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.
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.
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 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.
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
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.
3. Tumor resections
4. In vivo quantification of spontaneous lung metastasis
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.
Orthotopic injection of mouse cancer cell lines into the 4th mammary fat pad of mice is a reproducible and reliable procedure for inducing mouse primary tumors. Utilizing the EO771 cell line transduced with luciferase in the conditions described in this protocol, primary tumors become palpable and can be measured using calipers about 7 days after injection and reach approximately 150 mm3 in volume at around 14 days following initial injection (Figure 1). Bioluminescence growth of these lesions can also be monitored as desired. Following primary tumor resection, bioluminescence imaging is utilized to confirm appropriate resection (Figure 1D). Utilizing C57BL6 mice, significant lung metastasis bioluminescence signal is typically detected around 2 weeks following primary tumor resection and can be followed over time, although some variation can be found depending on the luciferase reporter type and mouse strain (Figure 2A,B). Finally, ex vivo bioluminescence imaging of the lung tissue at the endpoint provides accurate and sensitive quantification of lung metastatic burden that can be plotted to compare with other treatments (Figure 2C,D). Lung nodule counting under the stereoscope and hematoxylin and eosin histological analysis can be utilized to complement the quantification studies.
Figure 1: Orthotopic mammary gland injection and monitoring of the primary breast tumors until primary tumor resection. (A) Schematic of the overall procedure. EO771-TGL cells are injected into the 4th mammary fat pad at day 0 (green circle), surgically resected upon reaching 150 mm3 in size, with spontaneous lung metastasis visible by bioluminescence approximately 2 weeks after primary tumor resection (green lungs). (B) Typical primary tumor growth kinetics via caliper measurement following mammary gland injection of 150,000 EO771-TGL cells, n = 9, error bars depict standard error. (C) Bioluminescence image depicting a representative mouse with primary tumors of approximately 150 mm3 at the time they are resected. (D) Bioluminescence image depicting a representative mouse immediately after complete and successful tumor resection. Please click here to view a larger version of this figure.
Figure 2: Monitoring of lung metastasis after tumor resection. (A) Growth kinetics of spontaneous lung metastasis quantified by in vivo bioluminescence imaging of the thorax starting on the day of primary tumor resection, n = 5, error bars depict standard error. (B) Representative in vivo bioluminescence image of a mouse with spontaneous lung metastasis at day 5 and day 23 post resection. (C) Average range of ex vivo bioluminescence lung signal at the endpoint of the experiment, error bar depicts standard error. (D) Representative ex vivo bioluminescence images of dissected lungs at the endpoint of the experiment. Please click here to view a larger version of this figure.
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.
The authors have nothing to disclose.
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.)
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 |
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