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&#39;s policies</td>
		</tr>
		<tr>
			<td>Anesthetic agent</td>
			<td>See comments</td>
			<td>See comments</td>
			<td>Use approved materials in your institution&#39;s policies</td>
		</tr>
		<tr>
			<td>BALB/c Female Mice</td>
			<td>The Jackson Laboratory</td>
			<td>651</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&#39;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&#38;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% CO2 and 37 &#176;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>1 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&#39;s Phostphate Buffered Saline (DPBS)</td>
			<td>Fisher Scientific</td>
			<td>SH30028FS</td>
			<td></td>
		</tr>
		<tr>
			<td>1X Hank&#8217;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&#174; CRL&#173;2539&#8482;)</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 &#956;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...

Watch this Scientific Journal Video about Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model at JoVE.com

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

5.9K Views.  Virginia-Maryland College of Veterinary Medicine. We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

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

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

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

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

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

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

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

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 ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

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

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

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

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

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

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

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

Modeling Breast Cancer in Human Breast Tissue using a Microphysiological System

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs fail clinical trials. Therefore, new models are needed to improve our understanding of the disease. The NIH Microphysiological Systems (MPS) program was developed to improve the clinical translation of basic science discoveries and promising new therapeutic strategies. Here we present a method for generating MPS for breast cancers (BC-MPS). This model adapts a previously described approach of culturing primary human white adipose tissue (WAT) by sandwiching WAT between adipose-derived stem cell sheets (ASC)s. Novel aspects of our BC-MPS include seeding BC cells into non-diseased human breast tissue (HBT) containing native extracellular matrix, mature adipocytes, resident fibroblasts, and immune cells; and sandwiching the BC-HBT admixture between HBT-derived ASC sheets. The resulting BC-MPS is stable in culture ex vivo for at least 14 days. This model system contains multiple elements of the microenvironment that influence BC including adipocytes, stromal cells, immune cells, and the extracellular matrix. Thus BC-MPS can be used to study the interactions between BC and its microenvironment. 
We demonstrate the advantages of our BC-MPS by studying two BC behaviors known to influence cancer progression and metastasis: 1) BC motility and 2) BC-HBT metabolic crosstalk. While BC motility has previously been demonstrated using intravital imaging, BC-MPS allows for high-resolution time-lapse imaging using fluorescence microscopy over several days. Furthermore, while metabolic crosstalk was previously demonstrated using BC cells and murine pre-adipocytes differentiated into immature adipocytes, our BC-MPS model is the first system to demonstrate this crosstalk between primary human mammary adipocytes and BC cells in vitro.

This protocol describes the construction of an in vitro microphysiological system for studying breast cancer using primary human breast tissue with off the shelf materials.

Breast cancer (BC) remains a leading cause of death for women. Despite more than $700 million invested in BC research annually, 97% of candidate BC drugs ...

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

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

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

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

A Permanent Window for Investigating Cancer Metastasis to the Lung

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as the bone, brain, and lung. Although extensively studied, the mechanistic details of this process remain poorly understood. While common imaging modalities, including computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), offer varying degrees of gross visualization, each lacks the temporal and spatial resolution necessary to detect the dynamics of individual tumor cells. To address this, numerous techniques have been described for intravital imaging of common metastatic sites. Of these sites, the lung has proven especially challenging to access for intravital imaging owing to its delicacy and critical role in sustaining life. Although several approaches have previously been described for single-cell intravital imaging of the intact lung, all involve highly invasive and terminal procedures, limiting the maximum possible imaging duration to 6-12 h. Described here is an improved technique for the permanent implantation of a minimally invasive thoracic optical Window for High-Resolution Imaging of the Lung (WHRIL). Combined with an adapted approach to microcartography, the innovative optical window facilitates serial intravital imaging of the intact lung at single-cell resolution across multiple imaging sessions and spanning multiple weeks. Given the unprecedented duration of time over which imaging data can be gathered, the WHRIL can facilitate the accelerated discovery of the dynamic mechanisms underlying metastatic progression and numerous additional biologic processes within the lung.

Here, we present a protocol for the surgical implantation of a permanently indwelling optical window for the murine thorax, which enables high-resolution, intravital imaging of the lung. The permanence of the window makes it well-suited to the study of dynamic cellular processes in the lung, especially those that are slowly evolving, such as metastatic progression of disseminated tumor cells.

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as ...