Here we describe the model and approach to study functions of pulmonary alveolar macrophages in cancer metastasis. To demonstrate the role of these cells in metastasis, the syngeneic (4T1) model of breast cancer in conjunction with the depletion of alveolar macrophage with clodronate liposomes was used.
This paper describes the application of the syngeneic model of breast cancer (4T1) to the studies on a role of pulmonary alveolar macrophages in cancer metastasis. The 4T1 cells expressing GFP in combination with imaging and confocal microscopy are used to monitor tumor growth, track metastasizing tumor cells, and quantify the metastatic burden. These approaches are supplemented by digital histopathology that allows the automated and unbiased quantification of metastases. In this method the routinely prepared histological lung sections, which are stained with hematoxylin and eosin, are scanned and converted to the digital slides that are then analyzed by the self-trained pattern recognition software. In addition, we describe the flow cytometry approaches with the use of multiple cell surface markers to identify alveolar macrophages in the lungs. To determine impact of alveolar macrophages on metastases and antitumor immunity these cells are depleted with the clodronate-containing liposomes administrated intranasally to tumor-bearing mice. This approach leads to the specific and efficient depletion of this cell population as confirmed by flow cytometry. Tumor volumes and lung metastases are evaluated in mice depleted of alveolar macrophages, to determine the role of these cells in the metastatic progression of breast cancer.
The premetastatic niche is an important process in cancer metastasis defined as a set of alterations that occur in the organs that are targets for metastases prior to arrival of tumor cells1,2. Therefore, therapeutic targeting of this step of cancer progression may prevent metastases to the vital organs that cause approximately 90% of cancer-associated deaths. Although the concept of the premetastatic niche, also known as the “seed and soil” theory, was introduced more than a century ago, no experimental proof has been provided until recently, when the bone marrow-derived cells were demonstrated to contribute to the premetastatic soil1,3-7. Despite these developments, the premetastatic niche remains a largely understudied aspect of cancer pathophysiology and further research to identify cellular players and mechanisms involved is needed.
Here we report the in vivo approaches to study the role of alveolar macrophages in breast cancer metastases and the lung premetastatic niche. The alveolar macrophages arrive to the lungs early during the embryonic development and self-renew there during adulthood8. They also have important immunomodulatory and homeostatic functions including the protection of this organ from undesired inflammatory responses to the environmental innocuous antigens9. Therefore, we hypothesize that tumors exploit this physiological immunosuppression, imposed by alveolar macrophages, and, consequently, alveolar macrophages contribute to the lung premetastatic niche by suppressing antitumor immunity. This hypothesis is supported by our recent report demonstrating that the specific depletion of these cells reduces lung metastases and enhances antitumor T cell responses10.
For these studies we apply a well-established syngeneic model of breast cancer (4T1), which mimics stage IV metastatic breast cancer11; and has been previously reported in studies of the premetastatic niche6. To track metastasizing tumor cells in vivo we use 4T1 cells expressing GFP (4T1-GFP) in conjunction with animal imaging and confocal microscopy. We focus on the lung premetastatic niche, since this organ is one of the most common targets of hematogenous metastases of human malignancies12. To investigate functions of alveolar macrophages in the premetastatic niche, we use clodronate liposomes to deplete these cells13; and evaluate impact of this depletion on lung metastases. Of note, this method specifically depletes alveolar macrophages but no other phagocytic cells in the lungs or in circulation10.
All animal studies have been approved by Institutional Animal Care and Use Committee of Texas Tech University Health Sciences Center and followed the guidelines outlined in the "Guide for the Care and Use of Laboratory Animals" published by the National Institutes of Health. Use eight to twelve week old female BALB/c mice that are commercially available. Inject 1 x 105 4T1 or 1 x 105 4T1 cells expressing GFP, which can be purchased from various vendors, into the mammary fat pad.
1. Culture of 4T1 and 4T1-GFP Cells & Preparation of Tumor Cell Suspension for Injections14
2. Intranasal Administration of Liposomes10
NOTE: Perform all steps using sterile solutions in a laminar airflow (LAF) Bio-safety cabinet unless specified otherwise.
3. Mouse Sacrifice and Tissue Collection
NOTE: Use autoclaved and sterile instruments for mouse dissection. Euthanize mice while under anesthesia through the exsanguination and removal of the vital organs.
4. Lung Metastases Evaluation
5. Flow Cytometry (FACS) Analysis of Alveolar Macrophages in the Lungs
6. Analysis of FACS Data: Gating Strategies and the Identification of Cell Subpopulations
The injection of 4T1-GFP tumor cells into the mammary fat pad leads to the formation of mouse tumors (Figure 1A) that recapitulate the metastatic spread of human breast cancer, as metastases are rapidly formed in the lungs (Figure 2), liver, bones and brain of mice11. The stable transfection of 4T1 cells with GFP facilitates monitoring of tumor growth (Figure 1B), tracking metastasizing tumor cells and quantifying the metastatic burden (Figure 2B, 3B). In addition, imaging of tumors provides the additional information regarding several pathological features such as tumor cell death and tumor vasculature. The bright green fluorescence (Figure 1B-the middle panels) indicate the high GFP expression, which is usually associated with the viable tumor parenchyma. Whereas the lack of GFP in some tumor areas may indicate tumor cell death (Figure 1B-the right panel). GFP is also absent from the tortuous aberrant tumor blood vessels (Figure 1B-the right panel).
Since the majority of lung metastases are located on the lung surface, they can be observed under the regular dissection microscope. The metastatic lesions are usually clearly distinct from the surrounding parenchyma (Figure 2A). To verify gross observations and evaluate metastases that are deeper within the lung parenchyma, fluorescent imaging of lungs can be performed (Figure 2B), if GFP expressing cells were used for injection. Although lung autofluorescence is clearly visible in the images, bright GFP-derived fluorescence facilitates distinguishing metastases from surrounding lung parenchyma. Alternatively, routine histology (Figure 3A and Figure 4A) in conjunction with digital pathology algorithms (Figure 4B) provide good tools to verify and quantify metastases.
Multicolor FACS provides opportunity to characterize even rare cell populations through the use of multipole cell surface and/or cytoplasmic markers simultaneously. Alveolar macrophages are characterized by the lack of CD11b expression and expression of F4/80 and CD11c (Figure 5). The typical approach to identify these cells by flow cytometry involves: (i) selection of cell population based on morphology depicted by forward and side scatter properties, (ii) selection of viable CD45+ cells followed by (iii) gating of CD11bnegative cells, and (iv) gating of cells coexpressing F4/80 and CD11c (Figure 5A,B).
Figure 1. Monitoring Tumor Growth Through Animal Imaging. (A)White light images of the mouse injected with GFP expressing cells into the mammary fat pad at different time points after tumor cell injection. (B) Corresponding fluorescent images obtained through the use of GFP filter. Dashed lines mark tumor area. Note the lack of bright green fluorescence in some tumor areas on day 26 that may result from tissue necrosis or developing neovasculature. Scale bar, 10 mm. Please click here to view a larger version of this figure.
Figure 2. Evaluation of Lung Metastatic Burden. (A) Images of the lungs of mice injected with 4T1 cells into the mammary fat pad with the surface metastases (arrows). (B) Images of GFP+ lung metastases (bright green fluorescence) obtained through use of imaging microscope. Scale bar, 10 mm. Please click here to view a larger version of this figure.
Figure 3. Digital Histopathology and Confocal Microscopy in Imaging of Lung Metastases. (A) The scan of the hematoxylin and eosin (H&E) stained lung section from mice injected with 4T1 cells into the mammary fat pad (dark blue tissue-metastases). Scale bar, 4 mm. (B) Confocal microscopy of GFP+ lung metastases (bright green fluorescence). Scale bar, 200 µm. Please click here to view a larger version of this figure.
Figure 4. Quantification of Lung Metastatic Burden by Digital Histopathology. (A)The scan of hematoxylin and eosin (H&E) stained lung section (dark blue tissue-metastases). (B) The image of the lungs shown in (A) generated by a trainable histomorphology image analysis tool (software-"Genie"). Red color indicates the lung areas identified by "Genie" as metastases. Scale bar, 5 mm. Please click here to view a larger version of this figure.
Figure 5. Flow Cytometry Approaches for Identifying Alveolar Macrophages in the Lungs. (A)The representativeflow cytometrydot plotsillustrating the gating strategy to identify CD45+CD11bnegativeF4/80+CD11c+ alveolar macrophages. (B) Representative flow cytometry dot plotsillustrating effect of control or clodronate liposomes on lung alveolar macrophages. Numbers in plots represent percentages of gated cells. Please click here to view a larger version of this figure.
The recent insights into cancer biology and causative factors involved in carcinogenesis and tumor progression lead to development of genetically engineered mouse (GEM) models of cancer, in which tumors grow spontaneously, usually over a period of several months15. Although these tumor models appear to reflect better the natural history of human malignancies than xenografts or syngeneic models, much time required for tumor development and various degrees of malignant phenotype penetrance limit the use of these approaches in mechanistic studies. Therefore, the models utilizing immunocompetent mice injected orthotopically with syngeneic cells, although not perfect, are still helpful in addressing mechanistic questions pertaining to the tumor microenvironment and a role of the immune system in cancer. The 4T1 model has been extensively used for these purposes, as it recapitulates stage IV metastatic breast cancer in humans11.
The use of 4T1 tumor cells expressing fluorescent proteins offers opportunity to monitor tumor growth, changes in the tumor cell viability, and metastasis through animal imaging14. Although this is an elegant approach to track metastasizing cells, some concerns related to the potential immunogenicity of 4T1 cells expressing GFP, and the generation of antibodies specifically recognizing 4T1-GFP cells have been raised16. However, these antibodies were found to be present in mouse plasma three weeks or later after the implantation of tumor cells16. Therefore, considering rapid growth of tumors in this model and the fact that mice are usually sacrificed before a humoral response develops or shortly after, 4T1-GFP model appears to be a useful tool for tracking circulating tumor cells in short term studies. In addition, it has been noted that low GFP expression, which characterize cells that we used for our studies, induces tolerance to GFP peptides instead of causing lymphocyte activation17.
The potential problem associated with the low level of GFP expression is long exposure time required to image organs, which are target for metastases, or tissue sections, to detect endogenous GFP expression. For immunofluorescent microscopy this problem can be, however, overcome by staining sections with appropriate GFP antibodies
The critical aspect of these studies is also an appropriate technique of the orthotopic cell injection, as any leakage of the solution containing tumor cells reduces the number of cells injected and, consequently, affects the pace of tumor growth and metastatic potential. By adjusting the number of injected cells, one can modify speed of tumor growth and time interval between injection of tumor cells and metastatic progression of disease. In addition, it is important to consider high variability of results obtained through the majority of in vivo approaches including those discussed here. Therefore, a sufficient number of mice, individually determined through a power analysis, needs to be used. In our laboratory, we typically use ten mice per cohort.
The use of clodronate liposomes to deplete different macrophage populations has been extensively explored18. However, several concerns related to specificity of this approach are repeatedly raised. Although intravenous injections of clodronate-liposomes depletes several different type of phagocytes in different organs, intratracheal or intranasal administration affects mainly lung macrophages18. Furthermore, our recent study has demonstrated that intranasal instillation of clodronate-liposomes, according to the protocol established in our laboratory, leads only to the depletion of alveolar macrophages, typically located in the lumen of lung alveoli, but does not affect myeloid-derived suppressor cells and interstitial macrophages10.
The authors have nothing to disclose.
This research has been supported by Department of Defense grant TSA 140010 to M.K. and BC 111038 to M.M.M. Views and opinions of, and endorsements by the author(s) do not reflect those of the US Army or the Department of Defense.
4T1 cell line | American Type Culture Collection, Manassas, VA, USA | CRL 2539 | Tumor cells |
4T1-GFP cell line | Caliper life sciences/ Perkin Elmer, Waltham, MA, USA | BW128090 | Tumor cells |
RPMI | Corning, Corning, NY, USA | 10-040-CM | Media |
Heat inactivated FBS | Gibco (Thermo Scientific), USA | 10082147 | Media |
Penicillin Streptomycin | Fisher Scientific, Waltham, MA, USA | MT-300-02-CI | Media |
PBS | Fisher Scientific, Waltham, MA, USA | BP399-20 | Dilute with distilled water |
Trypsin 0.25% with EDTA | Hyclone, Logan, Utah, USA | SH30042.02 | Tissue culture supplies |
T75 cm2 flask | Fisher Scientific, Waltham, MA, USA | 12-565-32 | Tissue culture supplies |
15ml conical tube | BD falcon, Franklin Lakes, NJ, USA | 352096 | Tissue culture supplies |
50ml conical tube | BD falcon, Franklin Lakes, NJ, USA | 352098 | Tissue culture supplies |
60mm2 Petri dish | Fisher Scientific, Waltham, MA, USA | AS4052 | For lung imaging |
Isoflurane (Isothesia) | Butler Schein Animal health, Dublin, OH, USA | NDC 11695-6776-2 | Mouse anesthesia |
Clodronate liposomes | Formumax Scientific Inc, Palo Alto, CA, USA | F70101C-N | Macrophages depletion |
Control liposomes | Formumax Scientific Inc, Palo Alto, CA, USA | F70101-N | Control PBS-liposomes |
29 gauge insulin syringes (12.7 mm and 0.5 ml capacity)- Reli-On | Walmart, Bentonville, AR, USA | For tumor cell injection | |
Hair removal cream (Nair) | Walmart, Bentonville, AR, USA | ||
Paraformaldehyde solution (4%) | Affymetrix, Santa Clara, CA, USA | 19943-I Lt | Dilute to 4% or 1% using 1X PBS |
OCT compound | Fisher Scientific, Waltham, MA, USA | 230-730-571 | For freezing tissue in cryomolds |
Fluoro-Gel-II with DAPI | Electron Microscopy Sciences, Hatfield, PA, USA | 17985-51 | Mounting medium |
Sucrose | Sigma, St. Louis, MO, USA | S-9378 | Cryopreservation |
Collagenase P | Roche, Basel, Switzerland | 11249002001 | Components of tissue digestion buffer |
Dnase I | Roche, Basel, Switzerland | 10104159001 | Components of tissue digestion buffer |
Trypsin inhibitor | Sigma, St. Louis, MO, USA | T9253 | Components of tissue digestion buffer |
40 micron cell strainers | Fisher Scientific, Waltham, MA, USA | 22-363-547 | Used in tissue digestion to remove clumps |
Trustain FcX-Fc Block (CD16/CD32) | Biolegend, San Diego, CA, USA | 101320 | Antibodies for flow cytometry |
BV605 CD45 | Biolegend, San Diego, CA, USA | 103139 | Antibodies for flow cytometry |
PE CD11b | Biolegend, San Diego, CA, USA | 101207 | Antibodies for flow cytometry |
PE Cy7 F4/80 | Biolegend, San Diego, CA, USA | 123113 | Antibodies for flow cytometry |
APC/Cy7 CD11c | Biolegend, San Diego, CA, USA | 117323 | Antibodies for flow cytometry |
PerCPcy5.5 IA/IE (MHCII) | Biolegend, San Diego, CA, USA | 107625 | Antibodies for flow cytometry |
PE CD80 | Biolegend, San Diego, CA, USA | 104707 | Antibodies for flow cytometry |
AF647 CD86 | Biolegend, San Diego, CA, USA | 105019 | Antibodies for flow cytometry |
Fixable viability Dye eflour 506 | eBioscience, San Diego, CA,USA | 65-0866 | Antibodies for flow cytometry |
Cryostat | Leica Biosystems, Buffalo Grove, IL, USA | CM1850 | Cryosectioning |
UVP iBox Explorer | UVP Inc, Upland, CA, USA | Mouse and lung fluorescent imaging | |
Aperio Scanscope CS | Leica Biosystems, Buffalo Grove, IL, USA | Digital pathology | |
BD LSRFortessa | BD Biosciences, Franklin Lakes, NJ, USA | Flow cytometry/data acquisition | |
Nikon A1 confocal TE2000 microscope | Nikon Instruments Inc., Melville, NY 11747-3064, U.S.A. | Imaging and quantifying GFP fluorescence in lung cryosections | |
UVP visionworks software (Version 7.1RC3.38) | UVP Inc, Upland, CA, USA | Imaging software for iBOX | |
Aperio Imagescope software (v12.1.0.5029) | Leica Biosystems, Buffalo Grove, IL, USA | Imaging software for analysis of digital slides | |
Flow JO software (version 9.8.1) | Flow JO LLC, Ashland, OR, USA | Analysis of flow cytometric data | |
NIS Elements AR (version 4.20.01) 64 Bit | Nikon Instruments Inc., Melville, NY 11747-3064, U.S.A. | Acquisition and analysis of lung cryosections for GFP |