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.

Research

JoVE Journal

Cancer Research

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

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

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