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 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.
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).
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
2. Generation of High Titer Lentiviral Particles Carrying Traceable Markers
3. Generation of Patient-derived Xenografts
4. Tumor Dissection and Dissociation of PDX-derived Tumor Cells
5. Enrichment of Human Epithelial Cancer Cells
6. Transduction of PDX-derived Tumor Cells
7. Evaluation of Transduction Efficiency and Re-implantation of Labeled Cells in Immunocompromised Mice
8. Quality Control of Labeled PDXs
9. Experimental Metastasis Models with Labeled PDXs
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 "patchy" 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.
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 µm. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 µm. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Critical steps within the protocol:
The use of high titer lentiviral particles (>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 www.kottonlab.com. A gentle method for digestion of tumors and dissociation of cancer cells is critical for successful labeling and growth of labeled tumors. Extending the digestion time beyond three hours or increasing the digestion temperature to 37 °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 authors have nothing to disclose.
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.
DMEM/F12 (1:1) | Hyclone | SH30023.01 | |
bFGF | BD Biosciences | 354060 | |
EGF | BD Biosciences | 354001 | |
Heparin | Sigma | H4784 | |
B27 | Gibco/Thermo Fisher | 17504-44 | |
Anti-fungi-antibiotics | Hyclone | SV30010 | |
Accumax | Innovative Cell Technologies | AM-105-500 | Digestion Buffer |
FBS | Atlanta Biologicals | S11550 | |
HBSS Red Ca++/Mg++ free | Hyclone | SH30031.02 | |
Hepes | |||
10X PBS | Hyclone | SH30258.01 | |
Cultrex | Cultrex | 3433-005-01 | Basement Matrix Extract (BME) |
30C shaker | NewBrunswick Scientific CO. INC | Series 25 Incubator Shaker | |
70um filters | Falcon | 7352350 | |
scalpels | Fisher | 22079690 | |
Clorhexidine disinfectant | Durvet | NDC# 30798-624-35 | |
Red blood cell lysis reagent | Sigma | R7757 | |
Neuraminidase | Sigma | N7885-1UN | |
EpCAM (CD326+) microbeads* | Miltenyil Biotec | 130-061-101 | |
Lineage cell depletion Kit, mouse* | Miltenyil Biotec | 130-090-858 | |
MiniMACS Separator | Miltenyil Biotec | 130-042-102 | |
Mini MACS Magnetic Stand | Miltenyil Biotec | 130-042-303 | |
MS Columns | Miltenyil Biotec | 130-042-201 | MS or LS columns can be used, adjust to number of cells. |
Illumatool Tunable light system | Lightools research | Various | For in vivo fluorescence imaging |
Xenogen IVIS200 imaging device | Xenogen | Various | For in vivo luminiscence imaging |
Human Cytokeratin Clone MNF116 Monoclonal antibody | DAKO | M0821 | Pan-cytokeratin |
Epidermal Growth factor receptor antibody | Cell signaling | 4267S | EGFR |