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

Summary

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.

Abstract

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.

Introduction

Metastasis - 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 discussed^2,3,4.

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 time^5,6,7,8. 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 cachexia^9,10,11,12. 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 biology^10,11.

Protocol

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

1. 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).
2. Incubate at 37 °C and 5% CO₂ until 80%-90% confluent, with medium changes every 2-3 days as necessary.
3. 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 °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.
4. 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.
5. Collect a 50 µL sample for counting and then re-pellet the cells by spinning at 350 x g for 5 min.
   1. While the cells are centrifuging, add 50 µL of trypan blue to the 50 µL 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.
   2. Determine the volume required to resuspend cells at 6 x 10⁶ 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 × dilution factor × 1 x 10⁴ cells/mL
6. 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.

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.

1. Thaw growth factor-reduced basement-membrane matrix on ice and keep on ice until ready for injection.
2. 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.
3. 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.
4. Clean the prepared abdominal region using 70% ethanol and povidone-iodine solution.
5. 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.
6. Using forceps, hold the skin away from the peritoneum. Use ethanol-dipped sterile 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.
7. Resuspend the EO771 cell suspension using a manual pipette and transfer 100 µL 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 µL.
8. Draw up 100 µL of cell suspension into a 28G 0.5 mL U-100 insulin syringe and keep on ice.
9. Using forceps, lift the skin and gently grab and expose the right mammary fat pad. Inject 50 µL 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.
10. 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.
11. 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.
12. Upon surgical closure, transfer the mouse to a clean recovery cage. Monitor as needed until the animal rights itself and resumes normal behavior.
13. Monitor animals daily for the first 5 days post operation, assessing the animals' weight and any signs of distress, such as unkept fur, hunched back, and reddish-brown nasal or ocular discharge.
14. 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 >20% of their initial body weight or who meet the criteria for severe distress, as described by institution-specific IACUC guidelines.

3. Tumor resections

1. 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:
   πLW²/6
   1. 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.
2. 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. Administer 40 µL 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.
3. Remove prior surgical staples, if necessary, and clean the abdomen with 70% ethanol and povidone-iodine solution.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.
4. 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.
5. 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.
   1. For animals that have lost some blood during the procedure, administer a 300 µL injection of sterile 0.9% normal saline administered intraperitoneally following closure of the surgical site.
   2. 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.
6. Monitor the animals daily for the first 5 days post operation, assessing the animals' weight and any signs of distress, such as unkept fur, hunched back, and reddish-brown nasal or ocular discharge.
7. 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 >20% of their initial body weight or who meet the criteria for severe distress, as described by institution-specific IACUC guidelines.

4. In vivo quantification of spontaneous lung metastasis

1. 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.
2. Click Initialize to start the imaging instrument for warm-up while the animals are prepared for the procedure.
3. 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.
4. Inject the animals with 100 µL 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° 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.
5. 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%.
6. 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.
7. Measure photon flux as follows. Create a square ROI using 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.
8. 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.

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.

1. 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. Place the animal in a supine position in a nose cone attached to an anesthesia machine using a maintenance isoflurane rate of 2%.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. Place lung tissue in 4% paraformaldehyde in PBS overnight and store at 4 °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.

Representative Results

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 these lesions can also be monitored as desired. Following primary tumor resection, bioluminescence imaging is utilized to confirm appropriate resection (Figure 1D). Utilizing C57BL6 mice, significant lung metastasis bioluminescence signal is typically detected around 2 weeks following primary tumor resection and can be followed over time, although some variation can be found depending on the luciferase reporter type and mouse strain (Figure 2A,B). Finally, ex vivo bioluminescence imaging of the lung tissue at the endpoint provides accurate and sensitive quantification of lung metastatic burden that can be plotted to compare with other treatments (Figure 2C,D). Lung nodule counting under the stereoscope and hematoxylin and eosin histological analysis can be utilized to complement the quantification studies.

Figure 1: Orthotopic mammary gland injection and monitoring of the primary breast tumors until primary tumor resection. (A) Schematic of the overall procedure. EO771-TGL cells are injected into the 4th mammary fat pad at day 0 (green circle), surgically resected upon reaching 150 mm³ in size, with spontaneous lung metastasis visible by bioluminescence approximately 2 weeks after primary tumor resection (green lungs). (B) Typical primary tumor growth kinetics via caliper measurement following mammary gland injection of 150,000 EO771-TGL cells, n = 9, error bars depict standard error. (C) Bioluminescence image depicting a representative mouse with primary tumors of approximately 150 mm³ at the time they are resected. (D) Bioluminescence image depicting a representative mouse immediately after complete and successful tumor resection. Please click here to view a larger version of this figure.

Figure 2: Monitoring of lung metastasis after tumor resection. (A) Growth kinetics of spontaneous lung metastasis quantified by in vivo bioluminescence imaging of the thorax starting on the day of primary tumor resection, n = 5, error bars depict standard error. (B) Representative in vivo bioluminescence image of a mouse with spontaneous lung metastasis at day 5 and day 23 post resection. (C) Average range of ex vivo bioluminescence lung signal at the endpoint of the experiment, error bar depicts standard error. (D) Representative ex vivo bioluminescence images of dissected lungs at the endpoint of the experiment. Please click here to view a larger version of this figure.

Discussion

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 outgrowth in real time, but it is also clinically relevant as most patients undergo tumor resection or mastectomy in the clinical setting. Furthermore, this experimental setup permits the evaluation of the therapeutic benefit of pharmacological or genetic interventions in the neoadjuvant setting or the relevance of specific signaling pathways in the metastatic process^14,15.

This experimental model has a good number of additional advantages, as it is highly versatile. By using different host strains, it is amenable to the study of murine or human cell lines, albeit murine cell lines tend to have faster growth kinetics. Furthermore, a variety of reporters are now available that can be used to track the cells by different modalities, such as Firefly luciferase or Renilla luciferase for bioluminescence imaging, or TdTomato, mCherry, or eGFP for fluorescence¹⁶. The use of reporters is a significant part of the advantage of using this experimental setup. However, it is important to recognize that reporters might elicit adaptive immune responses, and whether or not this will influence the hypothesis being tested needs to be considered¹⁷.

Spontaneous models of cancer (known as genetically engineered mouse models-GEMMs) also undergo all stages of cancer progression, however, with a few exceptions, disseminated cells are not easily traceable, and primary tumors are usually multifocal and almost impossible to resect cleanly and with a single surgery. Therefore, in the tumor implantation, resection, and disseminated disease tracking setup described herein, the patterns of tumor growth and metastatic outgrowth are significantly more homogeneous in their development^2,3,4. This feature, combined with the fact that tracking disseminated tumor cells in spontaneous models requires sacrificing the animal to perform histological analysis at multiple time points, tremendously reduces the number of animals needed to observe specific patterns. However, the serial passage of tumor fragments or tumor organoids from GEMMs can easily be achieved by swapping this material for the cell lines used in this setup, making a pocket in the mammary gland, and inserting the tumor tissue instead of injecting cells. While still lacking reporters for real-time follow-up, this procedure facilitates the development of metastasis by starting with an individual harboring a single tumor with longer survival and also reduces the number of mice required for the analysis.

GEMMs tend to have very limited patterns of metastatic dissemination, mostly limited to the lymph nodes and lungs. Transplantable metastasis models can more closely resemble the metastatic dissemination patterns observed in humans². However, injection-based experimental models of metastasis require non-physiological numbers of cells to be injected systemically in order to initiate metastasis, which does not recapitulate spontaneous dissemination. That said, injection-based metastasis models excel at targeting metastasis to specific organs, with tail-vein injections commonly used to initiate lung metastasis and intracardiac or intracarotid inoculation used to initiate brain metastasis¹⁸. Notably, this protocol can be modified to include injection-based metastasis strategies following resection of the orthotopic primary tumor in order to model and study metastasis to organs where spontaneous colonization in short timelines is not observed, such as the brain, bone, or liver. The addition of this step to the above protocol also allows researchers to study the systemic effects of primary tumors on the metastatic cascade, particularly the development of the premetastatic niche.

The orthotopic transplantation model described in this protocol is not without its limitations. When utilizing established cell lines, one needs to be aware of the potential peculiarities accumulated over time through the expansion of cell lines in 2D culture prior to injection. This may lead to the development of orthotopic tumors that may not completely mimic the original tumor from which the cells were initially isolated or the phenotypic and genetic heterogeneity typically seen in human cancers⁴. Another issue is the genetic homogeneity of the host animals, which may be partially overcome by repeating experiments across multiple hosts. As previously mentioned, the use of immunocompetent mice also restricts the choice of cancer cell lines that can be used to establish orthotopic tumors to only those which are syngeneic to the same genetic background of mouse being used, of which there are a very limited number¹⁸. Additionally, the rapid growth of primary tumors implanted orthotopically may not fully recapitulate the kinetics of early phase metastasis seen clinically in human patients. Finally, as with all preclinical studies conducted in mice, there is always the formal possibility of the metastatic behavior observed being limited to the mouse microenvironment and not being able to be extrapolated to human studies. Therefore, validation in human archival or fresh tissues is the ultimate gold standard before translational studies.

The critical steps for the success of this protocol include the injection of the cell/basement-membrane matrix suspension into the mammary fat pad and the resection of the orthotopic primary tumor. As mentioned in the protocol above, avoiding backflow of the cell suspension out of the mammary tissue onto the peritoneum of the animal is key during injection to prevent the development of extra-mammary tumors. This is achieved by slightly delaying the withdrawal of the needle after injection to allow the suspension to start gelling and avoiding the application of excess pressure on the area close to the tumor to prevent leakage of the cell/basement-membrane matrix suspension. Extra-mammary tumor growth is more difficult to resect, leading to excessive morbidity or mortality post resection, and it contributes to background bioluminescence that confounds lung imaging results and decreases long-term survival. Ensuring complete resection of the primary tumor tissue is critical and can be assessed by acquiring a bioluminescence image of the animal immediately after the resection procedure. In situations where the primary tumor relapses before the end of the experiment, covering the abdominal region with a dark-colored paper can prevent confounding of the bioluminescence signal. Finally, the cell numbers used for establishing orthotopic tumors can be altered in order to slow or speed the kinetics of primary tumor development and account for differing growth rates between different cancer cell lines. The cell numbers described herein often result in the development of 150 mm³ primary tumors within 12-14 days after orthotopic injection. Success in establishing orthotopic tumors using 75,000 cells has also been achieved in our hands, though with slower kinetics. Additionally, this protocol describes orthotopic injection of tumor cells into mammary fat pads bilaterally, as this increases the number of cells that disseminate from the primary tumor and, therefore, the likelihood of minimal residual disease post resection of the primary tumors and spontaneous metastasis to the lungs. Unilateral injection of one mammary fat pad can also be utilized as an alternative, though the metastasis kinetics will likely differ.

In summary, this protocol describes a methodology for establishing and monitoring spontaneous metastasis of breast cancer to the lung. This protocol is versatile and can be modified by researchers, as described above, in order to study human cell lines or investigate systemic cancer effects on metastasis to specific organ systems.

Materials

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