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>Incub...

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+analysis+of+metastasis+in+transgenic+mouse+models.">The analysis of metastasis in transgenic mouse models.</a> Transgenic Research. 18 (1), 1-5 (2009).</li><li>Klein, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+progression+of+primary+tumours+and+metastases.">Parallel progression of primary tumours and metastases.</a> Nature Reviews Cancer. 9 (4), 302-312 (2009).</li><li>Pantel, K., Brakenhoff, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+metastatic+cascade.">Dissecting the metastatic cascade.</a> Nature Reviews Cancer. 4 (6), 448-456 (2004).</li><li>Sosa, M. S., Bragado, P., Aguirre-Ghiso, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+disseminated+cancer+cell+dormancy:+An+awakening+field.">Mechanisms of disseminated cancer cell dormancy: An awakening field.</a> Nature Reviews Cancer. 14 (9), 611-622 (2014).</li><li>Biswas, A. K., Acharyya, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+cachexia+in+the+context+of+metastatic+progression.">Understanding cachexia in the context of metastatic progression.</a> Nature Reviews Cancer. 20 (5), 274-284 (2020).</li><li>Liu, Y., Cao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characteristics+and+significance+of+the+pre-metastatic+niche.">Characteristics and significance of the pre-metastatic niche.</a> Cancer Cell. 30 (5), 668-681 (2016).</li><li>Peinado, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-metastatic+niches:+Organ-specific+homes+for+metastases.">Pre-metastatic niches: Organ-specific homes for metastases.</a> Nature Reviews Cancer. 17 (5), 302-317 (2017).</li><li>Psaila, B., Lyden, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metastatic+niche:+Adapting+the+foreign+soil.">The metastatic niche: Adapting the foreign soil.</a> Nature Reviews Cancer. 9 (4), 285-293 (2009).</li><li>Clark, N. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neoadjuvant+treatment+of+breast+cancer.+Annals+of+Oncology.">Neoadjuvant treatment of breast cancer. Annals of Oncology.</a> Official Journal of the European Society for Medical Oncology. 23, 231-236 (2012).</li><li>Serganova, I., Blasberg, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+with+reporter+genes:+Has+its+promise+been+delivered.">Molecular imaging with reporter genes: Has its promise been delivered.</a> Journal of Nuclear Medicine: Official Publication, Society of Nuclear Medicine. 60 (12), 1665-1681 (2019).</li><li>Grzelak, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elimination+of+fluorescent+protein+immunogenicity+permits+modeling+of+metastasis+in+immune-competent+settings.">Elimination of fluorescent protein immunogenicity permits modeling of metastasis in immune-competent settings.</a> Cancer Cell. 40 (1), 1-2 (2022).</li><li>Valiente, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+metastasis+cell+lines+panel:+A+public+resource+of+organotropic+cell+lines.">Brain metastasis cell lines panel: A public resource of organotropic cell lines.</a> Cancer Research. 80 (20), 4314-4323 (2020).</li></ol>

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

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

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

Watch this Scientific Journal Video about In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells at JoVE.com

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 ...

The systemic effects of primary tumors are now recognized to play a significant role in the process of metastatic dissemination. However, this is often missed in experimental metastasis assays. This model aims to provide researchers with a way to evaluate this effect.This protocol includes evaluation of spontaneous metastasis after the surgical removal of a primary tumor, which is clinically relevant. Mainly, it also takes advantage of bioluminescence imaging to monitor lung metastasis outgrowth in real time. This experimental setup can be extended to evaluate pharmacological or genetic interventions for breast cancer in the neoadjuvant setting, as well as the relevance of specific signaling pathways in the metastatic process.To begin, harvest the cells by aspirating the cell culture medium and wash with sterile PBS. Next, incubate with two milliliters of 0.25%Trypsin-EDTA solution for about two to three minutes at 37 degrees Celsius until the cells detach and then wash with eight millimeters of complete medium to quench the reaction. After aspirating the supernatant and washing the cells with PBS, resuspend the cells in PBS in the calculated volume necessary to dilute cells to 6 million cells per milliliter.Then transfer the cell suspension to 1.5-milliliter microcentrifuge tubes, and keep on ice until ready for injection. Shave the hair on the abdomen of the anesthetized mouse using electric clippers, and then place it in a supine position. Next, clean the prepared abdominal region using 70%ethanol and povidone-iodine solution.Using scissors, make a small midline incision through the abdominal skin at the level of the fourth mammary tissue, exposing but not penetrating through the underlying peritoneum. Then hold the skin away from the peritoneum using forceps and separate the skin away from the peritoneum with sterile saline-dipped cotton swabs, moving laterally to expose the right mammary fat pad, and repeat on the left side to expose the left mammary fat pad. After resuspending the E0771 cell suspension using a manual pipette, transfer 100 microliters to a new 1.5-milliliter microcentrifuge tube.Then add an equal volume of basement membrane matrix solution and mix well, taking care not to introduce bubbles. Afterward, keep the tube on ice. Draw 100 microliters of cell suspension into a 28-gauge 0.5-milliliter U-100 insulin syringe, and keep on ice.Using forceps, lift the skin and gently grab and expose the left mammary fat pad. Next, inject 50 microliters of cell suspension into the mammary fat pad and wait for three to five seconds prior to removing the syringe. Then release the skin from the forceps, allowing it to return naturally to its normal position, and close the skin incision, applying skin staples.To monitor the growth of the orthotopic breast tumor lesion, measure the primary tumor length and width using calipers three times per week. After anesthetizing, administer 40 microliters of meloxicam subcutaneously for pain control, and then place the mouse in a supine position. Next, remove prior surgical staples if necessary, and clean the abdomen with 70%ethanol and povidone-iodine solution.Using scissors, make a small midline incision through the abdominal skin at the level of the fourth mammary tissue, exposing but not penetrating through the underlying peritoneum. Then remove the orthotopic tumor by cutting through the normal mammary tissue, located proximally and distally to the tumor using scissors. Discard the tumor tissue into a biohazard bag while repeating the procedure with the contralateral tumor.Next, close the surgical site using one to three staples and transfer the mouse to a clean recovery cage with a warm heating pad underneath. Inject the animals with 100 microliters of D-luciferin solution using a retroorbital injection by inserting the needle in the medial canthus of the eye at a 45-degree angle from the nose until bony resistance is felt, ensuring that the needle is placed within the retroorbital venous sinus. Confirm successful injection by the lack of flush-back of any liquid upon delivery and wait two minutes prior to imaging.While waiting, transfer the animals to the nose cones located inside the bioluminescent imager in a supine position. To measure the photon flux, create a square ROI for each animal depicted in the image from under the ROI tool's dropdown menu by clicking the square ROI button. Reposition the automatically-generated ROI over the thorax of each animal by clicking and dragging with the mouse.And then click the measure ROI's button. Make a midline incision below the xiphoid process, cutting through the skin, musculature, and peritoneum to expose the lower part of the thoracic cavity using scissors until the diaphragm is visible. Afterward, puncture the diaphragm to collapse the lungs and then cut through the diaphragm.Cut through the rib cage on the right and left side, and then use a hemostat to grab the xiphoid process and move the rib cage out of the way, exposing the heart and lungs. Next, snip the right atrium using scissors. Then perfuse the animal with 10 milliliters 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.Next, identify the trachea and insert a 22-gauge needle syringe with three milliliters of 4%paraformaldehyde, holding it parallel to the trachea. Then deliver the solution at a slow pace until the lungs have fully inflated. Continue gently holding the trachea.Snip it with scissors above the forceps and start carefully lifting the tissue while removing all the connective tissue. Next, dissect the heart away from the lungs. Afterward, place lung tissue in 4%paraformaldehyde in PBS overnight and store at four degrees Celsius for fixation.Utilizing C57 black 6 mice, significant lung metastasis bioluminescence signal is typically detected around two 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. The 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. Lung nodule counting under the stereoscope and hematoxylin and eosin histological analysis can be utilized to complement the quantification studies.Slightly delaying needle withdrawal after mammary fat pad injection allows the matrix solution to gel, which is key to preventing leakage of the cell suspension and the development of extra-mammary tumors. We are now utilizing this technique in combination with various genetic knockout and knockin mouse models to further study the metastatic cascade, particularly the development of the pre-metastatic niche.

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Cancer Research

1.5K 조회수.  Virginia Commonwealth University School of Medicine. 원발성 종양의 전신 효과는 이제 전이성 전파 과정에서 중요한 역할을 하는 것으로 인식되고 있습니다. 그러나 이것은 실험적 전이 분석에서 종종 누락됩니다. 이 모델은 연구자에게 이 효과를 평가할 수 있는 방법을 제공하는 것을 목표로 합니다.이 프로토콜에는 임상적으로 관련성이 있는 원발성 종양을 외과적으로 제거한 후 자발적 전이에 ...