Name: labeling of breast cancer patient-derived xenografts with traceable reporters for tumor growth and metastasis studies
Uploaded: 2016-11-30T15:00:00.000+00:00
Duration: 9 min 53 s
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/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><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&lt;sup&gt;2+&lt;/sup&gt;/Mg&lt;sup&gt;2+&lt;/sup&gt; 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>30 &amp;#176;C shaker</td><td>NewBrunswick Scientific CO. INC</td><td>Series 25 Incubator Shaker</td><td></td></tr><tr><td>70 &amp;#956;m 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&amp;nbsp;</td><td>NDC# 30798-624-35</td><td></td></tr><tr><td>Red blood&amp;nbsp; 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&amp;nbsp;</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&amp;nbsp;</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

12.1K Views.  University of Colorado Anschutz Medical Campus.  We beschrijven een methode voor stabiele kenmerken van de patiënt afgeleide xenotransplantaten (PDXs) met lentivirale deeltjes uiten van groen fluorescerend eiwit en luciferase reporters. Deze werkwijze maakt het volgen van de groei van PDXs op de primaire locatie, alsmede detectie spontane en experimentele metastasen gebruik in vivo beeldvorming. 

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

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

Computed Tomography-guided Time-domain Diffuse Fluorescence Tomography in Small Animals for Localization of Cancer Biomarkers

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light absorption by tissue chromophores (e.g., hemoglobin, water, lipids, melanin) typically limits optical signal propagation through thicknesses larger than a few millimeters2. Compared to other visible wavelengths, tissue absorption for red and near-infrared (near-IR) light absorption dramatically decreases and non-elastic scattering becomes the dominant light-tissue interaction mechanism. The relatively recent development of fluorescent agents that absorb and emit light in the near-IR range (600-1000 nm), has driven the development of imaging systems and light propagation models that can achieve whole body three-dimensional imaging in small animals3.
Despite great strides in this area, the ill-posed nature of diffuse fluorescence tomography remains a significant problem for the stability, contrast recovery and spatial resolution of image reconstruction techniques and the optimal approach to FMI in small animals has yet to be agreed on. The majority of research groups have invested in charge-coupled device (CCD)-based systems that provide abundant tissue-sampling but suboptimal sensitivity4-9, while our group and a few others10-13 have pursued systems based on very high sensitivity detectors, that at this time allow dense tissue sampling to be achieved only at the cost of low imaging throughput. Here we demonstrate the methodology for applying single-photon detection technology in a fluorescence tomography system to localize a cancerous brain lesion in a mouse model.
The fluorescence tomography (FT) system employed single photon counting using photomultiplier tubes (PMT) and information-rich time-domain light detection in a non-contact conformation11. This provides a simultaneous collection of transmitted excitation and emission light, and includes automatic fluorescence excitation exposure control14, laser referencing, and co-registration with a small animal computed tomography (microCT) system15. A nude mouse model was used for imaging. The animal was inoculated orthotopically with a human glioma cell line (U251) in the left cerebral hemisphere and imaged 2 weeks later. The tumor was made to fluoresce by injecting a fluorescent tracer, IRDye 800CW-EGF (LI-COR Biosciences, Lincoln, NE) targeted to epidermal growth factor receptor, a cell membrane protein known to be overexpressed in the U251 tumor line and many other cancers18. A second, untargeted fluorescent tracer, Alexa Fluor 647 (Life Technologies, Grand Island, NY) was also injected to account for non-receptor mediated effects on the uptake of the targeted tracers to provide a means of quantifying tracer binding and receptor availability/density27. A CT-guided, time-domain algorithm was used to reconstruct the location of both fluorescent tracers (i.e., the location of the tumor) in the mouse brain and their ability to localize the tumor was verified by contrast-enhanced magnetic resonance imaging.
Though demonstrated for fluorescence imaging in a glioma mouse model, the methodology presented in this video can be extended to different tumor models in various small animal models potentially up to the size of a rat17.

Diffuse fluorescence tomography offers a relatively low-cost and potentially high-throughout approach to preclinical in vivo tumor imaging. The methodology of optical data collection, calibration, and image reconstruction is presented for a computed tomography-guided non-contact time-domain system using fluorescent targeting of the tumor biomarker epidermal growth factor receptor in a mouse glioma model.

Small animal fluorescence molecular imaging (FMI) can be a powerful tool for preclinical drug discovery and development studies1. However, light ...

Medicine

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

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 ...

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

Examining Tumor Cell Dissemination from Lung Metastases

Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional process wherein cells from the primary tumor disseminate and seed metastases, tumor cells in existing metastases can also redisseminate and give rise to new lesions in tertiary sites in a process known as &#34;metastasis-from-metastases&#34; or &#34;metastasis-to-metastasis seeding.&#34; Metastasis-to-metastasis seeding may increase the metastatic burden and decrease the patient&#39;s quality of life and survival. Therefore, understanding the processes behind this phenomenon is crucial to refining treatment strategies for patients with metastatic cancer.
Little is known about metastasis-to-metastasis seeding, due in part to logistical and technological limitations. Studies on metastasis-to-metastasis seeding rely primarily on sequencing methods, which may not be practical for researchers studying the exact timing of metastasis-to-metastasis seeding events or what promotes or prevents them. This highlights the lack of methodologies that facilitate the study of metastasis-to-metastasis seeding. To address this, we have developed - and describe herein - a murine surgical protocol for the selective photoconversion of lung metastases, allowing specific marking and fate tracking of tumor cells redisseminating from the lung to tertiary sites. To our knowledge, this is the only method for studying tumor cell redissemination and metastasis-to-metastasis seeding from the lungs that does not require genomic analysis.

Author Spotlight: Decoding Metastasis-to-Metastasis Seeding Using a New In Vivo Technique for Tracking Breast Cancer Spread 

We present a method for studying tumor cell redissemination from lung metastases involving a surgical protocol for the selective photoconversion of lung metastases, followed by the identification of redisseminated tumor cells in tertiary organs.

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional ...

High-Throughput Dissociation and Orthotopic Implantation of Breast Cancer Patient-Derived Xenografts

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which is essential for advancing knowledge of breast cancer (BC). Additionally, PDX models enable the study of BC systemic effects, which is not possible using in vitro models. Traditional methods for implanting BC xenografts typically involve anesthesia and sterile surgical procedures, which are time-consuming, invasive, and limit the scalability of PDX models in BC research. This protocol describes a simple and scalable method for the orthotopic implantation of BC PDXs in mice. The immunodeficient mouse strain NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) was used for PDX engraftment. Human BC samples obtained from IRB-consented patients were mechanically and enzymatically dissociated, then resuspended in a solution of basement membrane extract (BME) and RPMI 1640. Animals were restrained by scruffing, and depilatory cream was applied to remove hair from the fat pads at the fourth inguinal nipple, followed by injection. Approximately 2 million cells in a 100 &#181;L suspension were bilaterally injected orthotopically into the mammary fat pads using a 26 G needle. Notably, no anesthetic was required, and the total procedure time was under 5 min, from cell preparation to injection. After a growth period of several months, tumors were excised and processed for authentication. Validation included receptor status assessment using immunohistochemistry with specific antibodies for traditional BC receptors (i.e., ER, PR, HER2). Tumor morphology was confirmed with hematoxylin and eosin (H&amp;E) staining, which was interpreted by a pathologist. Genetic similarity to the patient sample was verified through bulk RNA sequencing and short tandem repeat (STR) analysis. This approach to PDX engraftment and validation supports the rigorous development of models and high-throughput tumor implantation, enabling well-powered studies across various BC subtypes.

This protocol describes an efficient, non-surgical method for the orthotopic implantation of breast cancer patient-derived xenografts in mice. The technique involves enzymatic tumor dissociation followed by direct injection into the mammary fat pads, enabling high-throughput implantation. Comprehensive validation ensures model fidelity, facilitating rigorous studies across various breast cancer subtypes.

Patient-derived xenografts (PDXs) provide a clinically relevant method for recapitulating tumor-involved cell types and the tumor microenvironment, which ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.

Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.

This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer ...

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

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