This work was supported by the Virginia-Maryland College of Veterinary Medicine (IA), the Virginia Tech Institute for Critical Technology and Applied Science Center for Engineered Health (IA), and National Institutes of Health R21EB028429 (IA).

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Virginia Tech and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Performing this protocol requires permission from the appropriate institutions and adherence to all appropriate guidelines.
1. Cell Culture
<ol>
	<li>Make complete culture media (RPMI + 10% Fetal Bovine Serum +1% Pen Strep). Revive 4T1 cells according to ATCC Protocol10 and incubate at 37 &#176;C and 5% CO2 in a T-25 flask until confluent. Change media the day after reviving to remove dead cells, and again if media is spent before cells are confluent enough to passage.</li>
	<li>Once the T-25 flask is confluent, passage cells to a T-75 flask by discarding media, washing flask with 5 mL of 1x Dulbecco&#8217;s Phosphate Buffered Saline (DPBS), and adding 500 &#956;L of Trypsin-EDTA. Incubate for 5-10 minutes at 37 &#176;C until cells detach.
	<ol>
		<li>Once detached, add 5 mL of warmed complete culture media to cells. Aspirate and transfer the 5 mL to a T-75 flask containing 15 mL of warmed complete culture media.</li>
	</ol>
	</li>
	<li>Passage cells in T-75 flasks at least four times. Do this once the flask is confluent by washing with 8 mL of 1x DPBS, adding 1 mL of Trypsin-EDTA for detaching cells, adding 10 mL of warmed media to cells, and diluting 1:6-1:8 into a new T-75 flask containing 20 mL of warmed complete culture media.</li>
	<li>Passage cells up to the appropriate number of T-150 flasks containing 40 mL of warmed complete culture media for the number of mice to be injected. Most studies will require multiple T-150 flasks to ensure enough cells for injection.</li>
	<li>When mice are ready to be injected (8 weeks old or weighing over 20 g, depending on the IACUC or institutional protocols), harvest cells by discarding media, washing each flask with 10 mL of 1x DPBS and adding 2 mL of trypsin-EDTA. Incubate for 5-10 minutes at 37 &#176;C until cells detach.</li>
	<li>Wash flask with 10 mL of complete media and transfer all contents (10 mL of media + 2 mL of trypsin-EDTA cell mixture) to the next flask. Continue to wash and collect cells from each flask using the same 10 mL of media to avoid using an excessive amount of media.
	<ol>
		<li>Once all flasks have been collected, transfer the contents into a 50 mL centrifuge tube. Collect a 10 &#956;L sample for counting in a microcentrifuge tube and centrifuge the 50 mL conical tube at 125 x g for 5 minutes.</li>
	</ol>
	</li>
	<li>While cells are being centrifuged, add 10 &#956;L of Trypan blue to 10 &#956;L of cell sample. Count cells using a hemocytometer. Once the total number of cells is determined, calculate the concentration of cells needed to inject mice for 1.2 x 106 cells per mouse (per 100 &#956;L).</li>
	<li>After centrifugation, decant media and resuspend cell pellet in correct amount of sterile 1x DPBS for 1.2 x 106 cells per 100 &#956;L. Split cell/DPBS mixture into microcentrifuge tubes for easy access with the syringe when aspirating cells for injection. Keep cells on ice and inject soon thereafter as cells will begin to die after being on ice for extended periods of time.</li>
</ol>
2. Injections
<ol>
	<li>Prepare cells for injection by tapping or gently mixing the microcentrifuge tube to resuspend the cells, and then aspirate 600 &#956;L into a 1 mL syringe. Turn the syringe upwards and pull the plunger down to bring cells away from the syringe opening. Tap the syringe to rid it of air bubbles.</li>
	<li>Attach the needle bevel up and dispense cells back into the microcentrifuge tube until only 500 &#956;L remain in the syringe. Put syringe flat on ice. 
	NOTE: 4T1 cells fall out of suspension quickly. Therefore, it is important to mix cells back into suspension by tapping frequently.</li>
	<li>Anesthetize 8 week old/&#62;20 g female BALB/c mouse using isoflurane or other approved anesthetic agent. Monitor the mouse&#8217;s breathing to assess depth of anesthesia.</li>
	<li>Once the mouse is properly anesthetized as indicated by lack of corneal reflex, place the mouse on its back. Using the thumb, pointer, and middle finger, gently hold down the mouse. Use the pointer and middle fingers to hold down the mouse&#8217;s upper body and thumb for its rear left leg. Be gentle but firm.</li>
	<li>With the bevel of the needle up, inject 100 &#956;L of cells subcutaneously into the mouse&#8217;s left abdominal mammary fat pad. Monitor for a good bleb and any leakage, and ensure the mouse wakes up and moves easily after injection.
	<ol>
		<li>Change needles between each mouse. 
		NOTE: Do not allow needle to enter the peritoneal cavity. This would cause the cancer to spread quickly and not be representative of the model. To ensure a subcutaneous injection, gently pull upwards on the needle when inserted in the left abdominal mammary fat pad. If the needle is easily lifted upwards, it is correctly positioned subcutaneously.</li>
	</ol>
	</li>
</ol>
3. Monitoring
<ol>
	<li>Monitor mice at least 3 times a week for weight, body condition score, tumor size, tumor condition, respiration, activity level, appearance, and movement. Once the tumor reaches 0.7-0.8 cm in diameter, begin to monitor daily.
	<ol>
		<li>Consider euthanasia when tumor size reaches 1.5 cm, or weight loss reaches 20%, or severe clinical decline in body condition score, tumor condition, respiration, activity level, appearance, or movement are observed based on institutional guidelines. 
		NOTE: Body condition score is crucial to monitor as body weight may increase as the tumor increases in size, negating body condition loss due to disease burden. Exact monitoring protocols will depend on the approved IACUC or institutional protocols.</li>
	</ol>
	</li>
</ol>
4. Necropsy
<ol>
	<li>Euthanize mouse using CO2 following institutional guidelines.</li>
	<li>Spray mouse with 70% ethanol to disinfect. Make an incision up the ventral midline of the mouse to expose the body cavity.</li>
	<li>Remove the kidney. Continue cutting up the midline until the diaphragm is visible. Use scissors to puncture the diaphragm to deflate the lungs. Trim the diaphragm to get better access to the cavity.</li>
	<li>Use blunt scissors to cut up the center of the ribcage. Pin ribcage back to expose the lung and heart.</li>
	<li>Perfuse the heart with 2 mL of non-sterile 1x DPBS by inserting a needle into the apex of the heart until it pools in the abdominal cavity where the kidney was removed.</li>
	<li>To remove the heart and lungs, use blunt scissors to cut the esophagus and trachea directly above the heart. Using forceps, begin to pull the heart away from the body and cut away at any connective tissue keeping it attached. The lungs will come out with the heart.</li>
	<li>Identify the multi-lobed (right) and single-lobed (left) lungs. Keep heart attached for reference, but once lungs are identified, cut the heart away.</li>
	<li>Label a 12 well plate containing 1x Hank&#8217;s Balanced Saline Solution (HBSS) in each well. Each mouse needs 2 wells. Place the multi-lobed (right) lung in the 12 well plate for metastasis evaluation and keep on ice. Keep the neighboring well empty for now. 
	NOTE: It is important to use the same lung (multi-lobed) from every mouse to ensure each sample is close in size. The single-lobed lung can then be used for other analysis, like histopathology. 
	NOTE: Samples are stable on ice or at 4 &#176;C for a few hours.</li>
</ol>
5. Processing Tissues
NOTE: All steps in this section should be done using sterile technique.
<ol>
	<li>Label 1 15 mL conical tube per mouse and add 2.5 mL of type IV collagenase mixture and 30 units of elastase to each tube. To make type IV collagenase mixture, dissolve 2 mg of type IV collagenase per mL 1x HBSS and sterile filter. This can be stored up to 12 months at -20 &#176;C and thawed when needed.</li>
	<li>Transfer the lung to the second, clean 1x HBSS well for that sample. Swirl using forceps to remove any remaining blood. Transfer clean lung to empty 3.5&#160;cm tissue culture plate. Mince lung with scissors. Rinse plate with 2.5 mL of 1x HBSS, transfer 1x HBSS and lung pieces into a 15 mL conical tube already containing collagenase/elastase cocktail (5 mL total).</li>
	<li>Incubate for 75 minutes at 4 &#176;C. Continue mixing samples during this time, so place tubes on a rocker or rotating wheel. During this incubation step, label 50 mL centrifuge tubes and 10 cm tissue culture plates for each mouse. If doing a dilution, label enough 10 cm tissue culture plates for the dilutions. 
	NOTE: Label the lid of the tissue culture plates. If labeling the plate itself, the writing will interfere with Fiji-ImageJ analysis.</li>
	<li>Bring volume of each tube up to 10 mL total with 1x HBSS. Pour contents over a 70 &#956;m cell strainer into a 50 mL conical tube for each sample. Use the plunger of a 1 mL syringe to gently grind the sample through the strainer to allow more cells to filter through.</li>
	<li>Centrifuge for 5 minutes at 350 x g at room temperature (RT). Discard the supernatant and wash pellet with 10 mL of 1x HBSS. Repeat this step twice.</li>
	<li>Resuspend pellet in 10 mL of 60 &#956;M 6TG complete culture media, either RPMI or IMDM. Plate samples in 10 cm cell culture plates, using a dilution scheme if desired. Incubate at 37 &#176;C, 5% CO2 for 5 days. 
	NOTE: 1:2, 1:10, and 1:100 are common dilutions that will need to be empirically determined based on study parameters. 
	CAUTION: 6TG is toxic. Use caution when handling and follow all Environmental Health and Safety guidelines for disposal.</li>
</ol>
6. Staining plates
<ol>
	<li>Pour culture media off plates into appropriate waste container. Fix cells by adding 5 mL of undiluted methanol per plate and incubate for 5 minutes at RT, making sure to swirl methanol so that it covers the entire plate. 
	CAUTION: Methanol is hazardous if ingested, inhaled, or is on skin. Use a fume hood for this step.</li>
	<li>Pour methanol off plates into appropriate waste container. Rinse plates with 5 mL of distilled water per plate and pour water into appropriate waste container. Add 5 mL of 0.03% methylene blue per plate and incubate for 5 minutes at RT, making sure to swirl methylene blue solution so that it covers the entire plate.</li>
	<li>Pour methylene blue into appropriate waste container. Rinse plates again with 5 mL of distilled water per plate. Turn plates upside down and blot against a paper towel to remove excess liquid. Place plate on its lid and let air dry overnight at RT. 
	NOTE: Metastatic colonies will be blue. Once plates are dried, they can be stored at RT indefinitely.</li>
</ol>
7. Image analysis
<ol>
	<li>Remove labeled lids from plates, taking care to ensure clear identification of samples. Line up all stained lung plates on a clean, light surface to take a picture of all of the plates in one image.</li>
	<li>Take a picture of the collection of plates in a well-lit area, making sure to minimize reflections as the plates are very reflective. Reflections in the plates will influence image analysis and therefore need to be avoided. 
	NOTE: Fiji-ImageJ has an upper limit of 2 gigapixels. Most modern smart phones will have sufficient cameras. Do not use a camera less than 8 megapixels. The camera used in this experiment was a 12.2 megapixel on a Google Pixel 2.</li>
	<li>Crop the image to include the plates, but exclude the lids or anything else in the background of the image. Upload the cropped image into Fiji-ImageJ.</li>
	<li>Change the image to black and white using the following commands: Image, Adjust, Color Threshold, Thresholding method: Default, Threshold color: B&#38;W, Threshold space: Lab. Unselect the Dark background box. The image should now be black and white. Black represents the light background, and white represents the blue metastatic colonies.</li>
	<li>Using the Circle tool on the Fiji-ImageJ toolbar, select the area to be analyzed. Draw one circle to use for all of the plates to ensure each plate is analyzed for the same-sized area. Choose a size that maximizes analyzed area on the plates while minimizing the background noise that appears on the edge of the plates. The size appears in the toolbar as it is drawn, so it is possible to make a perfect circle by monitoring the height and the width as the circle is drawn.</li>
	<li>Analyze the selected circle to determine what percentage of the area is white, which represents the area of the plate that has blue metastatic colonies. Use the following commands: 
	Analyze, Analyze Particles, Size (pixel2): 0-Infinity, Circularity: 0.00-1.00, Show: Nothing, and check the Summarize box. Hit OK.</li>
	<li>Record the % Area result. This is the percentage of the selected area that is white, and therefore represents the metastatic burden. 
	NOTE: It is recommended to either save the results in Fiji-ImageJ or copy/paste the entire results page into a separate document. If % Area results are unexpected or suspicious, it is then possible to see if any of the other measurements were also suspicious or if % Area was recorded incorrectly.</li>
	<li>Move the circle, without altering its size by grabbing it in its center, to the next plate in the picture. Repeat steps 7.6 and 7.7 for all plates in the picture.</li>
	<li>Repeat steps 7.1 &#8211; 7.8 on at least two more images. Once all plates and images have been analyzed, average the % Area results between different images for each plate to mitigate any inconsistencies between pictures.</li>
</ol>

<ol>
	<li>American Cancer Society. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Facts+&amp;+Figures.">Cancer Facts &amp; Figures.</a> American Cancer Society. , (2019).</li><li>Yousefi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ-specific+metastasis+of+breast+cancer:+molecular+and+cellular+mechanisms+underlying+lung+metastasis.">Organ-specific metastasis of breast cancer: molecular and cellular mechanisms underlying lung metastasis.</a> Cellular Oncology. 41 (2), 123-140 (2018).</li><li>Pulaski, B. A., Ostrand-Rosenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+4T1+breast+tumor+model.">Mouse 4T1 breast tumor model.</a> Current Protocols in Immunology. , (2001).</li><li>Pulaski, B. A., Ostrand-Rosenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduction+of+established+spontaneous+mammary+carcinoma+metastases+following+immunotherapy+with+major+histocompatibility+complex+class+II+and+B7.1+cell-based+tumor+vaccines.">Reduction of established spontaneous mammary carcinoma metastases following immunotherapy with major histocompatibility complex class II and B7.1 cell-based tumor vaccines.</a> Cancer Research. 58 (7), 1486-1493 (1998).</li><li>Aslakson, C. J., Miller, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+events+in+the+metastatic+process+defined+by+analysis+of+the+sequential+dissemination+of+subpopulations+of+a+mouse+mammary+tumor.">Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor.</a> Cancer Research. 52 (6), 1399-1405 (1992).</li><li>Sikpa, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+detection+and+quantification+of+breast+cancer+brain+metastases+in+an+animal+model+using+democratized+machine+learning+tools.">Automated detection and quantification of breast cancer brain metastases in an animal model using democratized machine learning tools.</a> Scientific Reports. 9 (1), 17333 (2019).</li><li>Valkonen, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis+detection+from+whole+slide+images+using+local+features+and+random+forests.">Metastasis detection from whole slide images using local features and random forests.</a> Cytometry A. 91 (6), 555-565 (2017).</li><li>Coutermarsh-Ott, S. L., Broadway, K. M., Scharf, B. E., Allen, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Salmonella+enterica+serovar+Typhimurium+VNP20009+and+VNP20009+with+restored+chemotaxis+on+4T1+mouse+mammary+carcinoma+progression.">Effect of Salmonella enterica serovar Typhimurium VNP20009 and VNP20009 with restored chemotaxis on 4T1 mouse mammary carcinoma progression.</a> Oncotarget. 8 (20), 33601-33613 (2017).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li><li>ATCC. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A.T.C.C.+4T1+(ATCC+CRL2539)+Product+Sheet.">A.T.C.C. 4T1 (ATCC CRL2539) Product Sheet.</a> ATCC. , (2020).</li></ol>

The authors have nothing to disclose.

As demonstrated, manually counting the metastatic colonies on each lung plate can be an inaccurate and imprecise method to quantify lung metastasis, demonstrating the need for a better means of quantification (Figure 2). Histopathological analysis differed slightly from both manual counting and Fiji-ImageJ analysis (Figure 2B and 4D), likely because the H&#38;E slides are not a representative sample of the entire organ. The protocol harvests an ...

One in eight women will be diagnosed with breast cancer in her lifetime, and yet despite multiple treatment options breast cancer is the second leading cause of cancer-related deaths in American women1. These women are not dying from the primary tumor in their breast. Instead, the metastatic burden is responsible for the mortality of this disease as it commonly spreads to the lung, bone, brain, liver, and lymph nodes2. Because of this, breast cancer models need to evaluate metastasis to contribute to curbing the mortality of this disease. The 4T1 murine breast cancer model is a superb protocol to accomplish this. The method described here offers an improvement to the 4T1 model by using Fiji-ImageJ to quantify lung metastasis, producing consistent and expeditious results.
The 4T1 model is well-established, with most labs using protocols such as those described by Pulaski and Ostrand-Rosenberg in 20013. The 4T1 cell line is 6-Thioguanine (6TG) resistant and representative of stage IV, triple negative breast cancer3,4,5. It is clinically relevant as it is an orthotopic model and spontaneously metastasizes to the same organs as in human breast cancer3,4. The 4T1 cells spontaneously metastasize at a predictable rate based on the quantity of cells injected3,4. Importantly, genetic differences between mice used here caused expected inter-individual variability in metastatic burden. To evaluate metastasis, tissues are harvested to collect and quantify cancer cells in distant sites using 6TG selection and methylene blue staining. The result is a collection of tissue culture plates with blue dots representing metastatic colonies. However, the Pulaski and Ostrand-Rosenberg protocol quantifies metastatic colonies by manually counting them, and therefore this has been the standard means of evaluating metastasis in this model. While this is easy for tissues with low metastatic burden, tissues like the lungs are often laden with metastases. As lung plates can be highly confluent, accurately and precisely quantifying metastatic colonies by manual counting is difficult and prone to human error. To better quantify metastatic burden, we describe using Fiji-ImageJ for a computer-based solution to human counting error. Histopathological analysis with hematoxylin and eosin (H&#38;E) staining is another means to quantify lung metastases, and interestingly has also been improved with Fiji-ImageJ software6,7. However, because histopathological analysis observes a single slice of the lung, it can be inaccurate and unrepresentative. This is because the 4T1 model causes several metastatic lesions throughout the organ that are not evenly distributed. While overall trends between histopathological analysis and manual counting can be similar8, individual values can differ and therefore histopathological analysis should not be used as the sole means of quantification. We demonstrate the benefit compared to histopathological analysis and the inconsistencies in manual counting between different counters, while also demonstrating the consistency of using Fiji-ImageJ. Additionally, we show that this method can reduce the incubation time from 10-14 days to 5 days, meaning researchers can analyze data from their study much sooner than when relying on manual counting.
This method is a collection of simple adjustments to the Pulaski and Ostrand-Rosenberg protocol3. Because the 4T1 model is widely used, and because lung metastasis is a critical parameter to measure in preclinical models, we believe this method can be widely used and is highly valuable to breast cancer researchers. The only additional supplies needed are a camera and access to a computer with Fiji-ImageJ, a free software used frequently in image analysis9. This method specifically focuses on lung metastasis, but it could be used for other tissues with significant metastatic burden.

<table><tbody><tr><td>Anesthesia chamber</td><td>See comments</td><td>See comments</td><td>Use approved materials in your institution&#039;s policies</td></tr><tr><td>Anesthetic agent</td><td>See comments</td><td>See comments</td><td>Use approved materials in your institution&#039;s policies</td></tr><tr><td>BALB/c Female Mice</td><td>The Jackson Laboratory</td><td>000651</td><td></td></tr><tr><td>Blunt scissors</td><td>Roboz</td><td>RS-6700</td><td></td></tr><tr><td>Calculator</td><td>Any</td><td>Any</td><td></td></tr><tr><td>Camera</td><td>Any</td><td>Any</td><td>Minimum of 8 megapixels</td></tr><tr><td>Centrifuge</td><td>Any</td><td>Any</td><td>Needs to be capable of 125 x g and 300 x g</td></tr><tr><td>CO2 euthanasia setup</td><td>See comments</td><td>See comments</td><td>Use approved materials in your institution&#039;s policies</td></tr><tr><td>Cold room, refrigerator, cold storage</td><td>Any</td><td>Any</td><td></td></tr><tr><td>Computer with Fiji-ImageJ</td><td>Any</td><td>Any</td><td>Needs to be capable of running Fiji-ImageJ</td></tr><tr><td>Counting Chamber</td><td>Fisher Scientific</td><td>02-671-10</td><td></td></tr><tr><td>Curved scissors</td><td>Roboz</td><td>RS-5859</td><td></td></tr><tr><td>Distilled water</td><td>Any</td><td>Any</td><td></td></tr><tr><td>Elastase</td><td>MP Biomedicals</td><td>100617</td><td></td></tr><tr><td>Electronic scale</td><td>Any</td><td>Any</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>R&amp;amp;D Systems</td><td>S11150</td><td></td></tr><tr><td>Forceps</td><td>Roboz</td><td>RS-8100</td><td></td></tr><tr><td>Ice</td><td>N/A</td><td>N/A</td><td></td></tr><tr><td>Incubator</td><td>See comments</td><td>See comments</td><td>Needs to be capable of 5% CO&lt;sub&gt;2&lt;/sub&gt; and 37 &amp;deg;C</td></tr><tr><td>Methanol</td><td>Fisher Scientific</td><td>A412SK-4</td><td></td></tr><tr><td>Methylene blue</td><td>Sigma-Aldrich</td><td>03978-250ML</td><td></td></tr><tr><td>Penicillin Streptomycin</td><td>ATCC</td><td>30-2300</td><td></td></tr><tr><td>Pins or needles</td><td>Any</td><td>Any</td><td>For pinning down mice during necropsy</td></tr><tr><td>Plastic calipers</td><td>VWR</td><td>25729-670</td><td></td></tr><tr><td>RMPI-1640 Medium</td><td>ATCC</td><td>30-2001</td><td></td></tr><tr><td>Rocker or rotating wheel</td><td>Any</td><td>Any</td><td></td></tr><tr><td>Sharp scissors</td><td>Roboz</td><td>RS-6702</td><td></td></tr><tr><td>Sterile disposable filter with PES membrane</td><td>ThermoFisher Scientific</td><td>568-0010</td><td></td></tr><tr><td>T-150 Flasks</td><td>Fisher Scientific</td><td>08-772-48</td><td></td></tr><tr><td>T-25 Flasks</td><td>Fisher Scientific</td><td>10-126-10</td><td></td></tr><tr><td>T-75 Flasks</td><td>Fisher Scientific</td><td>13-680-65</td><td></td></tr><tr><td>Tri-cornered plastic beaker</td><td>Fisher Scientific</td><td>14-955-111F</td><td>Used to weigh mice</td></tr><tr><td>Trypan blue</td><td>VWR</td><td>97063-702</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>ATCC</td><td>30-2101</td><td></td></tr><tr><td>Type IV collagenase</td><td>Sigma-Aldrich</td><td>C5138</td><td></td></tr><tr><td>3.5 cm tissue culture plates</td><td>Nunclon</td><td>153066</td><td></td></tr><tr><td>1 mL syringe</td><td>BD</td><td>309659</td><td></td></tr><tr><td>1.7 mL microcentrifuge tubes</td><td>VWR</td><td>87003-294</td><td></td></tr><tr><td>10 cm tissue culture plates</td><td>Fisher Scientific</td><td>08-772-22</td><td></td></tr><tr><td>12 well plate</td><td>Corning</td><td>3512</td><td></td></tr><tr><td>15 mL centrifuge tube</td><td>Fisher Scientific</td><td>14-959-70C</td><td></td></tr><tr><td>1X Dulbecco&#039;s Phostphate Buffered Saline (DPBS)</td><td>Fisher Scientific</td><td>SH30028FS</td><td></td></tr><tr><td>1X Hank&amp;rsquo;s Balanced Saline Solution (HBSS)</td><td>Thermo Scientific</td><td>SH3026802</td><td></td></tr><tr><td>27 g 1/2 in needles</td><td>Fisher Scientific</td><td>14-826-48</td><td></td></tr><tr><td>4T1 (ATCC&amp;reg; CRL&amp;shy;2539&amp;trade;)</td><td>ATCC</td><td>CRL-2539</td><td></td></tr><tr><td>50 mL centrifuge tube</td><td>Fisher Scientific</td><td>14-959-49A</td><td></td></tr><tr><td>6-Thioguanine</td><td>Sigma-Aldrich</td><td>A4882</td><td></td></tr><tr><td>70 &amp;mu;M cell strainer</td><td>Fisher Scientific</td><td>22-363-548</td><td></td></tr><tr><td>70% ethanol</td><td>Sigma Aldrich</td><td>E7023</td><td>Dilute to 70% with DI water</td></tr></tbody></table>

using computer-based image analysis to improve quantification of lung metastasis in the 4t1 breast cancer model

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

This method contains simple adjustments from the Pulaski and Ostrand-Rosenberg 4T1 protocol3 and can be visualized in Figure 1. When 3 separate researchers manually counted metastatic colonies for 12 lung plates (1:10 dilution), the results were very inconsistent between different counters (Figure 2A). All researchers were directed to &#8220;count the metastatic colonies that appear as blue dots&#8221;, yet the inconsistencies demonstrate...

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

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

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

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Research

JoVE Journal

Cancer Research

6.2K Views.  Virginia-Maryland College of Veterinary Medicine. Dit protocol kwantificeert longmetastase, dat is een agressieve en veel voorkomende oorzaak van borstkanker gerelateerde dood, met verhoogde precisie en efficiëntie in een preklinische borstkanker model. In vergelijking met de oorspronkelijke techniek, dit protocol geeft onderzoekers snellere, meer consistente resultaten. Het verlicht de fout bij het tellen van mensen door gebruiksvriendelijke computertechnologie.Deze techniek kan absoluut worden uitgebreid tot preklinisch onderzoek n...

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

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology.

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography ...

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, ...

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

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

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

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

Medicine

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

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

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

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

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 ...

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

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

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

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

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

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

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

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

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

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

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

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

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

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.

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

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