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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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.

Protokół

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. 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.
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 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.
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. Administer 40 µL of meloxicam (2 mg/mL) subcutaneously 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. 
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. 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 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.
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. Then, euthanize the mice by cervical dislocation.
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.

Wyniki

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 ...

Dyskusje

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 ...

Materiały

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