Name: combined use of tail vein metastasis assays and real-time in vivo imaging to quantify breast cancer metastatic colonization and burden in the lungs
Uploaded: 2019-12-19T16:00:00.000+00:00
Duration: 10 min 32 s
Description: Watch this Scientific Journal Video about Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs at JoVE.co

We thank Emily Norton for assisting with viral infections and critical reading of the manuscript. We also thank Ryan Kanai for help with acquiring images of the lungs and Kate E. Tubbesing for help with image analysis of the green metastases in the lungs. We thank the animal research facility staff for their support and for assistance in the preparation of this video. This work was supported by a Susan G. Komen Career Catalyst Grant that awarded to J.M.L. (#CCR17477184).

The authors have nothing to disclose.

Critical steps of the method 
It is critical to optimize the number of cells injected (step 3) for a given cell line and mouse strain as this can greatly influence the number of metastases that form and the length of the experiment. If too many cells are injected or the metastases grow for too long, the metastases may be difficult to count making the effects of the genetic manipulation difficult to assess. However, if too few cells are injected, few or no metastases may form. Thus, a preliminary experiment should be done using different numbers of cells to determine the optimal number and the length of time that the metastases grow. To ensure consistent numbers of metastases within a group, it is important inject equal numbers of viable cells into each mouse. Since cells can settle in both the tube and syringe, cells should be gently resuspended prior to loading the syringe, and cell suspensions should not be left in the syringe for too long (step 4.3.2). Cell viability decreases the longer the cells are in suspension, so the amount of time between trypsinization and injection of the cells should be no more than one hour (step 4.2.5). Air bubbles and large clumps of cells should not be injected since these can cause emboli that kill the mouse. In addition, drawing cells up through the needle can shear them, so the syringe should be loaded without the needle. To confirm that a relatively equal number of cells was injected, in vivo live animal images can be taken shortly after the injection (Figure 2B, Figure 4D).
Accurate and consistent quantification of the bioluminescent signal is important and dependent upon the cells taking up and metabolizing the D-luciferin. This can be influenced by many variables including, the dose of D-luciferin, timing of injection, mouse&#39;s body temperature, how anesthetized the mouse is when injected, and how a mouse is positioned in the anesthesia chamber prior to imaging. Therefore, it is critical to keep these steps consistent for all mice and imaging sessions. We used a heating pad to maintain consistent mouse body temperature while in the anesthesia chamber. Since the signal must penetrate the tissue for both bioluminescent and fluorescent detection, it is also critical to keep the mouse position consistent for each image (flat on its back works best for imaging of the lungs). Quantification of fluorescent images from the in vivo live animal imaging should always be done using the efficiency % units as this allows for appropriate comparison between images. Likewise, total flux (photons/second) should always be used when quantifying bioluminescent images. For analysis of the metastatic burden, the log10 transformation converts the growth curve (before maximum signal is reached) into a linear plot, which was necessary for the slope analysis.
Modifications, and troubleshooting of the method 
In the presented protocol a population of cells is first stably transduced with a fluorescent protein and luciferase, then that population is transduced with either a control vector or a vector targeting the gene of interest. This ensures that both cell populations have similar fluorescent protein and luciferase expression. However, as an alternative, viral vectors that simultaneously deliver two of these components could be utilized. Indeed, in the representative experiments we used ZsGreen expressing retroviral vectors to express the shRNAs in 4T1-luciferase cells. It is recommended that the expression of the luciferase and the fluorescent protein be assayed in all cell groups after the gene of interest is altered. Different levels of expression between groups can complicate the interpretation of the data, so it is best if the different groups have similar expression of both the fluorescent protein and luciferase. Additionally, red blood cells can be autofluorescent and may make it difficult to visualize the fluorescent metastases in the lungs. If this is the case, red blood cells can be cleared from the lungs by PBS perfusion during euthanasia to reduce autofluorescent background (appropriate euthanasia techniques and guidelines should be followed). Although the differences between control and knockdown groups in our representative experiments are dramatic, the inherent mouse-to-mouse variability that often exists in in vivo metastasis assays is evident in the control mice (Figure 3B and Figure 5B). When this variability is high, it can make it difficult to determine the magnitude of the knockdown effect. An alternative approach that could be used to reduce this variability is to label the control cells and knockdown cells with distinct fluorescent proteins and distinct luciferase enzymes and then mix the two populations in equal numbers and inject the mixture. For example, control cells stably expressing tomato and renilla luciferase could be mixed with knockdown cells expressing ZsGreen and firefly luciferase. In vivo live animal imaging devices can distinguish renilla luciferase from firefly luciferase, and tomato from ZsGreen, so either fluorescence or luminescence can be used to independently quantify each cell population. Indeed, dual luciferase imaging of 4T1 cells growing as primary tumors in live animals has been demonstrated using an in vivo live animal imaging device4. However, it is important to note that there may be overlap in fluorescent signal, thus a spectral unmixing step (see manufacturers guidelines) should be included in this approach. Additionally, the renilla luciferase and firefly luciferase should be imaged separately with an adequate time delay that allows for the first luciferase signal to no longer be detectable prior to imaging the second. Use of distinct fluorophores still enables the number and size of metastasis in the lungs to be quantified16. To test metastatic colonization and growth in other organs besides the lungs this protocol can be modified and used for intracardiac and portal vein injections9,10.
Limitations of the method 
Using an in vivo live animal imaging device to acquire real time metastatic burden in conjunction with fluorescently labeled cancer cells provides a powerful method to assay metastatic colonization and subsequent growth; however, there are limitations of this approach. Some genes may be required for innate cell proliferation rather than specific roles in metastasis. In vitro cell proliferation assays could be done prior to the in vivo experiment to determine if the gene disrupts in vitro growth. When imaging a live animal, the bioluminescent or fluorescent signal from metastases must be strong enough to pass through the tissue. Additionally, the optical properties (i.e., absorption and scattering) of the tissue also influence detection55. The excitation light source must be able to pass through tissue to excite fluorescently labeled cancer cells and bioluminescence has a larger dynamic range than fluorescence. Thus, although fluorescence can be used for live animal imaging, bioluminescence is preferred for detecting signal in internal organs. In addition, some immunocompetent mouse strains may reject cells expressing fluorescent proteins. Indeed, we found that cells expressing tomato, GFP, or mCherry were rejected or down regulated the fluorophore when growing in BALB/c mice (data not shown and15). However, as demonstrated here (Figure 3 and Figure 5) and previously15,16, ZsGreen-labeled cells are detectable in these mice. In cases where the fluorescent protein expression becomes undetectable, another way to quantify the relative metastatic colonization is to use qPCR to quantify either the fluorescent gene or another gene in the integrated viral vector15. Although an in vivo live animal imaging device allows you to detect changes in metastatic burden, it does not allow one to determine if these changes are due to altered size or number of metastases. It also does not provide spatial information about the location of the metastases. Furthermore, the strength of the signal can be influenced by how deep in the tissue it is.
The significance of the method and potential future applications 
There are many ways in which this approach can be modified to gain more or different information. A variety of cancer cell lines including human cancer cell lines or patient derived xenografts could be used in this assay. Other mouse models could also be used, including transgenic or knockout mice designed to test how targeting proteins made by stromal cells influences the metastatic colonization and growth of the injected cancer cells. If there are multiple candidate genes to be tested, this approach can be multiplexed because in vivo live animal imaging devices can detect and distinguish a wide range of fluorophores. This would reduce the number of mice required and increase the number of targets that can be tested. In vivo live animal imaging can also be combined with X-ray or CT9 imaging to provide a great deal more spatial information about the location of the metastases. Lastly, this approach can be modified to assay more specific steps within the metastatic colonization cascade. For example, the steps involved in tumor cell seeding (intravascular survival, extravasation, and post extravasation survival) can be distinguished by quantifying the number of tumor cells in the lungs at several time points within the first 3 days following injection (such as done here16). In this case, the lungs can also be stained with vascular markers and imaged ex vivo to determine the percentage of cancer cells that have extravasated at each time point56,57.
In summary, the use of real-time in vivo live animal imaging of fluorescent and luminescent cancer cells following tail vein injection enables a more detailed analysis of how a candidate gene or genes influences metastatic colonization and subsequent growth. This approach is faster and less expensive than autochthonous mouse models and avoids some of the caveats of spontaneous metastasis models. The use of both fluorescence and luminescence as a means to detect and quantify metastases gives researchers independent readouts that improve confidence in the results and allow for flexibility in the experimental design. The use of fluorescence to quantify metastasis size and number avoids the need for time-consuming and potentially costly histological processing and analysis, and allows for additional use of the whole tissues. The protocol can be used with a number of different techniques to manipulate the expression or function of the candidate gene, such as RNAi, transgene expression, or CRISPR/Cas-mediated editing, and can also be used to test small molecules, function blocking antibodies, or other potential therapeutic compounds. As mentioned above, several simple modifications can be made to enhance the assay and provide additional information. This approach is an ideal way to identify and test pro-metastatic genes that have therapeutic potential for the treatment of cancer.

Cancer remains the second leading cause of death worldwide1 and metastasis is responsible for the majority of these deaths2,3. However, a limited understanding of the molecular mechanisms that govern metastatic colonization and subsequent growth has hindered the development of effective treatments for metastatic disease. The identification of novel therapeutic targets requires an assay to test how perturbed expression or function of a candidate gene influences metastasis formation and growth. While autochthonous mouse models have their advantages, they are time-consuming and expensive to generate, making them more suited for target validation rather than target discovery. Transplant model systems in which the candidate gene is perturbed in cancer cells in vitro and then effects on metastatic potential are assessed in vivo, are less expensive and higher throughput than autochthonous models. In addition, viral vectors for stable delivery of RNAi, CRISPR/CAS9, and transgenes are widely available, making it relatively easy to perturb virtually any gene or genes of interest in a cancer cell lines. This approach can also be used to assay the role of candidate genes in metastatic colonization and growth in human cancer cell lines by transplanting the cells into immunocompromised or humanized mice.
The two types of assays used to test metastasis formation by transplanted cancer cells in vivo are spontaneous metastasis assays and experimental metastasis assays. In spontaneous metastasis assays4,5, cancer cells are injected into mice, allowed to form a primary tumor, and then spontaneous metastasis formation and subsequent growth are assayed. The strength of this model is that the cells must complete all steps of the metastatic process in order to form metastatic tumors. However, many cancer cell lines do not metastasize efficiently in spontaneous metastasis models, and any manipulation of the cells that impacts primary tumor growth can confound the results of the metastasis assay. Experimental metastasis assays, in which cancer cells are injected directly into circulation, are used to avoid these pitfalls. Common experimental metastasis assays include the tail vein injection6,7,8 (and demonstrated here), intracardiac injection9, and portal vein injection10.
The purpose of the protocol presented here is to provide an in vivo experimental metastasis assay that allows a researcher to monitor metastasis formation and growth in real time, as well as to quantify end point metastasis number and size in the lungs of the same mouse. To accomplish this, traditional experimental tail vein metastasis assays6,7,8 are combined with live animal imaging, using an in vivo imaging device9,11,12,13,14. Tumor cells stably expressing both luciferase and a fluorescent protein are injected into mice via the lateral tail vein and then the in vivo imaging device is used to measure changes in metastatic burden in the lungs over time (Figure 1). However, the in vivo live animal imaging device cannot distinguish or measure the size of individual metastases. Thus, at the end of the experiment, a fluorescent stereomicroscope is used to count the number and measure the size of the fluorescent metastases in the lungs without the need for sectioning and histology or immunohistochemistry (Figure 1). This protocol can be used to test how altering the expression or function of a candidate gene influences metastasis formation and growth. Potential therapeutic compounds such as small molecules or function blocking antibodies can also be tested.
To demonstrate this approach, we first performed a proof of concept experiment in which the essential replication factor, replication protein A3 (RPA3) is knocked down in metastatic mouse breast cancer cells. We show that mice injected with RPA3 knockdown cells have significantly less metastatic burden at every time point compared to mice injected with control cells. Analysis of the metastasis-containing lungs shows that this reduced metastatic burden is the result of significantly reduced metastatic colonization and impaired growth of the metastases that form. To further demonstrate this technique, we tested whether simultaneous knock down of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) impairs metastatic colonization or subsequent growth. YAP and TAZ are two related transcriptional co-activators that are the critical downstream effectors of the Hippo Pathway. We15,16 and others have implicated YAP and TAZ in metastasis (reviewed in17,18,19), suggesting that these proteins are good therapeutic targets. Consistently, we found that mice injected with YAP/TAZ knockdown cells had significantly reduced metastatic burden. Analysis of the lungs showed that the YAP/TAZ knockdown cells formed many fewer metastases and that the metastases that did form were smaller. These experiments demonstrate how experimental metastasis assays allow a researcher to quickly and inexpensively test the role of a candidate gene in metastasis formation and growth. They further show how the combined use of live animal imaging and fluorescent quantification of metastases in whole lungs allows the researcher to better understand the steps during metastatic colonization.

<table><tbody><tr><td>10% SDS-PAGE Gel</td><td></td><td></td><td>For western blot</td></tr><tr><td>2.5% Trypsin</td><td>Gibco</td><td>15090-046</td><td>Trypsin for tissue culture</td></tr><tr><td>96 well flat bottom white assay plate</td><td>Corning</td><td>3922</td><td>For measuring luciferase and renilla signal in cultured cells</td></tr><tr><td>Alcohol wipes</td><td></td><td></td><td>For sterolizing the injection site before tail vein injecitons</td></tr><tr><td>BALB/C mice (female, 6 weeks)</td><td>Taconic</td><td>BALB-F</td><td>For tail vein metastatic colonization and burden assays</td></tr><tr><td>BSA regular</td><td>VWR Ameresco</td><td>97061-416</td><td>For western blot</td></tr><tr><td>Cell lysis buffer</td><td>Cell Signaling</td><td></td><td>For collecting protien samples</td></tr><tr><td>Celltreat Syringe Filters, PES 30mm, 0.45 &amp;mu;m</td><td>Celltreat</td><td>40-229749-CS</td><td>For filtering viral supernatant</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; and euthanasia chamber</td><td></td><td></td><td>For euthanasing the mice</td></tr><tr><td>Dual-luciferase reporter assay kit</td><td>Promega</td><td>E1960</td><td>For measuring luciferase and renilla signal in cultured cells</td></tr><tr><td>Dulbecco&amp;amp;39;s phosphate buffered saline</td><td>Himedia</td><td>TS1006</td><td>For PBS</td></tr><tr><td>EDTA</td><td>VWR</td><td>97061-406</td><td>Used to dilute trypsin for tissue culture</td></tr><tr><td>FBS 100% US origin</td><td>VWR</td><td>97068-085</td><td>Component of complete growth media</td></tr><tr><td>Fujifilm LAS-3000 gel imager</td><td>Fujifilm</td><td></td><td>For western blot</td></tr><tr><td>GAPDH(14C10) Rabibit mAb</td><td>Cell Signaling</td><td>2118</td><td>For western blot</td></tr><tr><td>Goat anti-rabbit IgG (H+L) Secondary Antibody, HRP conjugate</td><td>Thermo Scientific</td><td>31460</td><td>For western blot</td></tr><tr><td>Human embryonic kidney cells, HEK-293FT</td><td>Invitrogen</td><td>R70007</td><td>Cell line used for packging virus</td></tr><tr><td>HyClone DMEM/High clucose</td><td>GE Healthcare life sciences</td><td>SH30243.01</td><td>Component of complete growth media</td></tr><tr><td>Hygromycin B, Ultra Pure Grade</td><td>VWR Ameresco</td><td>97064-810</td><td>For antibiotic selection of infected cells</td></tr><tr><td>I3-P/i3 Multi-Mode Microplate/EA</td><td>Molecular devices</td><td></td><td>For measuring luciferase and renilla signal in cultured cells</td></tr><tr><td>Imagej</td><td></td><td></td><td>Used for image analysis of lung metastases: threshold set to 25 &amp;amp; 100</td></tr><tr><td>Immuno-Blot PVDF Membrane</td><td>Biorad</td><td>1620177</td><td>For western blot</td></tr><tr><td>Isoflurane</td><td></td><td></td><td>For mouse anesthesia</td></tr><tr><td>IVIS Lumina XRMS In Vivo Imaging System (in vivo live animal imaging device)</td><td>PerkinElmer</td><td>CLS136340</td><td>For in vivo imaging of metastatic burden</td></tr><tr><td>Leica M205 FA &amp;amp; Lecia DCF3000 G (GFP and bright field filters)</td><td>Leica Microsystems</td><td></td><td>Microscope and camera for visualing, counting and taking pcitures of metastases in the lungs; 10X magnifacation, 3.5 sec exposure, 1.4 gain</td></tr><tr><td>L-Glutamine</td><td>Gibco</td><td>25030-081</td><td>Component of complete growth media</td></tr><tr><td>Lipofectamine 3000</td><td>Life technologies</td><td>L3000008</td><td>For YAP/TAZ-TEAD reporter transfection</td></tr><tr><td>Living Image 3.2 (image software program)</td><td>PerkinElmer</td><td></td><td>Software for IVIS</td></tr><tr><td>Mouse breast cancer cells, 4T1</td><td>Karmanos Cancer Institute</td><td>Aslakson, CJ et al.,1992</td><td>Mouse metastatic breast cance cell line</td></tr><tr><td>Multi-Gauge version 3.0</td><td>Fujifilm</td><td></td><td>Software for quantifying western blot band intensity</td></tr><tr><td>Opti-MEM (transfection buffer)</td><td>Gibco</td><td>31985-062</td><td>For packaging virus and transfection</td></tr><tr><td>Penicillin Streptomycin</td><td>Gibco</td><td>15140-122</td><td>Component of complete growth media</td></tr><tr><td>Pierce BCA protein assay kit</td><td>Thermo Scientific</td><td>23225</td><td>For quantifying protein concentration</td></tr><tr><td>Pierce Phosphatase Inhibitor Mini Tablets</td><td>Thermo Scientific</td><td>A32957</td><td>Added to cell lysis buffer</td></tr><tr><td>Pierce Protease Inhibitor Mini Tablets</td><td>Thermo Scientific</td><td>A32953</td><td>Added to cell lysis buffer</td></tr><tr><td>Polybrene (hexadimethrine bromide)</td><td>Sigma-Aldrich</td><td>45-H9268</td><td>For infection</td></tr><tr><td>Puromycin</td><td>Sigma-Aldrich</td><td>45-P7255</td><td>For antibiotic selection of infected cells</td></tr><tr><td>Rodent restrainer</td><td></td><td></td><td>For restraining mice during tail vein injeciton</td></tr><tr><td>SDS-PAGE running buffer</td><td></td><td></td><td>For western blot</td></tr><tr><td>TAZ (V3886) Antibody</td><td>Cell Signaling</td><td>4883</td><td>For western blot</td></tr><tr><td>TBST buffer</td><td></td><td></td><td>For western blot</td></tr><tr><td>TC20 automated cell counter</td><td>Bio-Rad</td><td></td><td>For counting cells</td></tr><tr><td>Vectors</td><td></td><td></td><td>See Table 1 for complete list of vectors</td></tr><tr><td>VWR Inverted Fluorescence Microscope</td><td>VWR</td><td>89404-464</td><td>For visualizing fluorescence in ZSGreen labeled cells</td></tr><tr><td>Western transfer buffer</td><td></td><td></td><td>For western blot</td></tr><tr><td>XenoLight D-Luciferin K+ Salt</td><td>PerkinElmer</td><td>122799</td><td>Substrate injected into mice for in vivo bioluminescent IVIS images</td></tr><tr><td>X-tremeGENE 9 DNA transfection reagent (lipid solution for transfection)</td><td>Roche</td><td>6365787001</td><td>For packaging virus</td></tr><tr><td>YAP (D8H1X) XP Rabbit mAb</td><td>Cell Signaling</td><td>14074</td><td>For western blot</td></tr></tbody></table>

combined use of tail vein metastasis assays and real-time in vivo imaging to quantify breast cancer metastatic colonization and burden in the lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

To demonstrate the above approach, we performed a proof of concept experiment in which a critical replication factor, RPA3 was knocked down in a metastatic mouse mammary carcinoma cell line (4T122). While the protocol describes labeling the cells with both luciferase and fluorescent proteins prior to genetic manipulation, we used a modified approach because THE RNAi vectors also deliver ZsGreen (Figure 2A). First, 4T1 cells were stably transduced with a lentiviral construct encoding firefly luciferase and hygromycin resistance (pHAGE-Luciferase-IRES-Hygro). After hygromycin selection, a luciferase assay was performed to confirm stable expression of the firefly luciferase (Figure 2A). Next, the 4T1-Luciferase cells were stably transduced with retroviral vectors that express ZsGreen and either a control miR30-based shRNA, or a miR30-based shRNA previously shown to effectively target mouse RPA316,23,24. The cells were then injected into the mice via the lateral tail vein and in vivo bioluminescent signal was measured twice a week to monitor the metastatic burden (Figure 2B,C). The log10 transformed signal for each mouse was plotted as metastatic burden over time (Figure 2D) and the slopes of each plot were determined (Figure 2E). These data show the rate of change in metastatic burden is significantly reduced with RPA3 knockdown compared to control cells. Although the differences are striking, these data alone do not allow us to determine if the reduced metastatic burden is the result of less metastases or due to slower growth of metastases. Therefore, the ZsGreen-labeled metastases in the lungs were also analyzed at the end of the experiment. Mice injected with RPA3 knockdown cells had almost no metastases in the lungs, whereas the control mice had numerous large metastases (Figure 3). Furthermore, the metastases that did form from RPA3 knockdown cells were clearly smaller than control 4T1 metastases (Figure 3A). Consistent with our manual counts (Figure 3B), image analysis software counted significantly less metastases in the RPA3 knockdown mice (Figure 3C). Additionally, the total metastatic burden in the lungs (sum of the areas of each metastasis in millimeters) was also drastically reduced by RPA3 knockdown (Figure 3D). Finally, the size of the individual metastases that formed in the RPA3 knockdown mice were generally much smaller than those in the control mice (Figure 3E). In addition to the large metastases in the control mice, we did observe numerous small metastases as well, but cannot determine if these are tumor cells that seeded the lung and did not grow or if they are tumor cells shed from one of the larger metastases that re-seeded the lung in a new location (Figure 3E). Collectively these data show that RPA3 is required for metastasis formation by 4T1 cells. These findings were expected because our previous work showed that RPA3 knock down cells had significantly reduced ability to form metastases in the lungs following tail vein injection16. However, this experiment demonstrates how the use live animal imaging and fluorescent quantification of metastasis number and size can be used to determine if changes in metastatic burden are due to differences in metastatic colonization or growth.
To demonstrate how this approach can be used to answer a more biologically relevant question, we asked if YAP and TAZ are required for metastatic colonization and subsequent growth in this syngeneic model of breast cancer. The expression and activity of YAP or TAZ are increased in many cancers compared to normal tissue and this is predictive of poor patient outcome25,26,27,28,29. Additionally, several studies have implicated YAP, TAZ, or TEADs, the critical YAP/TAZ-binding partners, in metastasis15,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52. Given this data we used our approach to test if YAP and TAZ are required for metastasis formation and growth. For this, we use a retroviral vector expressing tandem miR30-based shRNAs targeting both YAP and TAZ. As shown, this tandem shRNA effectively reduces YAP and TAZ protein expression and transcriptional activity in 4T1 cells (Figure 4A,B). 4T1-Luciferase cells were stably transduced with a ZsGreen-expressing version of this tandem YAP/TAZ shRNA vector or a control miR30-based shRNA (Figure 4C) and then assayed by tail vein injections in syngeneic BALB/c mice. As shown in Figure 4, the rate at which metastatic burden increased was significantly faster in the mice injected with control cells compared to the mice injected with YAP/TAZ knockdown cells (Figure 4D-F). Significantly fewer metastases formed in the mice injected with YAP/TAZ knockdown cells compared to mice with control cells whether counted manually (Figure 5A,B) or using the image analysis software (Figure 5C). Not only did many more metastases form in the control mice, but they were generally larger (Figure 5E). Consistently, the metastatic burden in the lungs (total metastasis area in mm) was also drastically reduced by YAP/TAZ knockdown (Figure 5D). It is important to note that when the metastases are large and close together, the software may be unable to differentiate distinct metastases. This was the case for a few metastases in control mice (mouse 3 and mouse 7). Thus, it is important to check the output of the image analysis software to ensure it is accurately measuring the area of single tumors. This is also why it is important to optimize the number of cells being injected and the length of the experiment. Collectively, these data show that YAP and TAZ are required to sustain the rate at which metastatic burden increases in a syngeneic murine model of metastatic breast cancer and that this is due to the inability of metastases to form and reduced growth of the few metastases that did form.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60687/60687fig01.jpg" /> 
Figure 1: Schematic of the protocol. All necessary viruses are first packaged in HEK-293FT cells. The desired cells for the study are then simultaneously infected with luciferase and fluorescent viruses that each express a unique mammalian drug selection gene. Infected cells are then expanded in the appropriate media containing both drugs to select for stably transduced cells expressing both luciferase and the florescent protein. Expression of both labels is confirmed as described in the protocol. The labeled cells are then transduced with a third virus a containing genetic manipulation (miR30 shRNA) and a third unique drug selection gene. After selection with the third drug, the cells are injected into the mice via the lateral tail vein. Metastatic burden is monitored in the mice throughout the experiment with an in vivo live animal imaging system. At the end of the experiment, mice are euthanized, and images of the whole lungs are taken using a fluorescent dissecting microscope, and then used to measure the number and size of metastases. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60687/60687fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60687/60687fig02.jpg" /> 
Figure 2: Luciferase-based in vivo live animal imaging shows that RPA3 knockdown reduces breast cancer metastatic burden. (A) Schematic showing how the cells were generated for this in vivo study. 4T1 cells were stably transduced with a lentivirus encoding luciferase and hygromycin resistance. Infected cells were selected with 600 &#956;g/mL of hygromycin B for one week and then luciferase activity was measured in parental or 4T1-luciferase cells using a luciferase reporter kit and plate reader (n = 1 experiment with replicate wells averaged). 4T1-Luciferase cells were then infected with retrovirus that deliver ZsGreen and miR30 shRNAs targeting either mCherry (control) or mRPA3. Puromycin selection (2.5 &#956;g/mL) for 3 days was used to select for stable integration of the virus. (B-E) 4T1-luciferase cells stably expressing ZsGreen and control (sh-mCherry) or mRPA3 (sh-mRPA3-431) shRNAs were injected into mice and imaged for bioluminescence on the indicated days as described in the protocol. (B&#38;C) Bioluminescent images for a representative mouse of each group on the indicated day post injection (D) Plot of the log10 transformed luciferase signal measured for each mouse over the course of the experiment. (E) Scatter plot with mean (solid line) for the slopes of each mouse in D (n = 6 for sh-control and 8 for sh-mRPA3-431). Statistical significance was tested using a Student&#39;s t test; *p &#8804; 0.05. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60687/60687fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60687/60687fig03.jpg" /> 
Figure 3: Quantification of fluorescent metastases reveals that RPA3 knockdown prevents metastatic colonization and subsequent growth of breast cancer cells. (A) Fluorescent (left) and brightfield (right) images of representative lobes from each mouse injected in Figure 2 21 days after injection. Asterisks (*) indicated green autofluorescent bronchi. (C-E) Image analysis software (see Table of Materials) was used to identify and measure objects using an intensity threshold of 25 to 100 and a size threshold of 0.01 mm2 to infinity. (B&#38;C) Scatter plot with the Mean (solid bar) of the total number of metastases in the lungs of each mouse when counted (B) by image analysis software (C). (D) The total area (mm) of lung that was measured with image analysis software is plotted as metastatic burden with the Mean indicated by a solid bar. (E) The size of each metastasis is plotted for every mouse. Control mice are indicated by the blue dots and mRPA3 knockdown mice by the red dots. Note, the scale bar is separated at 1 mm to make it easier to visualize the size and number of small metastases (n = 6 sh-control, 8 sh-mRPA3-431). Statistical significance was tested using a Student&#39;s t test; p = 0.06; ** p &#8804; 0.01. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60687/60687fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60687/60687fig04.jpg" /> 
Figure 4: YAP/TAZ knockdown reduces breast cancer metastatic burden. (A&#38;B) 4T1 cells stably expressing miR30-based control (mCherry) or tandem YAP/TAZ shRNAs (sh-mYAP1/mTAZ6) were analyzed by Western blot analysis (A) or transfected with a YAP/TAZ-TEAD luciferase reporter construct and assayed for YAP/TAZ-TEAD transcriptional activity as described previously15,16 (B) (n = 5 for control and 4 for YAP1/TAZ6). (C) Schematic showing how the cells were generated for the in vivo study. 4T1-Luciferase cells were infected with retroviruses that deliver ZsGreen and either a miR30 shRNA targeting mCherry (control) or tandem miR30 shRNAs targeting both mYAP and mTAZ. Infected cells were selected in Puromycin (2.5 &#956;g/mL) for 3 days. 4T1-luciferase cells stably expressing ZsGreen and control (sh-mCherry) or YAP/TAZ (sh-mYAP1/mTAZ1) shRNAs were then injected into mice and imaged for bioluminescence by on the injected days. (D&#38;E) Bioluminescent images for a representative mouse of each group on the indicated day post injection. (F) Plot of the log10 transformed luciferase signal measured for each mouse over the course of the experiment. (G) Scatter plot with Mean (solid bar) for the slopes of each mouse in F (n = 7 for sh-control and 8 for sh-mRPA3-431). Mean is indicated by a solid line. Statistical significance was tested using a Student&#39;s t test; p = 0.06; #, p &#8804; 0.01; ***. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60687/60687fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/60687/60687fig05.jpg" /> 
Figure 5: YAP and TAZ are required for metastatic breast cancer cells to colonize the lungs. (A) Fluorescent (left) and brightfield (right) images of representative lobes from each mouse injected in Figure 4 21 days after injection. (C-E) Images were processed with image analysis software as described in Figure 3. (B&#38;C) Scatter plot with the Mean (solid bar) of the total number of metastases in the lungs of each mouse when counted manually (B) or by image analysis software (C). (D) The total area of all metastases (mm) was measured with image analysis software and plotted as metastatic burden with the Mean (solid bar). (E) The size of each metastasis is plotted for every mouse. Control mice are indicated by the blue dots and sh-mYAP1/mTAZ6 knockdown mice by the red dots. Note, the scale bar is separated at 1 mm to better visualize the size and number of small metastases (n = 7 sh-control, 8 sh-mYA1/mTAZ6). Mean is indicated by a solid line. Statistical significance was tested using a Student&#39;s t test; *p &#8804; 0.05, **, p &#8804; 0.01; **. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60687/60687fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplemental Table 1: Table of vectors. All vectors used are listed and new vectors are described. Standard molecular biology techniques were used to clone new vectors and sequences for newly described 97-mer shRNA are included. Citations refer to: [1]53, [2]16, [3]54 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60687/Table_1.xlsx" target="_blank">Please click here to download this table.</a>
Supplemental Table 2: Example of in vivo live animal imaging data processing and analysis. Spreadsheet demonstrating how the raw data from the in vivo live animal images is converted for analysis as described in steps 6.7 and 6.8. Raw data acquired from the in vivo live animal imaging transformed using a log10 transformation. The rate of change in metastatic burden is calculated for each mouse by calculating the slope generated by the log10 transformed data. Examples include luciferase analysis from Figure 2. The columns are color-coded to indicate which data was used to generate each slope value and the formulas are imbedded in the spreadsheet. Double clicking the well will reveal the formula used to process the data. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60687/Table_2.xlsx" target="_blank">Please click here to download this table.</a>

Watch this Scientific Journal Video about Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs at JoVE.co

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

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Nanyang Technological University

Research

JoVE Journal

Cancer Research

10.8K Views.  Albany Medical College. Ons protocol kan worden gebruikt om snel en eenvoudig de rol van elk kandidaat-gen te testen in gemetastaseerde kolonisatie en groei. Het protocol maakt het ook mogelijk om realtime monitoring van metastase, vorming en groei mogelijk te maken, evenals kwantificering van metastasenummer en grootte bij dezelfde dieren zonder dat resectie of histologische kleuring nodig is. Plaat vier T1 cellen op 150.000 cellen per put in een 12-well plaat in complete groeimedia.Incubeer bij 37 graden Ce...

Video: Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

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 ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...

Discovery of Metastatic Regulators using a Rapid and Quantitative Intravital Chick Chorioallantoic Membrane Model

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be involved in differentially regulating cancer metastatic cascade genes and gene products dependent on the cancer type, tissue, and individual patient characteristics. These represent potentially important targets for genetic therapeutics and personalized medicine approaches. The development of rapid screening platforms is essential for the identification of these genetic targets.
The chick chorioallantoic membrane (CAM) is a highly vascularized, collagen rich membrane located under the eggshell that allows for gas exchange in the developing embryo. Due to the location and vascularization of the CAM, we developed it as an intravital human cancer metastasis model that allows for robust human cancer cell xenografting and real-time imaging of cancer cell interactions with the collagen rich matrix and vasculature. 
Using this model, a quantitative screening platform was designed for the identification of novel drivers or suppressors of cancer metastasis. We transduced a pool of head and neck HEp3 cancer cells with a complete human genome shRNA gene library, then injected the cells, at low density, into the CAM vasculature. The cells proliferated and formed single-tumor cell colonies. Individual colonies that were unable to invade into the CAM tissue were visible as a compact colony phenotype and excised for identification of the transduced shRNA present in the cells. Images of individual colonies were evaluated for their invasiveness. Multiple rounds of selections were performed to decreases the rate of false positives. Individual, isolated cancer cell clones or newly engineered clones that express genes of interest were subjected to primary tumor formation assay or cancer cell vasculature co-option analysis. In summary we present a rapid screening platform that allows for anti-metastatic target identification and intravital analysis of a dynamic and complex cascade of events.

This is an effective method to screen for suppressors or drivers of cancer metastasis. Cells, transduced with an expression library, are injected into the chicken chorioallantoic membrane vasculature to form metastatic colonies. Colonies having decreased or increased invasiveness are excised, expanded, reinjected to confirm their phenotype, and finally, analyzed using high throughput sequencing.

Recent advances in cancer research has illustrated the highly complex nature of cancer metastasis. Multiple genes or genes networks have been found to be ...

Modeling Brain Metastasis Via Tail-Vein Injection of Inflammatory Breast Cancer Cells

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are diagnosed with brain metastases each year. Significant progress has been made in improving survival outcomes for patients with primary breast cancer and systemic malignancies; however, the dismal prognosis for patients with clinical brain metastases highlights the urgent need to develop novel therapeutic agents and strategies against this deadly disease. The lack of suitable experimental models has been one of the major hurdles impeding advancement of our understanding of brain metastasis biology and treatment. Herein, we describe a xenograft mouse model of brain metastasis generated via tail-vein injection of an endogenously HER2-amplified cell line derived from inflammatory breast cancer (IBC), a rare and aggressive form of breast cancer. Cells were labeled with firefly luciferase and green fluorescence protein to monitor brain metastasis, and quantified metastatic burden by bioluminescence imaging, fluorescent stereomicroscopy, and histologic evaluation. Mice robustly and consistently develop brain metastases, allowing investigation of key mediators in the metastatic process and the development of preclinical testing of new treatment strategies.

We describe a xenograft mouse model of breast cancer brain metastasis generated via tail-vein injection of an endogenously HER2-amplified inflammatory breast cancer cell line.

Metastatic spread to the brain is a common and devastating manifestation of many types of cancer. In the United States alone, about 200,000 patients are ...

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several animal models have been generated, including the widely used orthotopic mouse models, where cancer cells are injected into the mammary fat pad. However, these models cannot help monitor tumor growth kinetics and metastatic colonization. Cutting-edge tools to monitor cancer cells in real time in mice will significantly advance the understanding of tumor biology. 
Here, breast cancer cell lines stably expressing luciferase and green fluorescent protein (GFP) were established. Specifically, this technique contains two sequential steps initiated by measuring the luciferase activity in vitro and followed by the implantation of the cancer cells into mammary fat pads of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. After the injection, both the tumor growth and metastatic colonization are monitored in real time by the noninvasive bioluminescence imaging system. Then, the quantification of GFP-expressing metastases in the lungs will be examined by fluorescence microscopy to validate the observed bioluminescence results. This sophisticated system combining luciferase and fluorescence-based detection tools evaluates cancer metastasis in vivo, which has great potential for use in breast cancer therapeutics and disease management.

Here, we describe a noninvasive monitoring method involving luciferase and green fluorescent protein expression in various breast cancer cell lines. This protocol provides a technique to monitor tumor formation and metastatic colonization in real time in mice.

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...

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 ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority of the practical and technical details are commonly omitted in published manuscripts which, therefore, makes it challenging to reproduce the models, particularly when it involves surgical techniques. Bioluminescence technology allows for the evaluation of small amounts of cancer cells even when a tumor is not palpable. Utilizing luciferase-expressing cancer cells, we establish a breast cancer orthotopic inoculation technique with a high tumorigenesis rate. Lung metastasis is assessed utilizing an ex vivo technique. We, then, establish a mastectomy model with a low local recurrence rate to assess the metastatic tumor burden. Herein, we describe, in detail, the surgical techniques of orthotopic implantation and mastectomy for breast cancer with a high tumorigenesis rate and low local recurrence rates, respectively, to improve breast cancer model efficiency.

We introduce a murine orthotopic breast cancer model and radical mastectomy model with bioluminescence technology to quantify the tumor burden to mimic human breast cancer progression.

In vivo mouse models to assess breast cancer progression are essential for cancer research, including preclinical drug developments. However, the majority ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

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