Name: in vivo imaging to measure spontaneous lung metastasis of orthotopically-injected breast tumor cells
Uploaded: 2022-06-23T14:00:00.000+00:00
Duration: 8 min 36 s
Description: Watch this Scientific Journal Video about In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells at JoVE.com

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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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 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 process14,15.
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 development2,3,4. 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.

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><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&amp;amp;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><td></td></tr></tbody></table>

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 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.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64002/64002fig01.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64002/64002fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64002/64002fig02.jpg" /> 
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. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/64002/64002fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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

Nanyang Technological University

Research

JoVE Journal

Cancer Research

1.6K Views.  Virginia Commonwealth University School of Medicine. Het is nu bekend dat de systemische effecten van primaire tumoren een belangrijke rol spelen in het proces van metastatische verspreiding. Dit wordt echter vaak over het hoofd gezien bij experimentele metastasetests. Dit model is bedoeld om onderzoekers een manier te bieden om dit effect te evalueren.Dit protocol omvat evaluatie van spontane metastase na de chirurgische verwijdering van een primaire tumor, die klinisch relevant is. Het maakt voornameli...

Video: In vivo beeldvorming om spontane longmetastasen van orthotopisch geïnjecteerde borsttumorcellen te meten

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Medicine

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

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

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

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

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

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 ...

A Permanent Window for Investigating Cancer Metastasis to the Lung

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

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

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

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Biology

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

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

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

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

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular and molecular processes that govern metastatic dissemination and growth are still elusive. Live imaging allows visualization of the dynamic and spatial interactions of cells and their microenvironment. Solid tumors commonly metastasize to the lungs. However, the anatomical location of the lungs poses a challenge to intravital imaging. This protocol provides a relatively simple and quick method for ex vivo live imaging of the dynamic interactions between tumor cells and their surrounding stroma within lung metastasis. Using this method, the motility of cancer cells as well as interactions between cancer cells and stromal cells in their microenvironment can be visualized in real time for several hours. By using transgenic fluorescent reporter mice, a fluorescent cell line, injectable fluorescently labeled molecules and/or antibodies, multiple components of the lung microenvironment can be visualized, such as blood vessels and immune cells. To image the different cell types, a spinning disk confocal microscope that allows long-term continuous imaging with rapid, four-color image acquisition has been used. Time-lapse movies compiled from images collected over multiple positions and focal planes show interactions between live metastatic and immune cells for at least 4 hr. This technique can be further used to test chemotherapy or targeted therapy. Moreover, this method could be adapted for the study of other lung-related pathologies that may affect the lung microenvironment.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

Metastasis is a major cause for cancer-related morbidity and mortality. Metastasis is a multistep process and due to its complexity, the exact cellular ...

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

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