We thank the members of the Y.D.S. laboratory. We would like to thank The Wohl Institute for Translational Medicine at the Hadassah Medical Center, Jerusalem, for providing the small animal imaging facility. This study was supported by Research Career Development Award from the Israel Cancer Research Fund.

All mouse experiments were carried out under the Hebrew University Institutional Animal Care and Use Committee-approved protocol MD-21-16429-5. In addition, the Hebrew University is certified by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).
1. Cell line maintenance
NOTE: The human breast cancer cell lines (MCF-7, MDA-MB-468, and MDA-MB-231) were used in this protocol.
<ol>
	<li>Culture all the breast cancer cell lines in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 &#176;C in a humidified carbon dioxide (5% CO2) incubator. 
	&#8203;NOTE: Check the cell density regularly; split and expand for future usage when they reach 70% confluency.</li>
</ol>
2. Virus preparation
<ol>
	<li>Treat the HEK293T cells with 1 mL of trypsin until they detach.</li>
	<li>Add 10 mL of DMEM (10% FBS) to neutralize the trypsin activity, and transfer the cell suspension to a new 15 mL centrifuge tube.</li>
	<li>Centrifuge at 150 &#215; g for 5 min to sediment the cells. Discard the supernatant after the centrifugation and add 1 mL of fresh DMEM medium.</li>
	<li>Determine the cell concentration and seed 1.2 &#215; 106 cells/well in a six-well plate.</li>
	<li>On the following day, prewarm all the necessary reagents, including transfection reagent, serum-free DMEM, envelope plasmid (VSV-G), lentivirus packaging plasmid (&#916;VPR), and pLX304 Luciferase-V5 blast plasmid or FUW GFP plasmid.</li>
	<li>In a 1.5 mL autoclaved centrifuge tube, mix 50 &#181;L of serum-free DMEM with 0.3 &#181;g of VSV-G plasmid, 1 &#181;g of &#916;VPR, and 1.2 &#181;g of pLX304 Luciferase-V5 blast plasmid or FUW GFP plasmid.</li>
	<li>After thoroughly mixing these plasmids with the medium, add 5 &#181;L of the transfection reagent, mix gently, and incubate the mixture at room temperature for 15 min.</li>
	<li>Following incubation, add the mixture dropwise to the HEK293T cells.</li>
	<li>After 24 h, replace the growth medium with 2 mL of (30% FBS) DMEM. On the following day (48 h post-transfection), harvest the medium containing the viruses (&#34;pLX304 Luciferase-V5 or the FUW GFP viruses&#34;). 
	NOTE: The use of 30% FBS enhances the virus production efficiency.</li>
	<li>To avoid any HEK293T cell residues, pass the virus-containing medium through a 0.45 &#181;m syringe filter or centrifuge at 150 &#215; g for 5 min, and collect the supernatant. 
	NOTE: For long-term storage, keep the working aliquots of the virus at -80 &#176;C.</li>
</ol>
3. Establishing cells stably expressing GFP and luciferase (&#34;GFP&#160;&#160;+&#160;Luc+ cells&#34;)
<ol>
	<li>The day before infecting the cells, seed 8 &#215; 105 cells per well in a six-well plate.</li>
	<li>After overnight incubation, replace the growth medium with fresh medium containing 8 &#181;g/mL of polybrene. Add 200 &#181;L of FUW GFP viruses dropwise to the cells.</li>
	<li>Optional: To enhance the virus efficiency, centrifuge the plate at 560 &#215; g for 30 min (37 &#176;C) (spin infection).</li>
	<li>Incubate the cells for 48 h, and verify the GFP expression by fluorescence microscopy.</li>
	<li>Sort the GFP-expressing cells by fluorescence-activated cell sorting (FACS) (GFP+) (Figure 1A).</li>
	<li>Infect the GFP-sorted cells with the pLX304 Luciferase-V5 blast viruses as described in step 3.2.</li>
	<li>Treat the cells with blasticidin (10 &#181;g/mL) 30 h post-infection to enrich for pLX304 Luciferase-V5 blast-expressing cells (GFP+, Luc+ cells). Then, every two days, replace with fresh medium containing blasticidin. Additionally, as a control, treat na&#239;ve cells with the same blasticidin-containing medium. 
	NOTE: A clear difference between the infected and control cells should be observed after a few days of blasticidin treatment. This effect is cell line-dependent and usually takes ~8-10 days. A poor survival rate will indicate a low viral production yield. If so, produce a new batch of viruses as low efficiency may affect future experiments.</li>
</ol>
4. Validating in vitro luciferase activity
<ol>
	<li>Grow the MCF-7, MDA-MB-468, and MDA-MB-231 GFP+ Luc+ cells in a 15 cm plate to 80% confluency. Harvest the cells by trypsinization, as described in steps 2.1-2.2.</li>
	<li>Seed an increasing number of cells in each well (0.1, 0.5, 1, 2, 3, 4 &#215; 104) into a black 96-well plate. 
	NOTE: Black 96-well plates are more suited for measuring luciferase levels as white or transparent plates will produce autoluminescence signals. As a control, use phosphate-buffered saline (PBS) alone in one well to ensure there is no autoluminescence from PBS.</li>
	<li>Fill all the wells with 100 &#181;L of DMEM and incubate for 16-24 h.</li>
	<li>Prepare luciferin solution in PBS at a 1.5 mg/mL concentration from the stock of 30 mg/mL. Aliquot the stock of luciferin solution and store at -20 &#176;C.</li>
	<li>Wash the cells once with PBS gently, add 100 &#181;L of luciferin solution into each well, and wait for 2 min. Finally, measure the luciferase activity in all the breast cancer cells using bioluminescence. 
	NOTE: Examining in vitro GFP and luciferase expression prior to animal experiments is crucial. Additionally, blank wells are used to subtract the background.</li>
</ol>
5. Injecting mice with GFP+ Luc+ cells
<ol>
	<li>Transfer 5 &#215; 106 (MCF-7 and MDA-MB-468) or 2 &#215; 106 (MDA-MB-231) GFP+ Luc+ cells into 200 &#181;L or 100 &#181;L PBS, respectively.</li>
	<li>Before injection, clean the sterile biological hood with 5% disinfectant solution (see the Table of Materials). Then, anesthetize the mice with filtered (0.2 &#181;m) air containing 4% isoflurane at an airflow rate of 1 L/min for 2&#8722;3 min. 
	NOTE: It is essential to confirm proper anesthetization; pinch the mouse&#39;s toe and look for any response.</li>
	<li>Place a cone over the anesthetized mouse head in a supine position. Apply vet ointment to its eyes to prevent dryness while under anesthesia.</li>
	<li>Wipe the abdominal area of the mouse, above the mammary gland, with ethanol using a cotton swab and lift the 4th mammary gland with forceps.</li>
	<li>Insert the needle 27 G x 3/4 (0.4 x 19 mm) under the fat pad and slowly inject 100 &#181;L of the cell suspension. 
	NOTE: A round bulge will appear under the skin. In addition, an improper injection may lead to deviation in the tumor growth rate or absence of tumor in the same experimental group.</li>
	<li>After the injection procedure, take the mice out of the hood and transfer them to a new cage. Monitor the mice until they return to consciousness. 
	&#8203;NOTE: Ensure that all the used needles and syringes are discarded in the sharps box.</li>
</ol>
6. Measuring the luciferase levels in GFP+ Luc+ mice
<ol>
	<li>Before the bioluminescence detection, restrain the conscious mouse by holding its neck with the left hand. Then, tilt the hand to the left, resulting in the mouse face upward with the lower body in a supine position.</li>
	<li>Inject 100 &#181;L of luciferin (30 mg/mL) intraperitoneally (i.p.) into the abdominal surface of the mouse in the lower-left abdominal quadrant using a 1.0 mL syringe with needle size 27 G x 3/4 (0.4 x 19 mm). 
	NOTE: The tip of the needle should not be inserted more than 3-5 mm from the abdominal wall, as it might penetrate visceral organs. Additionally, it is recommended to perform i.p. injection without anesthesia as luciferin distribution in the body of anesthetized mice is slower than in conscious mice. Thus, monitoring luciferase levels in conscious mice is a faster procedure.</li>
	<li>Keep the mouse for 7 min without anesthesia followed by 3 min within the anesthesia chamber before measuring the tumor kinetics. 
	NOTE: The incubation time varies between the different experiments, cell lines, and species to species. Thus, it is recommended to calibrate the incubation time before starting the experiments.</li>
	<li>Anesthetize the mice as described in steps 5.2-5.3.</li>
	<li>Open the software (Table of Materials) during the incubation, initialize the imaging system, and click the Initialize button. 
	NOTE: Be aware that the camera will take ~10 min to cool down and reach -90 &#176;C. Additionally, as background control, measure the luciferase levels in na&#239;ve mice (i.e., GFP+ mice injected with cells that do not express the luciferase gene).</li>
	<li>Keep the setup in auto exposure using the following settings: exposure time auto, 60 s; Binning Medium, F/Stop 1; Excitation filter blocked; Emission filter open. When Initialization ends, select Imaging Wizard | Bioluminescence and then click Next | Open Filter | select Mouse in the image subject.</li>
	<li>In Field View, select stage C (10 cm) and Subject Height: 1.50 cm.</li>
	<li>Click Image Setup Stage to C, and before clicking the Acquire button, ensure that the mouse is placed on the stage in the proper supine position.</li>
	<li>Close the door, click the Acquire button, and wait for an image to appear on the screen. 
	NOTE: With the Auto-Exposure option, a strong signal takes 5-20 s depending on the signal; a weak signal will take around 60 s.</li>
	<li>Repeat this step for the other mice.</li>
	<li>To detect lung metastasis, cover the strong signal of the primary tumor using thick black cardboard paper and expose only the ventral side of the lungs towards the camera. Capture the image using the same parameters described above.</li>
</ol>
7. Acquiring ex vivo image using bioluminescence and fluorescence
<ol>
	<li>Euthanize the mice using carbon dioxide (CO2) inhalation in the desiccator and dissect the mice using autoclaved scissors and forceps.</li>
	<li>Perfuse the mice using 0.9% saline, harvest the organs, and rinse with 1x PBS to eliminate bloodstains from the organ.</li>
	<li>Transfer the rinsed organ to a Petri dish and place it into the stage of the bioluminescence machine. Apply the same bioluminescence setting as described in steps 6.2-6.6. 
	NOTE: Due to the decrease in luciferase signal, this step is time-limiting. Thus, after euthanizing mice, immediately visualize the organ by bioluminescence.</li>
	<li>For GFP images, apply the same setting as described in step 6.3, using Fluorescence GFP filter instead of Bioluminescence.</li>
	<li>Take lung images using a stereo microscope to examine the presence of GFP+ colonies.</li>
</ol>
8. Bioluminescence data analysis
<ol>
	<li>Double-click the software and select Open from the File menu.</li>
	<li>Open all the files and minimize them. Ensure all the units are Radiance (Photons). Additionally, click Apply to All to keep the same parameters for all the images.</li>
	<li>From the View menu, select the Tool Palette, which will open a new window.</li>
	<li>From the Tool Palette window, select the ROI Tools tab and in Type, choose the Average Bkg ROI.</li>
	<li>From the ROI tools, select Circle, and draw a small circle on the thoracic area of the mouse.</li>
	<li>Double-click on the circle, select the Use as BKG for future ROIs option from the Background ROI tab, and click Done.</li>
</ol>
9. Measuring the total flux
<ol>
	<li>From the Tool Palette window, select the ROI Tools tab and in Type, choose the Measurement of ROI.</li>
	<li>From the ROI tools, select Circle, and draw a big circle on the primary tumor of the mouse.</li>
	<li>Right-click Copy ROI and right-click Paste ROI into every individual file of each mouse.</li>
	<li>Click Measure ROIs from the ROI Tools tab, which will open a new window named ROI Measurements. From this tab, keep the Measurements Types as Radiance (Photons) and Image Attributes as All Populated Values.</li>
	<li>Export the file as Measurements File (*.txt) or Csv (*.csv) format.</li>
	<li>Open the exported data in a spreadsheet and take the Total Flux (p/s) and the values for the weeks. 
	NOTE: This step can be used to measure different groups. For example, mice treated with a vehicle vs. those treated with a drug.</li>
	<li>Generate an XY plot, where the Time is presented along the X-axis and Total Flux (p/s) along the Y-axis. For each week, take the Total Flux (p/s) parameter for each sample group and determine any significant differences using the non-parametric Student&#39;s t-test.</li>
</ol>

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All authors have disclosed that they do not have any conflicts of interest.

Animal-based experiments are obligatory for cancer research7,8,9, and indeed many protocols have been developed3,6,10,11,12,13,14. However, most of these studies determined the biological effect onl...

Breast cancers are frequent types of cancer worldwide, with approximately 250,000 new cases diagnosed each year in the United States1. Despite its high incidence, a new set of anticancer drugs has significantly improved breast cancer patient outcomes2. However, these treatments are still inadequate, as many patients experience disease relapse and metastatic spread to vital organs2, which is the primary cause of patient morbidity and mortality. Therefore, one of the main challenges in breast cancer research is identifying the molecular mechanisms regulating the formation of distal metastases to develop new means to inhibit their development.
Cancer metastasis is a dynamic process in which cells detach from the primary tumor and invade neighboring tissues through the blood circulation. Thus, animal models in which the cells undergo a similar metastatic cascade can facilitate the identification of the mechanisms that govern this process3,4. Additionally, these in vivo models are essential for developing breast cancer therapeutic agents5,6. However, these orthotopic models cannot indicate the actual tumor growth kinetics as the effect is only determined upon termination. Therefore, we established a luciferase-based tool to detect tumor development and metastatic colonization in real time. Additionally, these cells express GFP to detect the metastatic colonies. This approach is relatively simple and does not involve any invasive procedures3. Thus, combining luciferase and fluorescence detection is a helpful strategy to advance the preclinical studies of breast cancer therapeutics and disease management.

<table><tbody><tr><td>1.7 mL eppendorf tubes</td><td>Lifegene</td><td>LMCT1.7B-500</td><td></td></tr><tr><td>10 &amp;micro;L tips</td><td>Lifegene</td><td>LRT10</td><td></td></tr><tr><td>1000 &amp;micro;L tips</td><td>Lifegene</td><td>LRT1000</td><td></td></tr><tr><td>15 mL tubes</td><td>Lifegene</td><td>LTB15-500</td><td></td></tr><tr><td>200 &amp;micro;L tips</td><td>Lifegene</td><td>LRT200</td><td></td></tr><tr><td>6 well cell culture plate</td><td>COSTAR</td><td>3516</td><td></td></tr><tr><td>96 well Plates BLACK flat bottom</td><td>Bar Naor</td><td>BN30496</td><td></td></tr><tr><td>Automated Cell Counters</td><td>Thermofisher</td><td>A50298</td><td></td></tr><tr><td>BD FACSAria III sorter</td><td>BD</td><td></td><td></td></tr><tr><td>BD Microlance 3 Needles 27 G (3/4&#039;&#039;)</td><td>BD</td><td>302200</td><td></td></tr><tr><td>BD Plastipak Syringes 1 mL x 120</td><td>BD</td><td>303172</td><td></td></tr><tr><td>Corning 100 mm x 20 mm Style Dish</td><td>CORNING</td><td>430167</td><td></td></tr><tr><td>Corning 150 mm x 20 mm Style Dish</td><td>CORNING</td><td>430599</td><td></td></tr><tr><td>Countess cell counting chamber slides</td><td>Thermofisher</td><td>C10228</td><td></td></tr><tr><td>Dulbecco&#039;s modified Eagle&#039;s medium (DMEM), high glucose, no glutamine</td><td>Biological Industries</td><td>01-055-1A</td><td></td></tr><tr><td>Eclipse 80i microscope</td><td>Nikon</td><td></td><td></td></tr><tr><td>eppendorf Centrifuge 5810 R</td><td>Sigma Aldrich</td><td>EP5820740000</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Biological Industries</td><td>04-127-1A</td><td></td></tr><tr><td>FUW GFP</td><td></td><td></td><td>Gifted from Dr. Yossi Buganim&#039;s lab (Hebrew University of Jerusalem)</td></tr><tr><td>HEK293T</td><td></td><td></td><td>Gifted from Dr. Lior Nissim&#039;s lab (Hebrew University of Jerusalem)</td></tr><tr><td>Isoflurane, USP Terrell</td><td>Piramal</td><td>NDC 66794-01-25</td><td></td></tr><tr><td>IVIS Spectrum In Vivo Imaging System</td><td>Perkin Elmer</td><td>124262</td><td></td></tr><tr><td>L-Glutamine Solution</td><td>Biological industries</td><td>03-020-1A</td><td></td></tr><tr><td>Living Image Software</td><td>PerkinElmer</td><td></td><td>bioluminescence measurement</td></tr><tr><td>MCF-7</td><td>ATCC</td><td>ATCC HTB-22</td><td></td></tr><tr><td>MDA-MB-231</td><td>ATCC</td><td>ATCC HTB-26</td><td></td></tr><tr><td>MDA-MB-468</td><td>ATCC</td><td>ATCC HTB-132</td><td></td></tr><tr><td>Pasteur pipettes</td><td>NORMAX</td><td>2430-475</td><td></td></tr><tr><td>PBS</td><td>Hylabs</td><td>BP655/500D</td><td></td></tr><tr><td>pCMV-dR8.2-dvpr</td><td>Addgene</td><td>#8455</td><td>Provided by David M. Sabatini&amp;rsquo;s lab (Whitehead institute, Boston, USA)</td></tr><tr><td>pCMV-VSV-G</td><td>Addgene</td><td>#8454</td><td>Provided by David M. Sabatini&amp;rsquo;s lab (Whitehead institute, Boston, USA)</td></tr><tr><td>Penicillin-Streptomycin Solution</td><td>Biological Industries</td><td>03-031-1B</td><td></td></tr><tr><td>Petri dish 90 mm (90x15)</td><td>MINI PLAST</td><td>820-090-01-017</td><td></td></tr><tr><td>Pipettes 10ml</td><td>Lifegene</td><td>LG-GSP010010S</td><td></td></tr><tr><td>Pipettes 25ml</td><td>Lifegene</td><td>LG-GSP010050S</td><td></td></tr><tr><td>Pipettes 5ml</td><td>Lifegene</td><td>LG-GSP010005S</td><td></td></tr><tr><td>pLX304 Luciferase-V5 blast plasmid</td><td>Addgene</td><td>#98580</td><td></td></tr><tr><td>Polybrene</td><td>Sigma Aldrich</td><td>#107689</td><td></td></tr><tr><td>Prism 9</td><td>GraphPad</td><td></td><td></td></tr><tr><td>Reagent Reservoirs</td><td>Bar Naor</td><td>BN20621STR200TC</td><td></td></tr><tr><td>SMZ18 Stereo microscopes</td><td>Nikon</td><td></td><td></td></tr><tr><td>Sodium Chloride</td><td>Bio-Lab</td><td>190359400</td><td></td></tr><tr><td>Syringe filters</td><td>Lifegene</td><td>LG-FPV403030S</td><td></td></tr><tr><td>Trypan Blue 0.5% solution</td><td>Biological industries</td><td>03-102-1B</td><td></td></tr><tr><td>Trypsin EDTA Solution B (0.25%), EDTA (0.05%)</td><td>Biological Industries</td><td>03-052-1a</td><td></td></tr><tr><td>Vacuum driven Filters</td><td>SOFRA LIFE SCIENCE</td><td>SPE-22-500</td><td></td></tr><tr><td>Virusolve</td><td></td><td>disinfectant</td><td></td></tr><tr><td>VivoGlo Luciferin, In Vivo Grade</td><td>Promega</td><td>P1043</td><td></td></tr><tr><td>X-tremeGENE HP DNA Transfection Reagent</td><td>Sigma Aldrich</td><td>#6366236001</td><td></td></tr></tbody></table>

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.

We generated breast cancer cell lines (MDA-MB-231, MCF-7, and MDA-MB-468) expressing GFP and luciferase vectors. Specifically, this was achieved by a sequential infection. First, the breast cancer cell lines were infected with a lentivirus vector expressing fluorescent GFP. The GFP-positive cells (GFP+) were sorted 2 days post-infection (Figure 1A,B) and infected with the pLX304 Luciferase-V5 vector. Then, blasticidin was used to select for luciferase to generate ...

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

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 ...

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Cancer Research

3.0K Views.  The Hebrew University-Hadassah Medical School. Met behulp van deze geavanceerde, niet-invasieve tool kunnen we real-time tumorgroeikinetiek en gemetastaseerde kolonisatie bij muizen volgen, wat een groot potentieel heeft in therapeutisch en ziektebeheer van borstkanker. Het combineren van luciferase en fluorescentiedetectie is een nuttige strategie om de preklinische studies van borstkankerprogressie en ziektebeheer te bevorderen. Dit kan worden toegepast voor geneesmiddelenstudies tegen kanker.Dit protocol is niet beperkt tot bors...

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

Monitoring Tumor Metastases and Osteolytic Lesions with Bioluminescence and Micro CT Imaging

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence imaging using IVIS Spectrum was employed to monitor the distribution and development of the tumor cells following the delivery procedure including DLIT reconstruction to measure the tumor signal and its location.
Development of metastatic lesions to the bone tissues triggers osteolytic activity and lesions to tibia and femur were evaluated longitudinally using micro CT. Imaging was performed using a Quantum FX micro CT system with fast imaging and low X-ray dose. The low radiation dose allows multiple imaging sessions to be performed with a cumulative X-ray dosage far below LD50. A mouse imaging shuttle device was used to sequentially image the mice with both IVIS Spectrum and Quantum FX achieving accurate animal positioning in both the bioluminescence and CT images. The optical and CT data sets were co-registered in 3-dimentions using the Living Image 4.1 software. This multi-mode approach allows close monitoring of tumor growth and development simultaneously with osteolytic activity.

An experimental mouse model of bone metastasis was established following intracardiac delivery of luciferase expressing mammary tumor cells. Tumor development and resulted osteolytic lesion were monitored longitudinally with bioluminescence and micro CT imaging.

Following intracardiac delivery of MDA-MB-231-luc-D3H2LN cells to Nu/Nu mice, systemic metastases developed in the injected animals. Bioluminescence ...

Medicine

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

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 ...

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.

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 ...

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 ...

Nanodrug Targeting Oncogenic miRNA-10b for Treating Metastatic Breast Cancer

Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have been shown to be associated with the metastatic potential of breast cancer and are implicated in tumor cell migration, invasion, and viability. However, it can be difficult to deliver an inhibitory RNA molecule to the tissue of interest. To overcome this challenge and deliver active antisense oligonucleotides to tumors, we utilized magnetic iron oxide nanoparticles as a delivery platform. These nanoparticles target tissues with increased vascular permeability, such as sites of inflammation or cancer. Delivery of these nanoparticles can be monitored in vivo by magnetic resonance imaging (MRI) due to their magnetic properties. Translation of this therapeutic approach into the clinic will be more accessible because of its compatibility with this relevant imaging modality. They can also be labeled with other imaging reporters such as a Cy5.5 near-infrared optical dye for correlative optical imaging and fluorescence microscopy. Here, we demonstrate that nanoparticles labeled with Cy5.5 and conjugated to therapeutic oligomers targeting oncogenic miRNA-10b (termed MN-anti-miR10b, or &#34;nanodrug&#34;) administered intravenously accumulate in metastatic sites, opening a possibility for therapeutic intervention of metastatic breast cancer.

Here, we describe the protocol for in vivo delivery of magnetic iron oxide nanoparticles carrying RNA oligomers to metastatic breast cancer in animal models, providing a clinically viable approach for the therapeutic silencing of oncogenic nucleic acids.

Metastatic breast cancer is a devastating disease with very limited therapeutic options, calling for new therapeutic strategies. Oncogenic miRNAs have ...

Bioluminescence-Based Tumor Quantification Method for Monitoring Tumor Progression and Treatment Effects in Mouse Lymphoma Models

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. Here we describe a method for bioluminescence imaging to obtain a semi-quantitative analysis of tumor burden and treatment response. This method is based on the calculation of a luminoscore, a value that allows comparisons of two animals from the same or different experiments. Current BLI instruments enable the calculation of this luminoscore, which relies mainly on the acquisition conditions (back and front acquisitions) and the drawing of the region of interest (manual markup around the mouse). Using two previously described mouse lymphoma models based on cell engraftment, we show that the luminoscore method can serve as a noninvasive way to verify successful tumor cell inoculation, monitor tumor burden, and evaluate the effects of in situ cancer treatment (CpG-DNA). Finally, we show that this method suits different experimental designs. We suggest that this method be used for early estimates of treatment response in preclinical small-animal studies.

Bioluminescence imaging is a well-known tool for localizing tumors and metastases, but quantification of these images often requires complex calculations and particular instruments. We describe the easy-to-use luminoscore method, based on precise acquisition conditions, requiring no calculations, and enabling tumor burden and treatment response to be monitored in mouse models.

Although bioluminescence imaging (BLI) shows promise for monitoring tumor burden in animal models of cancer, these analyses remain mostly qualitative. ...

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 ...

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

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