The authors would like to acknowledge support by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Bethesda MD, Cancer and Inflammation Program, and the Frederick National Laboratory - Small Animal Imaging Program, Leidos Biomedical Research, Inc, Frederick Maryland, USA.

Analysis of tumor growth was carried out using protocols approved by the Committee for the Ethics of Animal Experiments of the National Cancer Institute and adhered to the recommendations of the United States National Research Council&#39;s &#34;Guide for the Care and Use of Laboratory Animals&#34;, the United States Public Health Service&#39;s &#34;Guide for the Care and Use of Laboratory Animals&#34;, and the &#34;Policy on Humane Care and Use of Laboratory Animals&#34;.
1. Preparation of cells for implantation
NOTE: MDA-MB-231 human breast adenocarcinoma cells (commercially acquired) were stably transfected with the luciferase gene III and enhanced green fluorescent protein (GFP) marker by lentivirus and grown in the presence of the selection antibiotic (puromycin).
<ol>
	<li>Start the cell culture with 1 x 106 MDA-MB-231-Luc/GFP cells and add 15 mL of prewarmed (37 &#176;C) RPMI 1640 medium with 10% fetal bovine serum (FBS) in a T75 culture flask. Keep the cells growing in a temperature-controlled cell culture incubator at 37 &#176;C with 5% CO2. Replace the complete medium at least 2x a week. 
	NOTE: Do not disturb the cell growth and keep checking the cell growing conditions daily. The cells need to be subcultured when they reach 85%&#8722;90% confluency to maintain the proliferation phase.</li>
	<li>One week before the cell implantation step, remove the puromycin from the cell culture flask by first removing all the medium in the culture flask, then rinsing 2x with 10 mL of 1x phosphate buffered saline (PBS), and finally replacing the medium with complete medium without the puromycin. During the medium-adding and rinsing steps, do not disturb the cells. Remember not to use the complete medium with the selection antibiotics from now on for all medium replenishment steps.</li>
	<li>On the day of preparing the cells for implantation, remove all complete medium before trypsinization to avoid deactivating the trypsin. Add 5 mL of trypsin to the flask to trypsinize the cells. Monitor the cells and check that they detach from the culture flask.</li>
	<li>Once the majority of the cells detach, add 10 mL of complete medium to neutralize the trypsin activity. Next, transfer the cell suspension to a 50 mL centrifuge tube and centrifuge at ~150 x g to pellet the cells. Remove the supernatant after the centrifugation and then add 10 mL of 1x PBS to resuspend the cells and prepare for cell counting.</li>
	<li>Count the cells with a cell counter and adjust the cell concentration to 20 x 106 cells/mL in cold 1x PBS containing 25% basement membrane matrix. Inject 2 x 106 cells in 0.1 mL 1x PBS with 25% basement membrane matrix&#160;into the fourth mammary fat pad of each mouse. Bring extra needles and syringes for the cell implantation and keep the cell-basement-membrane-matrix mix on ice prior to implantation. 
	NOTE: There is dead space in the syringe and needle. Depending on the choice of needle and syringe length, type, and size, a tuberculin syringe with a 0.5 in 26 G needle can have up to 70 &#181;L dead space10. Prepare 40%&#8722;50% more cell implantation mix to compensate for the dead space and any other accidental loss.</li>
</ol>
2. Orthotopic breast cancer model and tumor size measurement
<ol>
	<li>Allow 6-week-old female athymic nude mice to acclimate to the housing facility for at least 1 week before the cell implantation. 
	NOTE: Using same age mice will reduce data variation.</li>
	<li>For each round, anesthetize five mice with isoflurane at 4% with filtered (0.2 &#181;m) air at a flow rate of 1 L/min for 3&#8722;4 min. Pay attention to the mouse respiratory rate and pattern change, and use a toe pinch to ensure that the mice are under proper anesthesia. Once they are no longer moving, remove one mouse and place a nose cone over its nose and mouth with the same anesthesia.</li>
	<li>Swab the left 4th mammary gland with alcohol. Using fine rat tooth forceps, lift the 4th mammary gland slightly to insert the 25 G needle. Make sure the needle is bevel up and then slightly pull the plunger to make sure the needle does not hit any blood vessels. If no blood enters the syringe, continue to slowly inject 100 &#181;L of the cell-basement-membrane-matrix mix into the mammary fat pad. 
	NOTE: A round, raised area will appear underneath the skin.</li>
	<li>Wait 10&#8722;15 s for the basement membrane matrix to harden, then remove the needle. Repeat the process with the remaining mice in the induction chamber. Place all used needles and syringes in a sharp box.</li>
	<li>Use the same mammary gland injection site for all mice. Keep a close eye on the injection site and note any cell implantation mix leaking out from the injection site. 
	NOTE: Within a few minutes, the mouse will start to regain consciousness.</li>
	<li>Allow the xenograft to grow freely without any interruption for 7&#8722;10 days prior to any tumor size measurement.</li>
	<li>Examine all mice for the xenograft size. Measure the tumor size with a caliper. Determine the tumor volume (mm3) with the following equation: 
	 
	<img src="/files/ftp_upload/60316/60316eq1.jpg" /> 
	 
	where L is the longest dimension and W is the shortest dimension perpendicular to L.</li>
	<li>Identify and remove outliers exceeding one standard deviation of the mean. Only use mice with comparable tumor sizes for experiments. Randomly divide the mice into different treatment groups and begin treatments.</li>
	<li>Measure the tumor size by caliper 2x a week. Keep note of all the physical changes of the xenografts (i.e., necrosis, laceration).</li>
</ol>
3. In vivo bioluminescence and ex vivo fluorescence imaging
NOTE: In this study, both two-dimensional bioluminescence and fluorescence imaging are conducted at the endpoint of the experiment. The bioluminescence imaging (BLI) was performed using a commercial preclinical optical scanner equipped with a 16-bit cooled CCD camera and heated imaging stage.
<ol>
	<li>Before imaging, anesthetize up to five tumor-bearing mice in the induction chamber by setting isoflurane at 3% with filtered (0.2 &#181;m) air at a flow rate of 1 L/min for 3&#8722;4 min. Determine anesthesia by a toe pinch. Ensure that the induction chamber is kept inside a vented hood to minimize staff exposure to isoflurane.</li>
	<li>Administer D-luciferin (150 mg/kg) to the anesthetized mice by an intraperitoneal route using a 27 G needle, and transfer an anesthetized mouse to the imaging chamber. At the scanner imaging chamber, maintain the isoflurane at 2%&#8722;2.5% with O2 as a carrier with a flow rate of 1 L/min. 
	NOTE: Maintain the body temperature of the mouse at ~37 &#176;C during the procedure by keeping a heated pad under the anesthesia induction chamber, imaging table (if the stage is not heated), and postprocedure recovery cage.</li>
	<li>Before imaging the study mice, obtain luciferin kinetics by imaging three extra tumor bearing mice (animals not assigned to any study groups) at 2 min intervals for a total of 40 min using the following parameters: excitation filter-blocked, emission filter-open, f/stop 1, FOV-D, medium binning (8 x 8), and auto exposure. Use the peak bioluminescence signal measured to define the optimal image acquisition time window for all subsequent time points.</li>
	<li>While performing metastasis imaging, shield the high BLI signal from the primary tumor by covering it with a sleeve cut out from a black glove. Acquire the image using the same parameters described in step 3.3 with the ventral side of the mouse facing the camera for lung metastasis imaging and the dorsal side of the mouse facing the camera for the brain metastasis imaging. 
	NOTE: It is essential to test a few different brands of black gloves to pick the one with minimal auto luminescence.</li>
	<li>Using a scanner and data analysis software, draw a standard shape region of interest (ROI) over the thoracic cavity or the brain ensuring that the entire organ of interest is covered to evaluate the metastatic burden. Quantify the bioluminescence (BL) output as total flux (photons/second).</li>
	<li>Immediately after BLI, euthanize the mouse via CO2 asphyxiation (as per ACUC guidelines and institution-approved animal protocols) and extract the lung and the brain with dissecting scissors and forceps for ex vivo imaging. 
	NOTE: The lung extracted for ex vivo imaging and for lung metastasis nodule counting cannot be performed on the same mouse due to incompatible procedures.</li>
	<li>Quickly rinse all organs with 1x PBS to remove any superficial bloodstains and place organs on a low-autofluorescence, black plastic plate. Transport organs to a multispectral fluorescence scanner equipped with spectral unmixing capability (solid-state liquid crystal wavelength tuning) with a 12-bit CCD camera for GFP detection.</li>
	<li>Acquire multispectral GFP images (excitation filter = 457 &#177; 23 nm; emission filter = 490 nm long pass) of the extracted organs and non-tumor bearing control mice by scanning through 500&#8722;720 nm at a step size of 10 nm. Use excised organs from non-tumor bearing control mice to correct for autofluorescence.</li>
	<li>After multispectral GFP imaging, determine the optimal imaging settings. Perform image acquisition of all target organs and conduct image analysis (i.e., generate a spectral library for autofluorescence and GFP for spectral unmixing procedure) according to the manufacturer&#39;s protocol.</li>
</ol>
4. Molecular detection of metastatic breast cancer cells
<ol>
	<li>After the ex vivo imaging, snap freeze the whole brain in liquid nitrogen and store at -80 &#176;C in a freezer until ready for the DNA extraction.</li>
	<li>For DNA extraction, put the whole brain in a 5 mL homogenizing tube with 2 mL of DNA lysis buffer. Use a homogenizer with the power setting at 50 to homogenize the snap-frozen whole brain tissue. Once the brain tissue is totally homogenized, transfer the 1 mL suspension (lysate) to a DNase-free and RNase-free 2 mL microcentrifuge tube and continue to isolate the DNA according to the manufacturer&#39;s protocol. 
	NOTE: To minimize carryover DNA contamination, thoroughly clean the homogenizer after each sample by first rinsing it with 75% ethanol in DNase-free and RNase-free water, then rinsing with 1 M NaOH solution, followed by a rinse with 75% ethanol in DNase-free and RNase-free water to effectively reduce and destroy any DNA residue.</li>
	<li>Add 0.5 mL of 100% ethanol of DNA lysis buffer to the lysate. Mix sample by inversion and store at room temperature for 3 min. 
	NOTE: Do not invert too many times or too vigorously. The DNA will be visible as a wool-like precipitate.</li>
	<li>Use a pipette tip to transfer the DNA to a fresh DNase-free and RNase-free 2 mL microcentrifuge tube. Let the sample air dry for 1 min. Add 1 mL 75% ethanol and invert the microcentrifuge tube 3&#8722;6x to wash the DNA sample. Discard the ethanol after each wash by pipetting. Repeat the wash step 2x.</li>
	<li>Air dry the DNA sample for 10 s. Then add 52 &#181;L of 8 mM NaOH to dissolve the DNA sample. Pipet 2 &#181;L of the DNA sample for DNA quantitation, and use the remaining&#160;50 &#181;L for a real-time PCR assay after concentration adjustment. 
	NOTE: NaOH helps to fully solubilize the DNA sample.</li>
	<li>Quantitate 2 &#181;L of each DNA sample with a spectrophotometer and use an absorbance ratio between A260/280 as a quality check reference. Calculate the DNA concentration with this formula: 
	 
	<img src="/files/ftp_upload/60316/60316eq2.jpg" /></li>
	<li>Adjust the DNA concentration to 50 ng/&#181;L. For each DNA sample, use 50 ng of DNA as the starting template for a real-time PCR assay. Use 8 mM NaOH solution or DNase-free and RNase-free water to dilute the DNA sample.</li>
	<li>Design the primer pair specific to the exogenous green fluorescence protein (GFP) sequence in-house using an open source primer design web portal for real-time PCR detection of the GFP tag in the metastatic cells that invaded and colonized distal sites. 
	NOTE: This study used F: 5&#39;-AGAACGGCATCAAGGTGAAC-3&#39;, R: 5&#39;-TGCTCAGGTAGTGGTTGTCG-3&#39;.</li>
	<li>Use a fast real-time PCR reagent and a real-time PCR machine that supports a fast real-time PCR protocol. In addition to the regular 30 cycle amplification steps, add a dissociation curve step to the end of the run for the detection of any nonspecific amplification. 
	NOTE: The following conditions were used for molecular detection: 95 &#176;C 2 min hot start, 40 cycles of 95 &#176;C 5 s, 60 &#176;C 15 s, and lastly a melting&#160;curve analysis. The GFP amplicon is 135 bp in size.</li>
	<li>Use a naive mouse brain DNA (not a MDA-MB-231 cell implant) as a negative control. Use DNA extracted from the MDA-MB-231-GFP/Luc cells as a positive control. Add all the PCR reagents without any DNA template as a no template control (NTC). 
	NOTE: A PCR control is needed for each PCR assay.</li>
</ol>
5. Metastatic breast cancer cell detection in lung
<ol>
	<li>For the mice subgroup chosen for the metastatic lung nodule counting, anesthetize mice with isoflurane (as in step 2.2) immediately after the ex vivo imaging, perform a cardiac puncture for blood collection, and then euthanize by cervical dislocation.</li>
	<li>Open the chest cavity with dissecting scissors and cut past the heart and thymus to expose the trachea.</li>
	<li>Insert a 10 mL syringe with a 22 G needle containing Bouin&#39;s solution into the trachea. Push the syringe plunger until the collapsed lungs are swollen with ~2 mL of the Bouin&#39;s solution. Remove the syringe and needle once the lungs have been inflated.</li>
	<li>Place the whole lung into a 15 mL tube containing Bouin&#39;s solution, gently invert the tube a few times, and allow to soak for 24 h at room temperature.</li>
	<li>After 24 h, remove the Bouin&#39;s solution from the tube, rinse the lung with water, and then place it back in the tube with 70% ethanol. Count metastasis (white patches or nodules) on the surfaces of the lungs using a dissecting microscope.</li>
</ol>
6. Data collection and analysis
<ol>
	<li>Record tumor volume data, as well as in vivo and ex vivo imaging data, on an electronic spreadsheet for easy data entry, collection, and management.</li>
	<li>At the end of the experiment, transfer all data to the electronic spreadsheet and statistical software for data plotting and statistical analyses.</li>
</ol>

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The authors have nothing to disclose.

For the study of TNBC in animals, two murine models have been developed: the MDA-MB-231 human breast adenocarcinoma cells in immune-compromised mice (i.e. athymic nude mice, NSG mice), and the 4T1 in immune-competent BALB/c mice. Both models have their advantages. The choice of the animal model for a study depends on the research goals. For example, the MDA-MB-231 model is a human TNBC cell line grown in immunocompromised mice that mimics immunosuppressed human breast cancer patients. On the other hand, the invasive phenotype of orthotopic 4T1 triple-negative murine breast cancer cells in BALB/c mice closely mimics the metastatic process as it occurs in stage IV human breast cancer patients. Unlike the intravenous cell injection approach, human MDA-MB-231 breast cancer cells were similarly injected into the mammary fat pad11,12 in the orthotopic breast cancer model11,13. The longer tumor growth and the acquired metastatic ability is more physiologically relevant, thus it is not an artificial metastatic cancer model4,14. Such a spontaneous metastasis model closely mimics human breast cancer development except for the initiation stage. This is a crucial model for in vivo drug screening and therapeutic efficacy assessment in metastatic breast cancer.
The tumor implantation site in the mouse plays a crucial role in providing a microenvironment that sustains tumor growth and the selection of metastatic phenotype similar to that which occurs in humans. The proximal lymph node and the presence of adipose tissue are the key factors affecting the disease progression of breast cancer15,16. In a human patient, the lymph node and adipose tissue are both key interacting factors affecting the malignancy and incidence of breast cancer17,18,19. Thus, the selection of the correct anatomical location for the injection site can highly impact the relevancy of the tumor model compared to the human disease. This study used the fourth mammary gland as the implantation site mainly due to the aforementioned requirements and that it is anatomically more accessible and easier to manipulate.
Different tumor volume calculation methods are available, and a researcher can choose any they see fit. The algorithm selected for this study is based on the findings by Faustino-Rocha et al., who compared different tumor volume calculation formulas and concluded that the formula below is the most accurate20.
<img src="/files/ftp_upload/60316/60316eq3.jpg" />
Basement membrane matrix is an important extracellular matrix used in various in vitro21 and in vivo22,23 assays. There are contradicting reports22,24,25 regarding the influence of the basement membrane matrix on xenograft malignancy. It seems to only affect the initial establishment of the xenograft and have no further effect on xenograft growth25. For the xenograft implantation described, the basement membrane matrix was mixed with the cancer cells to increase the cell/gel mix solution viscosity prior to implantation. The presence of the basement membrane matrix reduces the loss of mix solution from the injection site and keeps the mix solution at the implantation site, thus increasing the uniformity of the implanted xenograft volume.
The MDA-MB-231 cell line is a malignant immortalized human breast adenocarcinoma cell line and is a popular tool in breast cancer research because of its triple-negative status. The use of dual reporters (luciferase and GFP) cell line allows more flexibility in handling the in vivo and ex vivo imaging. It is well established that the bioluminescence signal possesses greater sensitivity, depth detectability, and superior contrast (signal-to-noise ratio) than GFP signals. Because of this, it is a widely used imaging modality for whole body imaging. Unfortunately, bioluminescence detection is limited by a narrow time window (~15&#8722;20 mins post luciferin injection) during which the signal detection is linear. BLI signal diminishes rapidly when the animals are euthanized. This becomes an experimental design issue if many mice need to be euthanized and harvesting multiple tissues or organs is required. In these studies, blood was harvested by direct heart puncture, the brain, primary tumor, lung, and affected lymph node were examined in 40 mice. By the time the organs were harvested and ready for ex vivo imaging, the bioluminescence signal was undetectable. Therefore, GFP detection is more suitable in these situations. The emission of the GFP signal is in the visible range, and at these wavelengths, signal absorption due to blood (i.e., hemoglobin) is significantly higher. Also, autofluorescence in the visible range due to NADH, lipo-pigments, and flavins results in a significant background that makes it difficult to distinguish between a low-level GFP signal and autofluorescence background. Employing a multispectral fluorescence imaging approach instead of traditional filter-pair imaging and using spectral unmixing algorithms helps identify true GFP signal in the organ of interest. Hence, by combining the strengths of bioluminescence imaging in whole body in vivo detection and multispectral GFP imaging in the ex vivo organ/tissue evaluations, quantifiable data can be maximized in a large cohort of mice.
No matter which approach is selected for an animal study, extracting all blood prior to organ/tissue retrieval is highly recommended, especially for studies targeting the metastatic burden. This protocol detects GFP signals from the whole blood samples obtained from the mice (data not shown) in the orthotopic breast cancer model by real-time PCR assays. Minimizing the blood volume in organs/tissues will reduce the false positive signal in the target organs.
In conclusion, the orthotopic breast cancer model using the MDA-MB-231-Luc/GFP cells is a highly relevant animal model that closely mimics the human TNBC patient condition. This model is essential for studying, monitoring, and assessing therapeutic efficacy in a tumor microenvironment similar to human beings. The use of dual reporter cell lines further enhances the practicality of this orthotopic breast cancer model.

Approximately one in eight women in the United States will develop invasive breast cancer during her lifetime, and 10%&#8722;20% of these women will be diagnosed with the aggressive triple negative breast cancer (TNBC) subtype. While primary lesions can be surgically removed in most cases, the subclinical micrometastasis and chemoresistance make it an intractable disease. Importantly, most patients with metastatic TNBC eventually relapse, even if they underwent treatment in the early stage1. Thus, cancer heterogeneity, micrometastasis, and therapeutic resistance are three major challenges limiting the successful clinical outcome of TNBC patients. Hence, there is an urgent need to better understand the polymorphic molecular background of TNBC and develop effective therapeutic agents that limit metastatic disease.
Tumor metastasis is a multistep process where the tumor cell controls and usurps its microenvironment to promote its own dissemination via membrane degradation and tumor cell escape from the primary lesion, via entry into (i.e., intravasation) and exit from (i.e., extravasation) the vasculature, and ultimately adaptation and colonization within distal tissue beds2. Animal models have been developed to study breast cancer metastasis, where two methodologies are commonly implemented: direct blood circulation injection and orthotopic implantation. Commonly employed methods for direct blood circulation injection include tail vein injection, while other approaches including direct cardiac injection3, direct brain injection4, and direct liver injection5 have also been employed. The direct blood circulation injection is often referred to as an artificial metastasis model, which is quick and easy but less physiologically accurate because it circumvents tumor escape from the primary lesion and intravasation6,7,8. When compared to direct injection models, the orthotopic breast cancer model takes longer for the occurrence of detectable metastatic lesions in remote organs such as the lung, but it is more physiologically relevant because it closely mimics the multistep metastatic process as it occurs in humans. Importantly, a 2013 study9 compared the tail vein injection and orthotopic models and found that the breast cancer cells injected into the tail vein and those isolated from lung metastatic lesions after tail vein injection exhibited similar global gene expression profiles. In contrast, the global gene expression profile of orthotopically injected breast cancer cells was dramatically different than that of lung metastatic lesions arising from orthotopically injected cells9. These observations suggest that the orthotopic model is more physiologically relevant, because the metastatic lesions undergo a selection process similar to the multistep process of metastasis as it occurs in humans.
This work describes an orthotopic breast cancer (MDA-MB-231-Luc/GFP) model in nude mice that was optimized in our laboratory for imaging detection techniques as well as the identification of novel biomarkers and development of targeted chemotherapeutic agents.

<table><tbody><tr><td>Bouins Solution</td><td>Sigma</td><td>HT10132-1L</td><td>Lung metastatic nodule staining</td></tr><tr><td>D-Luciferin, Potassium Salt</td><td>GoldBio</td><td>LUCK-1G</td><td>Luciferase substrate</td></tr><tr><td>DNAzol</td><td>ThermoFisher</td><td>10503027</td><td>DNA extraction Kit</td></tr><tr><td>Excel</td><td>Microsoft</td><td></td><td>Spreadsheet software</td></tr><tr><td>homogenizer</td><td>Virtis</td><td>Cyclone Virtishear</td><td>For tissue homogenization</td></tr><tr><td>IVIS SPECTRUM scanner</td><td>Perkin Elmer</td><td></td><td>fluorescence and BLI imaging system</td></tr><tr><td>Maestro GNIR-FLEX fluorescence scanner</td><td>Perkin Elmer</td><td></td><td>fluorescence imaging system</td></tr><tr><td>MatriGel Matrix</td><td>Corning</td><td>356234</td><td>Store at -20C and keep old (4 C) when in use.</td></tr><tr><td>MDA-MB-231 / Luciferase-2A-GFP Stable Cell Line</td><td>GenTarget</td><td>SC044</td><td>Dual Reporter human breast cancer cell line</td></tr><tr><td>Microscope</td><td>ThermoFisher</td><td>EVOS</td><td>histology image capture</td></tr><tr><td>Phosphate-Buffered Saline</td><td>ThermoFisher</td><td>10010049</td><td>rinse buffer</td></tr><tr><td>Primer3</td><td>MIT</td><td></td><td>Primer Design</td></tr><tr><td>Prism</td><td>GraphPad</td><td></td><td>Statistical Analysis Software</td></tr><tr><td>Puromycin</td><td>ThermoFisher</td><td>A1113803</td><td>Antibiotics</td></tr><tr><td>RPMI 1640 media</td><td>ThermoFisher</td><td>61870127</td><td>Culture media</td></tr><tr><td>SeniFAST SYBR Lo-ROX kit</td><td>Bioline</td><td>BIO-94020</td><td>Fast Real-Time PCR Reagent</td></tr><tr><td>StepOne Plus Real-Time PCR system</td><td>ThermoFisher</td><td></td><td>Real-Time PCR machine</td></tr></tbody></table>

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.

Tumor volume measurement by caliper is a well-established method to assess treatment efficacy (Figure 1). The number of cells implanted, use of basement membrane matrix, microbiome, facility cleanliness, and injection site are the key factors impacting the tumor growth rate.
Unlike the GFP imaging, BLI required the administration of luciferase substrate at least 15&#8722;20 min prior to BLI imaging. Furthermore, the mouse strain, cell line, treatments, and luciferase reporter all affect the bioluminescence signal levels. Thus, to obtain comparable signals between the study groups, a pre-imaging BLI kinetic study must be conducted to determine the best imaging time frame (Figure 2).
Due to the superior signal-to-background ratio26,27, the whole body in vivo bioluminescence imaging was quite sensitive in detecting low-level metastasis signal compared to the GFP approach (Figure 3). Furthermore, the bioluminescence signal provided deeper penetration than GFP by a few millimeters. However, bioluminescence signal production requires ATP, which is a limiting factor in evaluating ex vivo tissue samples. If the animals are processed quickly, then BLI could be a viable approach to obtain quantitative results on metastasis. One way to achieve this is by processing one animal at a time. However, this approach was not feasible for this study because a large cohort of mice was used. BLI signal was not detected when the organs were imaged around 45 min post luciferin injection. The use of a dual reporter cell line is useful in this situation. Due to the stable nature of the GFP molecule, researchers would have sufficient time to process the carcass prior to capturing the GFP signals from target organs (Figure 4).
Figure 5 provides a real-life example of how caliper tumor size measurements could distort the data interpretation, because the xenograft lost most of the viable tumor cell content but still maintained its large mass and shape. Histopathological examination of those xenografts indicated necrosis inside the mass (Figure 5B).
For researchers who challenge the imaging results regarding the brain metastasis in the orthotopic breast cancer model, molecular detection by real-time PCR can provide convincing evidence (Figure 6). In this case, the exogenous GFP DNA sequence was the best way to address this problem, because the transfected GFP DNA sequence does not naturally exist in humans or rodents.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60316/60316fig1.jpg" /> 
Figure 1: Tumor volume measurement. Tumor volume/loading measurement with a caliper started on day 10 after xenograft implantation and then was measured 2x a week until the end of the experiment. Error bars are calculated by SEM value. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60316/60316fig1large.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/60316/60316fig2.jpg" /> 
Figure 2: Determination of optimal image acquisition window. Left: One mouse with a large xenograft and one mouse with a small xenograft were selected for the optimal luciferin kinetic range determination. Right: Luciferin kinetic curve obtained by acquiring the images every 2 min for 40 min. A plateau was observed between 15&#8722;22 min, which was used as an optimal image acquisition time for all subsequent imaging. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60316/60316fig2large.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/60316/60316fig3.jpg" /> 
Figure 3: Whole body BLI imaging. L: Whole body BLI imaging. M: Ventral view with the lower body covered with a sleeve of a black glove. R: Dorsal view with the lower body covered with a sleeve of a black glove. An axillary lymph node metastasis is detected in the ventral view by BLI imaging. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60316/60316fig3large.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/60316/60316fig4.jpg" /> 
Figure 4: Ex vivo BLI/GFP signal detection. GFP signals were detected in the brain and lung from the same mouse 20 min after the mouse was sacrificed. BLI signal was not detected 45 min post luciferin injection. No GFP signal was detected in naive mouse brain and lung. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60316/60316fig4large.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/60316/60316fig5.jpg" /> 
Figure 5: Advantages of optical imaging techniques over caliper measurements. (A) Representative image showing in vivo GFP signal detection in a primary tumor. Only a minor portion of the xenograft showed GFP signal, suggesting that a significant portion of the mass was stroma. This cannot be determined by traditional caliper measurements and can lead to an inaccurate conclusion. This example highlights the importance of optical imaging for the animal model study. (B) Histopathology section of the same mouse showing that the necrotic region (i.e., inner region) also contributed to the tumor size calculation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60316/60316fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/60316/60316fig6.jpg" /> 
Figure 6: Melting curve analysis. The melt curve plot showing the specificity of GFP amplicons. DNA from the brain obtained from the MDA-MB-231-Luc/GFP-implanted mice and the DNA isolated from the MDA-MB-231-Luc/GFP cells grown in culture (positive control) were assayed. The real-time PCR assay was performed to assess the GFP content in the brain tissue. (A,B) The melting curves of the positive control and the DNA extracted from mouse brain, respectively. (C) The overlapped curves of panels A and B, indicating the peak signals from the mouse brain DNAs are specific for the exogenous GFP sequence. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/60316/60316fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model at JoVE.com

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

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 ...

studying-triple-negative-breast-cancer-using-orthotopic-breast-cancer

Nanyang Technological University

Research

JoVE Journal

Cancer Research

17.4K Views.  National Cancer Institute at Frederick. 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.

Video: Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

Orthotopic Injection of Breast Cancer Cells into the Mice Mammary Fat Pad

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, xenografts, genetic manipulation, chemical reagents induction, etc.) have various pathological characters and play important roles in investigating certain aspects of diseases. Although no single model can totally mimic the whole human disease progression, orthotopic organs disease models with a proper stromal environment play an irreplaceable role in understanding diseases and screening for potential drugs. In this article, we describe how to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and follow the metastasis to distant organs. With the proper features of primary tumor growth, breast and nipple pathological changes, and a high occurrence of other organs&#39; metastasis, this model maximumly mimics human breast cancer progression. Primary tumor growth in situ, long-distant metastasis, and the tumor microenvironment of breast cancer can be investigated by using this model.

Here, we present a protocol to implant breast cancer cells into the mammary fat pad in a simple, less invasive, and easy-to-handle way, and this mouse orthotopic breast cancer model with a proper mammary fat pad environment can be used to investigate various aspects of cancer.

A proper animal model is crucial for a better understanding of diseases. Animal models established by different methods (subcutaneous injections, ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

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 ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Medicine

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 ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer ...

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 ...

Orthotopic Injection: Implanting Tissue Specific Cancer Cells into an Adult Mouse

Source: Katsuta, E., et al. <a href="https://www-jove-com.remotexs.ntu.edu.sg/v/57849/generating-murine-orthotopic-metastatic-breast-cancer-model">Generating a Murine Orthotopic Metastatic Breast Cancer Model and Performing Murine Radical Mastectomy.</a>&#160;J. Vis. Exp.&#160;(2018).
The video demonstrates the breast cancer orthotopic inoculation technique in a mouse model. This technique produces a high tumorigenesis rate with less variability in tumor size and shape compared to subcutaneous or non-surgical injection.