Work in the Bos lab is supported by the Susan G. Komen Foundation (CCR18548205 P.D.B.), V Foundation (V2018-22 P.D.B.), and American Cancer Society (RSG-21-100-01-IBCD P.D.B.)

All animal procedures and protocols described here were approved by the Institutional Animal Care and Use Committee of Virginia Commonwealth University.
1. Preparation of cells for injection
<ol>
	<li>Thaw luciferase-transduced EO771 cells13 from liquid nitrogen storage and plate 1 x 106 cells in a 10 cm tissue-culture dish in complete cell culture medium (RPMI1640 + 10% FBS + 1% penicillin/streptomycin + 1% amphotericin B).</li>
	<li>Incubate at 37 &#176;C and 5% CO2 until 80%-90% confluent, with medium changes every 2-3 days as necessary.</li>
	<li>To harvest cells, aspirate cell culture medium and wash with 1x PBS. Incubate with 2 mL of 0.25% Trypsin-EDTA solution for about 2-3 min at 37 &#176;C until the cells detach and then wash with 8 mL of complete medium to quench the reaction. 
	NOTE: Prolonged cell exposure to trypsin will result in stripping of the cell surface proteins from the membrane and, ultimately, cause cell death.</li>
	<li>Transfer the contents to a 10 mL centrifuge tube and pellet the cells by spinning at 350 x g for 5 min. Aspirate the supernatant and resuspend the cells in 10 mL of 1x PBS.</li>
	<li>Collect a 50 &#181;L sample for counting and then re-pellet the cells by spinning at 350 x g for 5 min.
	<ol>
		<li>While the cells are centrifuging, add 50 &#181;L of trypan blue to the 50 &#181;L sample and count the number of viable cells using a hemacytometer. Viable cells with intact cell membranes will exclude the dye and remain clear after the addition of trypan blue, while dying/dead cells will allow the dye to enter the cytoplasm and turn blue.</li>
		<li>Determine the volume required to resuspend cells at 6 x 106&#160;viable cells/mL (viable cell concentration [viable cells/mL]) by using the following formula: 
		Average number of viable cells in the 4 sets of 16 squares &#215; dilution factor &#215; 1 x 104&#160;cells/mL</li>
	</ol>
	</li>
	<li>Aspirate the supernatant and resuspend the cells in sterile 1x PBS in the calculated volume necessary to dilute cells to 6 x 106 cells/mL. Transfer the cell suspension to 1.5 mL microcentrifuge tubes and keep on ice until ready for injection.</li>
</ol>
2. Mammary fat pad injections
NOTE: The present protocol can be used with any mouse strain, but given our interest in the immune microenvironment, we utilize C57BL6 mice. Female, virgin mice of 6-8 weeks old are typically used for breast cancer studies, as parity enhances tumorigenesis processes.
<ol>
	<li>Thaw growth factor-reduced basement-membrane matrix on ice and keep on ice until ready for injection.</li>
	<li>Anesthetize mice in an induction chamber using 4%-5% isoflurane. Confirm a sufficient plane of anesthesia by assessing for the lack of toe-pinch reflex and lower the gas to 2% isoflurane for maintenance during the procedure. 
	CAUTION: Isoflurane is an odorless inhaled anesthetic that is a known irritant to the eyes and skin and toxic to the central nervous system. It should be used in an environment with adequate ventilation. Long-term or chronic exposure to isoflurane may have adverse health effects. Veterinary anesthesia equipment that is properly calibrated, utilizes gas scavenging systems, and is frequently maintained by veterinary staff should be used.</li>
	<li>Shave the hair on the abdomen of the mouse using electric clippers and then place in a supine position in a nose cone attached to an anesthesia machine using a maintenance isoflurane rate of 2%. Apply ophthalmic ointment to each eye of the animal to prevent corneal injury.</li>
	<li>Surgically scrub the abdominal region three times in a circular motion using alternating rounds of 70% ethanol and povidone-iodine solution. Confirm a sufficient plane of anesthesia by assessing for the lack of toe-pinch reflex.</li>
	<li>Using scissors, make a small midline incision (usually ~1 cm) through the abdominal skin at the level of the 4th mammary tissue, exposing but not penetrating through the underlying peritoneum. 
	NOTE: Other procedures for tumor cell implantation in the mammary gland, such a subcutaneous injection or intraductal inoculation, can be utilized. While somewhat invasive, this surgical procedure is straightforward to learn and master, and visualizing the fat pad significantly improves the accuracy, with little risk of injection outside the mammary gland, which is critical for the subsequent effective removal of the tumor.</li>
	<li>Using forceps, hold the skin away from the peritoneum. Use sterile saline-dipped cotton swabs to separate the skin away from the peritoneum, moving laterally to expose the right mammary fat pad. Repeat on the left side to expose the left mammary fat pad.</li>
	<li>Resuspend the EO771 cell suspension using a manual pipette and transfer 100 &#181;L to a new 1.5 mL microcentrifuge tube. Add an equal volume of basement-membrane matrix solution and mix well, taking care not to introduce bubbles, and keep on ice. The final cell suspension will now contain 150,000 cells per 50 &#181;L.</li>
	<li>Draw up 100 &#181;L of cell suspension into a 28G 0.5 mL U-100 insulin syringe and keep on ice.</li>
	<li>Using forceps, lift the skin and gently grab and expose the right mammary fat pad. Inject 50 &#181;L of cell suspension into the mammary fat pad and wait for 3-5 s prior to removing the syringe from the mammary fat pad to allow the matrix to begin to solidify and decrease the chance of the cell suspension backflowing through the injection site. A small bubble will form by the end of the injection.</li>
	<li>Release the skin from the forceps, allowing the mammary fat pad to return naturally to its normal position. Repeat the procedure with the left mammary fat pad, placing the syringe containing the cell suspension back on ice between injections.</li>
	<li>Close the skin incision, applying skin staples. The length of the incision will determine the number of skin staples required, with most procedures requiring one to two staples per incision. Make sure there is a minimum distance of 0.5 cm between staples.</li>
	<li>Upon surgical closure, transfer the mouse to a clean recovery cage. Monitor as needed until the animal rights itself and resumes normal behavior. Administer 40 &#181;L of meloxicam (2 mg/mL) subcutaneously&#160;for pain control every 24 h for 3 days. Alternatively, a slow-release formulation dose of an opioid like buprenorphine can be administered at the time of the procedure, which will last for 72 h.&#160;</li>
	<li>Monitor animals daily for the first 5 days post operation, assessing the animals&#39; weight and any signs of distress, such as unkept fur, hunched back, and reddish-brown nasal or ocular discharge.</li>
	<li>Check the animals for signs of wound infection, such as surgical site erythema or necrosis and guarding of the incision site, and humanely sacrifice animals who lose &#62;20% of their initial body weight or who meet the criteria for severe distress, as described by institution-specific IACUC guidelines.</li>
</ol>
3. Tumor resections
<ol>
	<li>Use calipers to monitor the growth of the orthotopic breast tumor lesion 3 times/week by taking measurements of the primary tumor length (L) and width (W). Calculate tumor volume using the formula: 
	&#960;LW2/6
	<ol>
		<li>Determine tumor resection empirically depending on the specific cell line injected. Always remove tumors at the smallest possible size to reduce chances of re-growth. For the EO771 cells described in this protocol, perform tumor resection when the tumors reach 150 mm3 in volume. 
		NOTE: The optimal time of resections needs to be determined for individual cell lines, but 150 mm3 is a good starting point, as the procedure can efficiently excise all the tumor once the user is experienced.</li>
	</ol>
	</li>
	<li>Anesthetize the mice in an induction chamber using 4% isoflurane. Administer 40 &#181;L of meloxicam (2 mg/mL) subcutaneously for pain control, and then place the mouse in a supine position in a nose cone attached to an anesthesia machine using a maintenance isoflurane rate of 2%. Apply ophthalmic ointment to each eye of the animal to prevent corneal injury. 
	NOTE: Analgesic needs to be administered every 24 h for 3 days. Alternatively, a slow-release formulation dose of an opioid like buprenorphine can be administered at the time of the procedure, which will last for 72 h.</li>
	<li>Remove prior surgical staples, if necessary, and surgically scrub the abdominal region three times in a circular motion using alternating rounds of 70% ethanol and povidone-iodine solution. Confirm a sufficient plane of anesthesia by assessing for the lack of toe-pinch reflex. Using scissors, make a small midline incision (usually ~1 cm) through the abdominal skin at the level of the 4th mammary tissue, exposing but not penetrating through the underlying peritoneum.</li>
	<li>Use blunt dissection to separate the orthotopic tumor from the peritoneum and the overlying skin. Remove the orthotopic tumor by cutting through the normal mammary tissue located proximally and distally to the tumor using scissors and discard the tumor tissue into a biohazard bag. Repeat with the contralateral tumor. If bleeding occurs, quickly cauterize the vasculature. 
	NOTE: If orthotopic tumors have infiltrated into the peritoneum, evidenced by a tumor that is not well-circumscribed or easily separable from the peritoneum by blunt dissection, animals should be sacrificed, as removal of the tumor will not be complete, and it will regrow, leading to confounding of the background bioluminescence signal and morbidity.</li>
	<li>Use one to three staples to close the surgical site and transfer the mouse to a clean recovery cage with a warm heating pad underneath to improve the recovery of the animal following the tumor resection procedure. Monitor as needed until the animal rights itself and resumes normal behavior.
	<ol>
		<li>For animals that have lost some blood during the procedure, administer a 300 &#181;L injection of sterile 0.9% normal saline administered intraperitoneally following closure of the surgical site.</li>
		<li>If needed, image the animals at this point for bioluminescence signal, as described in step 4., to assess the completeness of tumor resection and baseline minimal residual disease. If resection was incomplete and there is remaining bioluminescence signal in the primary tumor region, humanely sacrifice the animal, as growth of the remaining tumor cells may result in confounding of the background bioluminescence signal.</li>
	</ol>
	</li>
	<li>Monitor the animals daily for the first 5 days post operation, assessing the animals&#39; weight and any signs of distress, such as unkept fur, hunched back, and reddish-brown nasal or ocular discharge.</li>
	<li>Check the animals for signs of wound infection, such as surgical site erythema or necrosis and guarding of the incision site. Humanely sacrifice animals who lose &#62;20% of their initial body weight or who meet the criteria for severe distress, as described by institution-specific IACUC guidelines.</li>
</ol>
4. In vivo quantification of spontaneous lung metastasis
<ol>
	<li>Perform in vivo imaging of the animals on the day of tumor resection to establish a baseline signal, and then 2-3 times/week thereafter to assess the growth of spontaneous metastatic lung tumor lesions.</li>
	<li>Click Initialize to start the imaging instrument for warm-up while the animals are prepared for the procedure.</li>
	<li>Anesthetize the mice in an induction chamber using 4% isoflurane and confirm a sufficient plane of anesthesia by assessing for the lack of toe-pinch reflex. Confirm that oxygen and isoflurane anesthesia is flowing to the imaging instrument.</li>
	<li>Inject the animals with 100 &#181;L of D-luciferin solution (15 mg/mL in sterile PBS) using a retro-orbital injection by inserting the needle in the medial canthus of the eye at a 45&#176; angle from the nose. Insert the needle until bony resistance is felt, at which point withdraw the needle by ~1 mm prior to injecting D-luciferin solution to ensure the needle is placed within the retroorbital venous sinus. 
	NOTE: D-luciferin solution can be delivered by other routes, such as intraperitoneal or subcutaneous injection, however, the kinetics of substrate metabolization will be longer and the distribution to different organs heterogeneous.</li>
	<li>Confirm successful injection by the lack of flush back of any liquid upon delivery and wait 2 min prior to imaging.While waiting, transfer the animals to the nose cones located inside the bioluminescence imager in a supine position and decrease the isoflurane to a maintenance rate of 2%.</li>
	<li>Before acquiring a bioluminescence image of the animal, ensure that the checkbox next to Photograph is checked to simultaneously acquire a photograph of the animal using medium binning and f/stop 8. Ensure the checkbox next to Overlay is checked to overlay the photograph with the bioluminescence image. Set the exposure time to 1 min, with medium binning, f/stop = 1, and capture an image by clicking on Acquire.</li>
	<li>Measure photon flux as follows. Create a square ROI using&#160;the ROI tools dropdown menu for each animal depicted in the image, by clicking the Square ROI button. Reposition the automatically generated ROI over the thorax of each animal by clicking and dragging with the mouse.</li>
	<li>Click the Measure ROIs button and ensure that data are displayed as radiance (photons/s) and not counts, so that images acquired with different exposure times can be compared.</li>
</ol>
5. Collection of lung tissues for histological analysis
NOTE: Animals can be sacrificed as described below at any experimental time point or when animals meet the criteria for humane sacrifice, according to institution-specific IACUC guidelines. In our experience, mice reach the humane endpoint approximately 21-28 days after resection of the primary tumors.
<ol>
	<li>Anesthetize the mice in an induction chamber using 4% isoflurane and confirm a sufficient plane of anesthesia by assessing for the lack of toe-pinch reflex. Then, euthanize the mice by cervical dislocation.</li>
	<li>Use scissors to make a midline incision below the xyphoid process, cutting through the skin, musculature, and peritoneum to expose the lower part of the thoracic cavity, until the diaphragm is visible. Puncture the diaphragm to collapse the lungs and then cut through the diaphragm.</li>
	<li>Cut through the ribcage on the right and left side and then use a hemostat to grab the xyphoid process and move the ribcage out of the way, exposing the heart and lungs. Snip the right atrium using scissors.</li>
	<li>Perfuse the animal with 10 mL of ice-cold PBS through the left ventricle and assess the completeness of perfusion by confirming that fluid flowing from the right atrium turns clear and that the liver turns a pale-yellow color.</li>
	<li>Identify the trachea and insert a 22G needle syringe with 3 mL of 4% paraformaldehyde, holding it parallel to the trachea. Deliver the solution at a slow pace until the lungs have fully inflated. Hold the trachea with forceps over the needle, and slowly remove the needle to prevent backflow. 
	NOTE: Threading a suture beneath the trachea prior to injection of fixative, followed by tying of the suture around the trachea after injection, may be another option for preventing backflow of the fixative.</li>
	<li>Continue gently holding the trachea, snip it with scissors above the forceps, and start carefully lifting the tissue while removing all the connective tissue. Dissect the heart away from the lungs.</li>
	<li>Place lung tissue in 4% paraformaldehyde in PBS overnight and store at 4 &#176;C for fixation. Transfer the tissue to PBS containing 0.05% sodium azide for long-term storage or process further for histological analysis, as needed.</li>
</ol>

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	<li>Gupta, G. P., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+metastasis:+Building+a+framework.">Cancer metastasis: Building a framework.</a> Cell. 127 (4), 679-695 (2006).</li><li>Bos, P. D., Nguyen, D. X., Massagu&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+metastasis+in+the+mouse.">Modeling metastasis in the mouse.</a> Current Opinion in Pharmacology. 10 (5), 571-577 (2010).</li><li>Francia, G., Cruz-Munoz, W., Man, S., Xu, P., Kerbel, R. 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+models+of+advanced+spontaneous+metastasis+for+experimental+therapeutics.">Mouse models of advanced spontaneous metastasis for experimental therapeutics.</a> Nature Reviews Cancer. 11 (2), 135-141 (2011).</li><li>G&#243;mez-Cuadrado, L., Tracey, N., Ma, R., Qian, B., Brunton, V. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+metastasis:+Progress+and+prospects.">Mouse models of metastasis: Progress and prospects.</a> Disease Models &amp; Mechanisms. 10 (9), 1061-1074 (2017).</li><li>Husemann, Y., Klein, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analysis+of+metastasis+in+transgenic+mouse+models.">The analysis of metastasis in transgenic mouse models.</a> Transgenic Research. 18 (1), 1-5 (2009).</li><li>Klein, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+progression+of+primary+tumours+and+metastases.">Parallel progression of primary tumours and metastases.</a> Nature Reviews Cancer. 9 (4), 302-312 (2009).</li><li>Pantel, K., Brakenhoff, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+metastatic+cascade.">Dissecting the metastatic cascade.</a> Nature Reviews Cancer. 4 (6), 448-456 (2004).</li><li>Sosa, M. S., Bragado, P., Aguirre-Ghiso, J. 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The authors have nothing to disclose.

As the early nature of metastatic dissemination and the systemic effects of cancer become more widely recognized, the need for models in which both of these critical factors are taken into consideration becomes a necessity. This protocol allows researchers to monitor minimal residual disease and outgrowth of lung metastasis occurring spontaneously from primary breast tumors, accounting for the systemic effects of cancer that influence the metastatic process. Primary tumor removal is necessary to evaluate lung metastasis ...

Metastasis&#160;- the spread of cancer cells from the primary tumor to other parts of the body - remains the cause of death in more than 90% of cancer patients. This process is complex, involving migration of the tumor cells out of the primary tumor and intravasation into the circulation, survival in the blood, extravasation and survival in the target organ, re-instauration of the proliferative state, and outgrowth1. Spontaneous and transplantable murine cancer models have been used to investigate early or late stages of metastasis, each presenting its own advantages and disadvantages, which have been thoroughly discussed2,3,4.
Unlike previously thought, tumor cells abandon the primary tumor at early stages during tumor development, sometimes remaining dormant in distant tissues for what can be long periods of time5,6,7,8. In addition, there is mounting evidence of the strong systemic effects the primary tumor has on disease outcomes, often manifested through the secretion of soluble factors and exosomes that condition the metastatic soil or stimulate muscle wasting during cachexia9,10,11,12. For these reasons, modeling the length of the metastatic process in the initial presence of the primary tumor has become essential for achieving a more complete understanding of the biology driving these processes and testing potential new interventions aimed at disrupting or delaying the process.
In this work, a protocol is described for quantifying lung metastasis arising spontaneously from traceable cell lines injected orthotopically in the mammary gland, a process that models all of the above steps in the metastatic cascade. Transplantable models of metastasis are known to be more representative of human metastasis as compared to spontaneous, genetically-driven cancer models, thus improving clinical translatability2. Furthermore, this protocol utilizes bioluminescence imaging to study the growth and progression of spontaneous lung metastasis from primary breast tumors in real time within living animals, thus improving efficiency over more traditional histology-based assessments of metastatic dissemination. This protocol also includes evaluation of spontaneous metastasis after the surgical removal of the primary tumor, which is both clinically relevant and allows researchers to study the effect of minimal residual disease on the metastatic process. Finally, the use of immunocompetent mice confers the advantage of allowing for an intact immune system to shape the metastatic process, as is the case in human biology10,11.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin/EDTA</td>
			<td>Hyclone</td>
			<td>SH30042.01</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5ml microcentrifuge tubes</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
			<td></td>
		</tr>
		<tr>
			<td>10% povidone-iodine solution</td>
			<td>Medline</td>
			<td>MDS093906</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml centrifuge tubes</td>
			<td>VWR</td>
			<td>89039-666</td>
			<td></td>
		</tr>
		<tr>
			<td>1X PBS</td>
			<td>Hyclone</td>
			<td>SH30256.01</td>
			<td></td>
		</tr>
		<tr>
			<td>28G 0.5ml U-100 Insulin Syringe</td>
			<td>BD Biosciences</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Gemini Bio-products</td>
			<td>400-104</td>
			<td></td>
		</tr>
		<tr>
			<td>Cautery Kit</td>
			<td>Braintree Scientific</td>
			<td>DEL2</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Luciferin Potassium</td>
			<td>SydLabs</td>
			<td>MB102</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Koptec</td>
			<td>V1001</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>R&#38;D Systems</td>
			<td>S11150H</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisherbrand</td>
			<td>16-100-110</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth factor-reduced Matrigel</td>
			<td>Corning</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Covetus</td>
			<td>29405</td>
			<td></td>
		</tr>
		<tr>
			<td>IVIS Spectrum 200</td>
			<td>Perkin Elmer</td>
			<td>124262</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam (2mg/ml)</td>
			<td>Zoopharm LLC</td>
			<td>N/A</td>
			<td>By veterinary prescription</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gemini Bio-products</td>
			<td>400-109</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI1640</td>
			<td>Hyclone</td>
			<td>SH30027.01</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Miltex</td>
			<td>5-300</td>
			<td></td>
		</tr>
		<tr>
			<td>Silk sutures</td>
			<td>Braintree Scientific</td>
			<td>SUT-S 103</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical staples</td>
			<td>Reflex7</td>
			<td>203-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue</td>
			<td>Gibco</td>
			<td>15250-061</td>
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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.

Orthotopic injection of mouse cancer cell lines into the 4th mammary fat pad of mice is a reproducible and reliable procedure for inducing mouse primary tumors. Utilizing the EO771 cell line transduced with luciferase in the conditions described in this protocol, primary tumors become palpable and can be measured using calipers about 7 days after injection and reach approximately 150 mm3 in volume at around 14 days following initial injection (Figure 1). Bioluminescence growth of ...

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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.

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 ...

in-vivo-imaging-to-measure-spontaneous-lung-metastasis-orthotopically

Research

JoVE Journal

Cancer Research

1.5K Views.  Virginia Commonwealth University School of Medicine. 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.

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

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

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 ...

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many transgenic mouse models, often fail to recapitulate the key aspects of human malignancies. Thus, alternative models that better represent the heterogeneity of patients' tumors and their metastases are being developed. Patient-derived xenograft (PDX) models in which surgically resected tumor samples are engrafted into immunocompromised mice have become an attractive alternative as they can be transplanted through multiple generations,and more efficiently reflect tumor heterogeneity than xenografts derived from human cancer cell lines. A limitation to the use of PDXs is that they are difficult to transfect or transduce to introduce traceable reporters or to manipulate gene expression. The current protocol describes methods to transduce dissociated tumor cells from PDXs with high transduction efficiency, and the use of labeled PDXs for experimental models of breast cancer metastases. The protocol also demonstrates the use of labeled PDXs in experimental metastasis models to study the organ-colonization process of the metastatic cascade. Metastases to different organs can be easily visualized and quantified using bioluminescent imaging in live animals, or GFP expression during dissection and in excised organs. These methods provide a powerful tool to extend the use of multiple types of PDXs to metastasis research.

We describe a method for stable labeling of patient-derived xenografts (PDXs) with lentiviral particles expressing green-fluorescent protein and luciferase reporters. This method allows for tracking the growth of PDXs at the primary site, as well as detecting spontaneous and experimental metastases using in vivo imaging systems.

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the tumor microenvironment and respond adequately to tumor antigens. The recent development of improved imaging approaches, such as 2-photon microscopy, and the use of powerful mouse tumor models have shed light on some of the mechanisms that regulate anti-tumor T cell activities. Whereas such systems have provided valuable insights, they do not always predict human responses. In human, our knowledge in the field mainly comes from a description of fixed tumor samples from human patients, as well as in vitro studies. However, in vitro models lack the complex three-dimensional tumor milieu and, therefore, are incomplete approximations of in vivo T cell activities. Fresh slices made from explanted tissue represent a complex multi-cellular tumor environment that can act as an important link between co-cultured studies and animal models. Originally set up in murine lymph nodes1 and previously described in a JoVE article2, this approach has now been transposed to human tumors to examine the dynamics of both plated3&#160;as well as resident&#160;T cells4.
Here, a protocol for the preparation of human lung tumor slices, immunostaining of resident CD8 T and tumor cells, and tracking of CD8 T lymphocytes within the tumor microenvironment using confocal microscopy is described. This system is uniquely placed to screen for novel immunotherapy agents favoring T cell migration in tumors.

Ex Vivo Imaging of Resident CD8 T Lymphocytes in Human Lung Tumor Slices Using Confocal Microscopy

This protocol describes a method to image resident tumor-infiltrating CD8 T cells labeled with fluorescently coupled antibodies within human lung tumor slices. This technique permits real-time analyses of CD8 T cell migration using confocal microscopy.

CD8 T cell are key players in the fight against cancer. In order for CD8 T cells to kill tumor cells they need to enter into the tumor, migrate within the ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

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 ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

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 ...