the column outside of the magnetic field. Place a new polypropylene tube underneath and elute the EpCAM+ fraction by adding 500 &#181;l of ME buffer, three times. Flush out the magnetically labeled cells by firmly pushing the plunger into the column. Collect effluent containing the enriched EpCAM+ tumor cell fraction and keep on ice. Proceed to step 6.</li>
	</ol>
	</li>
</ol>
6. Transduction of PDX-derived Tumor Cells
<ol>
	<li>Centrifuge dissociated tumor cells at 300 g x 5 min and resuspend in 2 ml mammosphere media. Count viable cells using trypan blue exclusion as in 4.11.</li>
	<li>Prepare 10 ml of mammosphere media containing 8 &#181;g/ml polybrene (2 &#181;l of stock polybrene per ml of media).</li>
	<li>Determine the volume needed for 2 x 105 viable tumor cells. Centrifuge cells at 300 x g for 5 min, and resuspend pellet in 2 ml of polybrene containing media. Plate 2 x 105 viable tumor cells per well in a 6-well ultra-low adherence tissue culture plate. 
	NOTE: This is an optimal number of cells required for transduction with one lentiviral vector. 
	CAUTION! Lentiviral particles and all consumables used with lentiviral particles should be handled following institutional procedures for recombinant DNA biohazards. 
	NOTE: Lentiviral transduction of primary breast cells strongly favors myoepithelial cells which can result in poor labeling of luminal cells and selection of subpopulations of tumor cells during labeling21.</li>
	<li>Incubate the virus with 200 mU/ml neuraminidase at 37 &#176;C for 1 hr prior to transduction to increase the binding of viral particles to different primary cell subpopulations and correct for this potential bias21.</li>
	<li>Add lentiviral particles at 10 MOI (2 x 106 TU for 2 x 105 viable cells) if PDX-dissociated cells are depleted from Lin+ mouse cells or enriched in EpCam+ cells. Add lentiviral particles at 30 MOI (6 x 106 TU for 2 x 105 viable cells) if using non-enriched PDX-dissociated cells. 
	NOTE: The increased MOI allows for efficient transduction even in the presence of large amounts of debris and dead cells in crude extracts. Keep one well with unlabeled cells to serve as a control for viability and transduction efficiency.</li>
	<li>Swirl to mix virus with cells. Incubate at 37 &#176;C, 5% CO2 for up to 96 hr. Add 500 &#181;l of fresh mammosphere media 24 hr after transfection. Cells will not attach to plates, do not aspirate media.</li>
</ol>
7. Evaluation of Transduction Efficiency and Re-implantation of Labeled Cells in Immunocompromised Mice
<ol>
	<li>Monitor expression of the traceable marker (GFP) every 24 hr using a fluorescent microscope at 10X magnification. 
	NOTE: GFP expression can be observed as early as 24 hr after infection but in most PDXs GFP expression is clearly visible after 72 hr (Figure 1).</li>
	<li>Estimate the efficiency of transduction by evaluating the percentage of GFP+ cells within the total well. 
	NOTE: Since cultures contain tumor cells, residual stromal cells, and dead cells at various degrees, wells with as low as 10% GFP+ cells can be implanted in a host mouse.</li>
	<li>Transfer transduced cells from 6-well plates into a 15 ml conical tube. Add 1 ml of mammosphere media to the well to collect all the cells left behind. Place on ice.</li>
	<li>Add 10 ml of HBSS/hepes to transduced cells and centrifuge at 300 x g for 5 min, 4 &#176;C. Carefully aspirate supernatant and resuspend in 50 &#181;l basement matrix extract (BME) on ice. 
	NOTE: BME aliquots must be thawed on ice, 1 - 2 hr prior to use. BME will solidify at room temperature; keep on ice at all times.</li>
	<li>Load BME-embedded cells into a 0.5 ml insulin syringe, keep on ice, and bring to the animal facility for reimplantation into NSG mice.</li>
	<li>Anesthetize female 4 - 8 week old NSG mice, using 5% isoflurane/95% oxygen for 5min, followed by 2% isoflurane/98% oxygen or according to approved animal research ethics approved protocol.</li>
	<li>When animal is unresponsive to pain stimuli (toe-pinch), clean the injection area with betadine followed by ethanol wipes, and inject the 50 &#181;l of cells in the 4th mammary fat pad. Provide mice with analgesia to alleviate injection discomfort as indicated in approved protocol.</li>
	<li>Allow cells to form tumors (2 - 12 weeks) and monitor GFP and/or luciferase activity using a tunable light system and/or in vivo luciferase imaging system (Figure 2). 
	NOTE: Follow guidelines for anesthesia, analgesia, tumor burden measurements and euthanasia criteria as approved by institutional animal research ethics committee.</li>
</ol>
8. Quality Control of Labeled PDXs
<ol>
	<li>Determine the efficiency of labeling by assessing luciferase and GFP expression in labeled tumors. 
	NOTE: Follow institutional animal research ethics committee approved procedures for in vivo bioluminescence imaging (Figures 2, 3).
	<ol>
		<li>Dissect tumors from euthanized mice when tumor size has reached 1.0 - 1.5 cm diameter (or before according to approved protocols, as described in 4). Dissection should be performed under a tunable light system (or an equivalent system that allows GFP fluorescence evaluation).</li>
		<li>From each tumor, qualitatively estimate the percentage of GFP+ tumor using microscopy. If less than 100%, dissect GFP+ portion only. Dissect a 3 mm disk and fix in 10% formalin for paraffin embedding and histological analysis, save a small piece for RNA or other desired analyses, and save as much of the tumor as possible in individual 1.5 ml cryotubes containing 90% FBS/10% DMSO for cryopreservation.</li>
	</ol>
	</li>
	<li>Re-implant pieces of labeled tumor into new recipient NSG mice18 to expand and use in experimental metastasis studies.</li>
	<li>Perform standard immunohistochemical staining of paraffin embedded tissue obtained from tumors prior to transduction and at each generation thereafter to verify that labeled PDXs remain histologically similar to the original PDXs. 
	NOTE: The markers used for quality control vary with each PDXs. For breast cancer PDXs, expression of estrogen receptor, progesterone receptor, Epidermal growth factor receptor 2 (HER2), epidermal growth factor receptor 1 (HER1 or EGFR), pan-cytokeratin (pan-CK) are recommended for initial screening.</li>
</ol>
9. Experimental Metastasis Models with Labeled PDXs
<ol>
	<li>Following the steps described in section 4, dissociate tumor cells from a labeled PDX. If tumor is highly vascularized or rich in mouse stroma, perform enrichment of cancer cells as described in 4.5 or 4.6.</li>
	<li>Count viable cells using trypan blue exclusion, and dilute 250,000 cells in 100 &#181;l PBS (Ca2+, Mg2+ free) per mice. Keep cells on ice. For injection of multiple mice, adjust the numbers accordingly (and prepare an excess of at least one extra injection to account for pipetting errors).</li>
	<li>Bring cells on ice to the appropriate animal procedure room and proceed to intra-cardiac injection according to approved IACUC protocol. 
	NOTE: Detailed protocols for intra-cardiac injection of cancer cells have been described elsewhere22,23.</li>
	<li>Track and quantify metastatic spread over time using bioluminescent imaging. Visualize macroscopic metastases at different organs using bioluminescence and/or GFP imaging of isolated organs at necropsy24 (Figure 6).</li>
</ol>

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R., Masood-Campbell, S. K., Elefteriou, F., Sterling, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+bone+metastasis.">Models of bone metastasis.</a> J Vis Exp. (67), e4260 (2012).</li><li>Kang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+TGFbeta+Signaling+in+Mouse+Models+of+Cancer+Metastasis.">Imaging TGFbeta Signaling in Mouse Models of Cancer Metastasis.</a> Methods Mol Biol. 1344, 219-232 (2016).</li><li>Jenkins, D. E., Hornig, Y. S., Oei, Y., Dusich, J., Purchio, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescent+human+breast+cancer+cell+lines+that+permit+rapid+and+sensitive+in+vivo+detection+of+mammary+tumors+and+multiple+metastases+in+immune+deficient+mice.">Bioluminescent human breast cancer cell lines that permit rapid and sensitive in vivo detection of mammary tumors and multiple metastases in immune deficient mice.</a> Breast Cancer Res. 7 (4), R444-R454 (2005).</li><li>Lawson, D. 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=Single-cell+analysis+reveals+a+stem-cell+program+in+human+metastatic+breast+cancer+cells.">Single-cell analysis reveals a stem-cell program in human metastatic breast cancer cells.</a> Nature. 526 (7571), 131-135 (2015).</li></ol>

The authors have nothing to disclose.

Critical steps within the protocol:
The use of high titer lentiviral particles (&#62;108 TU/ml) is a critical step in the success of this protocol, as allows careful control of the media composition during in vitro transduction. While multiple methods for production of high-titer viral particles have been well described18,19; this protocol uses lentiviral particles produced as described in detail at <a href="http://www.kottonlab.com">www.kottonlab.com</a>. A gentle method for digestion of tumors and dissociation of cancer cells is critical for successful labeling and growth of labeled tumors.&#160; Extending the digestion time beyond three hours or increasing the digestion temperature to 37&#160;&#176;C increases the number of dissociated cells but decreases cell viability and success of labeling in most tumors.
Modifications and troubleshooting:
This protocol should be considered dynamic and requires adaptation for unique PDX with careful monitoring at each step. While this standard protocol works well for most xenografted tumors, small variations may be necessary to optimize labeling of different PDXs. For example, the abundance and composition of stroma and extracellular matrix usually differ among PDXs, which can affect the time needed for dissociation and the yield of tumor cells obtained thereafter. Large tumors that become necrotic and highly vascularized tumors can be difficult to label due to debris and excessive mouse blood cells. The use of Lin+ depletion and epithelial EpCam+ cell enrichment described in this protocol improves the efficiency of transduction in such PDXs. However, expression of EpCam+ cells varies even in tumors from epithelial origin, thus its expression must be verified in each PDX prior to use as method for cell enrichment.
Limitations of the technique:
Transduction of dissociated cells and their temporary culture in vitro could result in selection of cell subpopulations, which will then give rise to labeled tumors fundamentally different from the parental PDXs. The pre-incubation of lentiviral particles with neuraminidase is recommended to increase the binding of viral particles to cell subpopulations21 difficult to transduce, thus decreasing the transduction bias that could be introduced at this step. In our experience, tumors reconstituted by transduced cells closely resemble the features of the parental PDXs not only histologically, but also using hierarchical clustering analysis of mRNA expression (RNA seq, data not shown). Quality control in terms of labeling efficiency and tumor fidelity should be performed for each PDX at every passage. Since PDX labeling requires expansion of the tumor in vivo and tumor drift could occur after repeated passaging, transduction should be performed at the earliest passage possible.
Significance of the technique:
This protocol describes how breast cancer PDXs can be fluorescently a bioluminescently labeled in vitro and re-implanted in vivo for tracking of orthotopic and metastatic tumor growth. While prior studies have used a similar strategy to label PDX-derived organoids18, the current protocol includes variations in tumor digestion and labeling that result in a high transduction efficiency of multiple PDXs.
Future applications or directions after mastering this technique:
The ability to stably express traceable markers (GFP, luciferase), and to overexpress or knockdown specific genes (i.e., pSIH1-H1-copGFP-T2A-puro can be used to deliver shRNAs) allows the use of PDXs to answer fundamental questions about tumor growth and metastasis in a similar fashion to established cell lines. Specifically, this protocol demonstrates the use of labeled PDXs in experimental metastasis models to study organ-colonization steps in the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, and GFP expression used to guided dissections and visualize metastases in excised organs. This represents a powerful tool to facilitate the use of PDXs for metastasis research.

The development of patient-derived tumor xenografts (PDXs), where surgically resected tumor samples are engrafted directly into immune-compromised mice, offers several advantages over standard cell-line xenograft models and represents a major advance in cancer research1,2. PDXs can be maintained and expanded by successive passages with minimal alteration of the genetic and biological characteristics of the tumor grown at the first passage; and more accurately reflect tumor heterogeneity than xenografts derived from human cancer cell lines3-8. These models are now extensively used as a platform for personalizing cancer therapeutics9,10, as a preclinical platform in drug development6,11 and as an experimental tool for studying cancer biology4,12.
Most PDXs are implanted and propagated subcutaneously, which feasibly allows measurement of tumor growth over time using calipers. However, metastatic disease has been more difficult to model using PDXs. Specifically for breast cancer, xenografts with metastatic capacity to different organs have been described3,5,13, but the frequency of spontaneous dissemination to metastatic sites is extremely low. Where reported, the identification and quantification of metastatic burden relies in laborious histological examination of target organs post-mortem. Cancer cell lines expressing bioluminescent (luciferase, Luc) or fluorescent (Green Fluorescent Protein, GFP) gene reporters are commonly used in experimental models of breast cancer metastases to brain, lung, bone and liver after intracardiac, tail-vein, intrafemoral and splenic injection14-16. While these models bypass dissemination from the primary tumors, they are valuable to study the mechanisms of organ tropism and metastatic colonization. However, cells derived from primary patient tumors and PDXs can have low transfection or transduction rates using standard procedures. One alternative is to establish PDX-derived cell lines in vitro17, which can be then labeled using conventional tissue culture protocols. This approach however, is not suitable for labeling most PDXs, for which cell-line derivation is difficult and can change the phenotype of the cells. Here we present a protocol for transduction of PDX-dissociated tumor cells with lentiviral vectors suitable for in vivo imaging. In addition, we describe experimental metastasis using intracardiac injection of dissociated luc-GFP labeled PDX cells in immunocompromised mice.
A basic protocol for transduction of PDX-dissociated organoids with gene-reporter expressing lentivirus has previously been described 18. In the current protocol we describe additional methods to enrich for human tumor cells and obtain near 100% transduction efficiency, as well as the use of labeled PDXs for detecting experimental breast cancer metastases. This protocol can be adapted for labeling multiple cancer types of PDXs with various luminescent and fluorescent markers as well as modulation of gene expression (i.e., shRNA knockdown of genes of interest).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM/F12 (1:1)</td>
			<td>Hyclone</td>
			<td>SH30023.01</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>BD Biosciences</td>
			<td>354060</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>BD Biosciences</td>
			<td>354001</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H4784</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco/Thermo Fisher</td>
			<td>17504-44</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-fungi-antibiotics</td>
			<td>Hyclone</td>
			<td>SV30010</td>
			<td></td>
		</tr>
		<tr>
			<td>Accumax</td>
			<td>Innovative Cell Technologies</td>
			<td>AM-105-500</td>
			<td>Digestion Buffer</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Atlanta Biologicals</td>
			<td>S11550</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS Red Ca++/Mg++ free</td>
			<td>Hyclone</td>
			<td>SH30031.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td>Hyclone</td>
			<td>SH30258.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex</td>
			<td>Cultrex</td>
			<td>3433-005-01</td>
			<td>Basement Matrix Extract (BME)</td>
		</tr>
		<tr>
			<td>30C shaker</td>
			<td>NewBrunswick Scientific CO. INC</td>
			<td>Series 25 Incubator Shaker</td>
			<td></td>
		</tr>
		<tr>
			<td>70um filters</td>
			<td>Falcon</td>
			<td>7352350</td>
			<td></td>
		</tr>
		<tr>
			<td>scalpels</td>
			<td>Fisher</td>
			<td>22079690</td>
			<td></td>
		</tr>
		<tr>
			<td>Clorhexidine disinfectant</td>
			<td>Durvet&#160;</td>
			<td>NDC# 30798-624-35</td>
			<td></td>
		</tr>
		<tr>
			<td>Red blood&#160; cell lysis reagent</td>
			<td>Sigma</td>
			<td>R7757</td>
			<td></td>
		</tr>
		<tr>
			<td>Neuraminidase</td>
			<td>Sigma</td>
			<td>N7885-1UN</td>
			<td></td>
		</tr>
		<tr>
			<td>EpCAM (CD326+) microbeads*</td>
			<td>Miltenyil Biotec</td>
			<td>130-061-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Lineage cell depletion Kit, mouse*</td>
			<td>Miltenyil Biotec</td>
			<td>130-090-858</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS Separator&#160;</td>
			<td>Miltenyil Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini MACS Magnetic Stand</td>
			<td>Miltenyil Biotec</td>
			<td>130-042-303</td>
			<td></td>
		</tr>
		<tr>
			<td>MS Columns</td>
			<td>Miltenyil Biotec</td>
			<td>130-042-201</td>
			<td>MS or LS columns can be used, adjust to number of cells.</td>
		</tr>
		<tr>
			<td>Illumatool Tunable light system</td>
			<td>Lightools research</td>
			<td>Various</td>
			<td>For in vivo fluorescence imaging</td>
		</tr>
		<tr>
			<td>Xenogen IVIS200 imaging device</td>
			<td>Xenogen</td>
			<td>Various</td>
			<td>For in vivo luminiscence imaging</td>
		</tr>
		<tr>
			<td>Human Cytokeratin Clone MNF116 Monoclonal antibody</td>
			<td>DAKO</td>
			<td>M0821</td>
			<td>Pan-cytokeratin&#160;</td>
		</tr>
		<tr>
			<td>Epidermal Growth factor receptor antibody</td>
			<td>Cell signaling</td>
			<td>4267S</td>
			<td>EGFR</td>
		</tr>
	</tbody>
</table>

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.

This method describes the transduction of PDX-dissociated breast cancer cells using high titer lentiviral vectors pSIH1-H1-copGFP-T2A-puro and pHAGE-EF1aL-luciferase-UBC-GFP-W. These vectors express a fluorescent marker that allows estimating the efficiency of transduction in vitro, as early as 24 hr after infection (Figure 1a). For most PDXs, expression of GFP will be delayed up to 72 hr after infection (Figure 1b), at this time the formation of cell aggregates is commonly observed. Transduction can be achieved after enriching for human cancer cells (i.e., using Lin+ cell depletion, Figure 1a) or in crude PDX-dissociated cells (Figure 1b). Enrichment of tumor epithelial cells is recommended for highly vascularized tumors were mouse blood cells represent a significant percentage of the total number of viable cells.
Once transduction efficiency is verified in vitro, labeled cells are collected and suspended in extracellular matrix and re-implanted in NSG mice. Labeled tumor cells regenerate tumors that can be tracked using bioluminescence or fluorescence (Figure 2). Transduction efficiency must be evaluated following tumor formation, and it will vary depending on the susceptibility of different PDXs to in vitro transduction. A successfully labeled PDX will be near 100% GFP+ at first generation post-transduction (Figure 2b,c) and will remain near 100% GFP+ in subsequent passages (Figure 3). However, some PDXs will show &#34;patchy&#34; distribution of GFP+ cells (Figure 4), indicating a suboptimal in vitro transduction. In these cases, tumors can be dissected under a fluorescence-viewing system to select GFP+ areas that can be re-implanted for expansion of the labeled subpopulation, or tumors can be dissociated and GFP+ cells can be isolated using FACS followed by re-implantation in NSG mice.
Since PDX dissociation and transduction can result in the selection of cell subpopulations within the tumor, investigators need to verify that labeled tumors closely resemble the parental tumors from which they were derived. PDXs should be stained by IHC at each generation to demonstrate that critical markers such EGFR, hormone receptors (i.e., estrogen receptor) and pan-cytokeratins (PanCK, markers of epithelial cells) are conserved in labeled PDXs. Figure 5 shows staining for EGFR and panCK in a triple negative breast cancer PDX (hormone receptor staining is not shown as this PDX lacks estrogen and progesterone receptor).
Labeled PDXs can be used to track spontaneous metastatic spread as well as experimental metastatic spread by seeding cells directly into the circulation. Spontaneous metastases from breast cancer PDXs occur less frequently, but have been reported by several groups3,13,25. Experimental models of metastases provide an alternative to study organ tropism and organ-colonization steps in the metastatic cascade. Cells dissociated from labeled tumors are injected intracardially and metastatic burden is tracked using bioluminescent imaging. As shown in Figure 6, a PDX transduced with pHAGE-EF1aL-luciferase-UBC-GFP-W injected at 250,000 cells/mouse formed metastases in lungs and liver that were easily identified with luciferase imaging in vivo, and GFP expression in organs ex vivo.
<img alt="Figure 1" src="/files/ftp_upload/54944/54944fig1.jpg" /> 
Figure 1:Tracking Transduction Efficiency in Dissociated PDX Cells. A) PDX-dissociated cells enriched for human epithelial cells (Lin+ depletion) 24 hr after transduction with pSIH1-H1-copGFP-T2A-puro lentiviral particles. B) PDX-dissociated cells from a separate experiment, without epithelial cell enrichment, 72 hr after transduction with pHAGE-EF1aL-luciferase-UBC-GFP-W. Both panels show live cells. BF: Bright field images, bar represents 50 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54944/54944fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54944/54944fig2.jpg" /> 
Figure 2: Establishment of Labeled PDXs in Host Mice. Cells transduced with pHAGE-EF1aL-luciferase-UBC-GFP-W for 72 hr were implanted in the mammary fat pad of a female NSG mouse. The tumor was allowed to grow for 14 weeks after injection. A) Luciferase reporter activity is assessed by in vivo imaging after intraperitoneal injection of luciferin. B) GFP expression can be observed through the intact skin using a fluorescence viewing system. C) GFP expression should also be verified at dissection to determine the extent of tumor labeling. This example shows ubiquitous GFP expressing tumor. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54944/54944fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54944/54944fig3.jpg" /> 
Figure 3: Labeled PDXs Retain Traceable Markers after Multiple Passages In Vivo. A breast cancer PDX labeled with pHAGE-EF1aL-luciferase-UBC-GFP-W is shown 3 passages after transduction. GFP expression remains at nearly 100% in the passaged tumor. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54944/54944fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54944/54944fig4.jpg" /> 
Figure 4. Example of a PDXs with Suboptimal Transduction Efficiency. A) Breast cancer PDX labeled with pHAGE-EF1aL-luciferase-UBC-GFP-W was passaged after a suboptimal transduction took place. The resulting tumors contain mixed populations of GFP+ and GFP- tumor cells. The left tumor is not suitable for further propagation. The right tumor can be propagated by dissecting the GFP+ regions under a fluorescence viewing system. B) Regions of GFP+ cells in mixed tumors can be easily identified under fluorescent light. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54944/54944fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54944/54944fig5.jpg" /> 
Figure 5. Quality Control of PDX Features after Transduction and Passaging. Expression of EGFR and Pan-cytokeratin (PanCK) in paraffin embedded tissue from a triple negative breast cancer PDX F2-7 at passage 2 (P2, prior to transduction), the tumor generated immediately after transduction (P2-i0), and the following passage (P2-i1). 20X images, Bars represents 100 &#181;m. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54944/54944fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54944/54944fig6.jpg" /> 
Figure 6. Experimental Metastasis Using Labeled PDXs. A PDX expressing pHAGE-EF1aL-luciferase-UBC-GFP-W was dissociated for 3 hr as described in 4, and 250,000 cells were injected in the left cardiac ventricle of NSG mice. A) Metastatic growth can be traced in live animals using luminescence imaging. Metastatic burden of this breast cancer PDX can be observed in B) liver and C) lungs using ex-vivo imaging of dissected organs under a fluorescence viewing system. Bars represents approximately 1cm. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54944/54944fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies at JoVE.com

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

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 ...

labeling-breast-cancer-patient-derived-xenografts-with-traceable

Nanyang Technological University

Research

JoVE Journal

Cancer Research

12.0K Views.  University of Colorado Anschutz Medical Campus. 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.

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

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

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 Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

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 ...

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 stable, thereby preserving molecular, genetic, and histological features, as well as heterogeneity of the original tumor. However, using these models to perform a multitude of experiments, including drug screening, is prohibitive both in terms of cost and time. Three-dimensional (3D) culture systems are widely viewed as platforms in which cancer cells retain their biological integrity through biochemical interactions, morphology, and architecture. Our team has extensive experience culturing PDX cells in vitro using 3D matrices composed of hyaluronic acid (HA). In order to separate mouse fibroblast stromal cells associated with PDXs, we use rotation culture, where stromal cells adhere to the surface of tissue culture-treated plates while dissociated PDX tumor cells float and self-associate into multicellular clusters. Also floating in the supernatant are single, often dead cells, which present a challenge in collecting viable PDX clusters for downstream encapsulation into hydrogels for 3D cell culture. In order to separate these single cells from live cell clusters, we have employed density step gradient centrifugation. The protocol described here allows for the depletion of non-viable single cells from the healthy population of cell clusters that will be used for further in vitro experimentation. In our studies, we incorporate the 3D cultures in microfluidic plates which allow for media perfusion during culture. After assessing the resultant cultures using a fluorescent image-based viability assay of purified versus non-purified cells, our results show that this additional separation step substantially reduced the number of non-viable cells from our cultures.

This protocol demonstrates methods to enable extended in vitro culture of patient-derived xenografts (PDX). One step enhances overall viability of multicellular cluster cultures in 3D hydrogels, through straightforward removal of non-viable single cells. A secondary step demonstrates best practices for PDX culture 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 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.

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 ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately represent the disease. Patient derived xenograft (PDX) models of CNS metastasis have been shown to better represent the phenotypic and molecular characteristics of the human disease, as well as better reflect the heterogeneity and clonal dynamics of human patient tumors compared to historic cell line models. There are multiple sites that can be used to implant patient-derived tissue when setting up preclinical trials, each with their own advantages and disadvantages, and each suited for studying different aspects of the metastatic cascade. Here, the protocol describes how to establish PDX models and present three different approaches for utilizing CNS metastasis PDX models in pre-clinical studies, discussing each of their applications and limitations. These include flank implantation, orthotopic injection in the brain, and intracardiac injection. Subcutaneous flank implantation is the easiest to monitor and, therefore, most convenient for pre-clinical studies. In addition, metastases to brain and other tissues from flank implantation were observed, indicating that the tumor has undergone multiple steps of metastasis, including intravasation, extravasation, and colonization. Orthotopic injection in the brain is the best option for recapitulating the brain tumor microenvironment and is useful for determining the efficacy of biologics to cross the blood-brain barrier (BBB) but bypasses most steps of the metastatic cascade. Intracardiac injection facilitates metastasis to the brain and is also useful for studying organ tropism. While this method forgoes earlier steps of the metastatic cascade, these cells will still have to survive circulation, extravasate, and colonize. The utility of a PDX model, is therefore, impacted by the route of tumor inoculation and the choice of which one to utilize should be dictated by the scientific question and overall goals of the experiment.

Central nervous system metastasis PDX models represent the phenotypic and molecular characteristics of human metastasis, making them excellent models for preclinical studies. Described here is how to establish PDX models and the inoculation routes that are best used for preclinical studies.

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

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 ...

Establishment and Culture of Patient-Derived Breast Organoids

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor organoids developed in our laboratory consist of a mix of multiple tumor-derived cell populations, and thus represent a better approximation of tumor cell diversity and milieu than the established 2D cancer cell lines. Organoids serve as an ideal in vitro model, allowing for cell-extracellular matrix interactions, known to play an important role in cell-cell interactions and cancer progression. Patient-derived organoids also have advantages over mouse models as they are of human origin. Furthermore, they have been shown to recapitulate the genomic, transcriptomic as well as metabolic heterogeneity of patient tumors; thus, they are capable of representing tumor complexity as well as patient diversity. As a result, they are poised to provide more accurate insights into target discovery and validation and drug sensitivity assays. In this protocol, we provide a detailed demonstration of how patient-derived breast organoids are established from resected breast tumors (cancer organoids) or reductive mammoplasty-derived breast tissue (normal organoids). This is followed by a comprehensive account of 3D organoid culture, expansion, passaging, freezing, as well as thawing of patient-derived breast organoid cultures.

A detailed protocol is provided here for establishing human breast organoids from patient-derived breast tumor resections or normal breast tissue. The protocol provides comprehensive step-by-step instructions for culturing, freezing, and thawing human patient-derived breast organoids.

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...