Name: labeling of breast cancer patient-derived xenografts with traceable reporters for tumor growth and metastasis studies
Uploaded: 2016-11-30T15:00:00
Description: 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

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/...

<ol>
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S., 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=Tumor+grafts+derived+from+women+with+breast+cancer+authentically+reflect+tumor+pathology,+growth,+metastasis+and+disease+outcomes.">Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes.</a> Nat Med. 17 (11), 1514-1520 (2011).</li><li>Kabos, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+luminal+breast+cancer+xenografts+retain+hormone+receptor+heterogeneity+and+help+define+unique+estrogen-dependent+gene+signatures.">Patient-derived luminal breast cancer xenografts retain hormone receptor heterogeneity and help define unique estrogen-dependent gene signatures.</a> Breast cancer research and treatment. 135 (2), 415-432 (2012).</li><li>Zhang, X., 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=A+renewable+tissue+resource+of+phenotypically+stable,+biologically+and+ethnically+diverse,+patient-derived+human+breast+cancer+xenograft+models.">A renewable tissue resource of phenotypically stable, biologically and ethnically diverse, patient-derived human breast cancer xenograft models.</a> Cancer Res. 73 (15), 4885-4897 (2013).</li><li>Lum, D. 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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+human+primary+tumorgraft+models:+comparisons+with+traditional+oncology+preclinical+models+and+the+clinical+relevance+and+utility+of+primary+tumorgrafts+in+basic+and+translational+oncology+research.">Overview of human primary tumorgraft models: comparisons with traditional oncology preclinical models and the clinical relevance and utility of primary tumorgrafts in basic and translational oncology research.</a> Curr Protoc Pharmacol. , (2012).</li><li>Marangoni, E., 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=A+new+model+of+patient+tumor-derived+breast+cancer+xenografts+for+preclinical+assays.">A new model of patient tumor-derived breast cancer xenografts for preclinical assays.</a> Clin Cancer Res. 13 (13), 3989-3998 (2007).</li><li>Garrido-Laguna, I., 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=Tumor+engraftment+in+nude+mice+and+enrichment+in+stroma-+related+gene+pathways+predict+poor+survival+and+resistance+to+gemcitabine+in+patients+with+pancreatic+cancer.">Tumor engraftment in nude mice and enrichment in stroma- related gene pathways predict poor survival and resistance to gemcitabine in patients with pancreatic cancer.</a> Clin Cancer Res. 17 (17), 5793-5800 (2011).</li><li>Landis, M. 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J., 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=Patient-derived+tumour+xenografts+as+models+for+oncology+drug+development.">Patient-derived tumour xenografts as models for oncology drug development.</a> Nat Rev Clin Oncol. 9 (6), 338-350 (2012).</li><li>Zhang, H., 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=Patient-derived+xenografts+of+triple-negative+breast+cancer+reproduce+molecular+features+of+patient+tumors+and+respond+to+mTOR+inhibition.">Patient-derived xenografts of triple-negative breast cancer reproduce molecular features of patient tumors and respond to mTOR inhibition.</a> Breast Cancer Res. 16 (2), R36 (2014).</li><li>Liu, H., 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=Cancer+stem+cells+from+human+breast+tumors+are+involved+in+spontaneous+metastases+in+orthotopic+mouse+models.">Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models.</a> Proc Natl Acad Sci U S A. 107 (42), 18115-18120 (2010).</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=Analysis+of+cancer+stem+cell+metastasis+in+xenograft+animal+models.">Analysis of cancer stem cell metastasis in xenograft animal models.</a> Methods Mol Biol. 568, 7-19 (2009).</li><li>Thibaudeau, L., 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=Mimicking+breast+cancer-induced+bone+metastasis+in+vivo:+current+transplantation+models+and+advanced+humanized+strategies.">Mimicking breast cancer-induced bone metastasis in vivo: current transplantation models and advanced humanized strategies.</a> Cancer Metastasis Rev. 33 (2-3), 721-735 (2014).</li><li>Francia, G., Cruz-Munoz, W., Man, S., Xu, P., Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+advanced+spontaneous+metastasis+for+experimental+therapeutics.">Mouse models of advanced spontaneous metastasis for experimental therapeutics.</a> Nat Rev Cancer. 11 (2), 135-141 (2011).</li><li>Powell, E., 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=p53+deficiency+linked+to+B+cell+translocation+gene+2+(BTG2)+loss+enhances+metastatic+potential+by+promoting+tumor+growth+in+primary+and+metastatic+sites+in+patient-derived+xenograft+(PDX)+models+of+triple-negative+breast+cancer.">p53 deficiency linked to B cell translocation gene 2 (BTG2) loss enhances metastatic potential by promoting tumor growth in primary and metastatic sites in patient-derived xenograft (PDX) models of triple-negative breast cancer.</a> Breast Cancer Res. 18 (1), (2016).</li><li>DeRose, Y. S., 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=Patient-derived+models+of+human+breast+cancer:+protocols+for+in+vitro+and+in+vivo+applications+in+tumor+biology+and+translational+medicine.">Patient-derived models of human breast cancer: protocols for in vitro and in vivo applications in tumor biology and translational medicine.</a> Curr Protoc Pharmacol. , (2013).</li><li>Wang, X., McManus, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivirus+production.">Lentivirus production.</a> J Vis Exp. (32), (2009).</li><li>Indumathi, S., 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=Lineage+depletion+of+stromal+vascular+fractions+isolated+from+human+adipose+tissue:+a+novel+approach+towards+cell+enrichment+technology.">Lineage depletion of stromal vascular fractions isolated from human adipose tissue: a novel approach towards cell enrichment technology.</a> Cytotechnology. 66 (2), 219-228 (2014).</li><li>Hines, W. C., Yaswen, P., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+breast+cancer+requires+identification+and+correction+of+a+critical+cell+lineage-dependent+transduction+bias.">Modelling breast cancer requires identification and correction of a critical cell lineage-dependent transduction bias.</a> Nat Commun. 6, 6927 (2015).</li><li>Campbell, J. P., Merkel, A. 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. 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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.kottonla...

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...

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 ...

The overall goal of this transduction protocol is to stably introduce reporter genes in patient-derived xenografts, or PDXs, to facilitate their use in experimental and spontaneous metastasis models. This method can help answer key questions in cancer research, such as the extent of excutaneous metastasis from patient-derived xenografts. The main advantage of this technique is that labeled PDX can be track, using in vivo imaging, facilitating the assessment of organized, specific metastasis without the extensive histological analysis post-mortem.Demonstrating this protocol will be Jessica Finlay-Schultz, a post-doc, who routinely applies this protocol for the label of breast cancer patient-derived xenografts. Using aseptic technique, begin by using a scalpel to cut the skin around the tumor. Then, pull the skin with forceps and use a clean scalpel to separate the skin from the tumor.When the tumor is completely exposed, use a clean set of forceps and scalpel to transfer the tissue into five milliliters of wash medium on ice. Next, discard the medium and wash the tumor two times with 10 milliliters of HBSS, supplemented with HEPES. Place the rinsed tumor in a sterile, pre-weighed 60 centimeter tissue culture plate, and weigh the plate to estimate the tumor mass.Using a scalpel, cut the tumor in half. Then cut a three millimeter disc from one of the halves, and place the disc in 10%formalin for 24 hours. Add 200 microliters of HBSS with HEPES to the rest of the tumor, and use forceps and a clean scalpel to mince the tissue into the smallest possible pieces.Transfer the fragments into a steril, 50 milliliter conical tube, and add at least one milliliter of digestion buffer, supplemented with antimicotics and antibiotics per 100 milligrams of tumor. After three hours at 30 degrees Celsius, and 125 to 200 rotation per minute, add 35 milliliters of wash medium to stop the digestion, and filter the resulting tissue slurry through a 70 micron nylon mesh into a new 50 milliliter conical tube, to remove any undigested tissue. Collect the cells by centrifugation, and re-suspend the pellet in one milliliter of red blood cell lysis buffer, for five minutes at room temperature.At the end of the lysis, restore the tonicity with 10 milliliters of wash buffer, and strain the cells through a 40 micron filter to remove any clumps. Centrifuge the cells again, and re-suspend the pellet in five milliliters of wash buffer on ice. Then count the number of viable cells by trypan blue exclusion.To enrich for the human epithelial cancer cells using lineage plus Mel cell depletion, transfer up to one times ten to the seventh cells into a new five milliliter polypropylene tube, and add two milliliters of epithelial enrichment buffer to the cells. Collect the cells by centrifugation, and use a pipette to completely remove the supernatant. Next, re-suspend the pellet in 40 microliters of ice cold epithelial enrichment buffer, and add 10 microliters of biotin antibody cocktail per one times ten to the seventh cells.Mix by gentle pipetting, and incubate the cell solution for 10 minutes at four degrees Celsius. At the end of incubation, mix the cells with 30 microliters of epithelial enrichment buffer per one times ten to the seventh cells. Followed by the addition of 20 microliters of antibiotin microbeads with gentle mixing.After 15 minutes at four degrees Celsius, wash the cells in three milliliters of cold epithelial enrichment buffer, and carefully aspirate the supernatant. Re-suspend the pellet in 500 microliters of epithelial enrichment buffer on ice, and place a column in a magnetic separator. Rinse the column with 0.5 milliliters of epithelial enrichment buffer.And place a new polypropylene collection tube under the column. Then slowly add the labeled cells over the column. Collecting the enriched, unlabeled, lineage negative, tumor cell fraction affluent, followed by three column rinses with 500 microliters of fresh epithelial enrichment buffer.To transduce the tumor cells, centrifuge the isolated cells for re-suspension in two milliliters of mammosphere medium. After counting, centrifuge the cells again, and re-suspend the pellet in 2 milliliters of mammosphere medium, supplemented with 20 microliters of polybrene. Next, plate two times ten to the fifth viable tumor cells per well in an ultra low adherence six well tissue culture plate, and add lentiviral particles to the cells at a multiplicity of infection of 10.Swirl the plate to mix the virus with the cells. And incubate the co-cultures at 37 degrees Celsius and 5%carbon dioxide for up to 96 hours. Adding 500 microliters of fresh mammosphere medium after the first 24 hours.When at least 10%of the cells are GFP positive, transfer the transduce cells from at least one well into a 15 milliliter conical tube on ice, and wash the well with one milliliter of mammosphere medium. Pool the wash with the cells, and centrifuge the tumor cells in 10 milliliters of HBSS plus HEPES. Finally, re-suspend the cells in 50 microliters of basement matrix extract on ice.And load the embedded cells into a 0.5 milliliter insulin syringe for re-implantation. Some high titer vial vectors express a fluorescent marker that allows estimation of the transduction efficiency in vitro, as early as 24 hours after infection. For most PDXs, the GFP expression will be delayed up to 72 hours after infection.At which point, the formation of cell aggregates is commonly observed. After extracellular matrix suspension, and re-implantation, the labeled tumor cells regenerate tumors that can be tracked using bioluminescence or fluorescence. A successfully labeled PDX will be nearly 100%GFP positive at the first generation post transduction, and will remain so in subsequent passages.However, some PDX's will exhibit a patchy distribution of GFP positive cells, indicating a suboptimal in vitro transduction. Immunohistochemical tumor tissue staining reveals that the expression of critical markers, such as epidermal growth factor receptor, and pan cytokeratins, are conserved even after several passages and re-implantations. Experimental models of metastasis provide an alternative to studying organ tropism and organ colonization steps in the metastatic cascade.Indeed, a transduced PDX injected at 2.5 times ten to the fifth cells per mouse, forms metastasis in the liver that are easily identified with luciferase imaging in vivo. And in the lungs that can be visualized by fluorescence microscopy ex vivo. Once mastered, the dissociation and labeling of the PDXs takes about 4 hours.The actual expansion of the tumor can take 8 to 12 weeks depending on the tumor, and system used. When attempting this procedure it's important to use a high titer virus with a reporter gene, so you can monitor both transfection efficiency and tumor growth in vivo. Following this procedure, other methods such as in vivo metastasis studies can be performed to answer other questions on tumor progression, metastasis, and response to treatments.This technique paves the way for breast cancer researchers to explore multiple aspects of tumor progression, using novel PDX models that better represent tumor heterogeneity. After watching this video, you should have a good understanding of how to label dissociated tumor cells with high titer lentiviral particles. Which will allow you to both trace tumor cells in vivo as well as potentially manipulate the expression of genes of interest.Don't forget that working with lentiviral particles can be extremely hazardous, and you should always take proper precautions when performing this procedure.

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

Research

JoVE Journal

Cancer Research

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 ...

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture models. PDOs have been successfully generated from a variety of human tumors to recapitulate the architecture and function of tumor tissue accurately and efficiently. However, PDOs are unsuitable for an in vitro high-throughput assay system (HTS) or cell analysis using 96-well or 384-well plates when evaluating anticancer drugs because they are heterogeneous in size and form large clusters in culture. These cultures and assays use extracellular matrices, such as Matrigel, to create tumor tissue scaffolds. Therefore, PDOs have a low throughput and high cost, and it has been difficult to develop a suitable assay system. To address this issue, a simpler and more accurate HTS was established using PDOs to evaluate the potency of anticancer drugs and immunotherapy. An in vitro HTS was created that uses PDOs established from solid tumors cultured in 384-well plates. An HTS was also developed for assessment of antibody-dependent cellular cytotoxicity activity to represent the immune response using PDOs cultured in 96-well plates.

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

A highly accurate in vitro high-throughput assay system was developed to evaluate anticancer drugs using patient-derived tumor organoids (PDOs), similar to cancer tissues but are unsuitable for in vitro high-throughput assay systems with 96-well and 384-well plates.

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...