Studying the Role of Alveolar Macrophages in Breast Cancer Metastasis

Summary

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.

Abstract

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.

Introduction

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 cells^1,2. 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 soil^1,3-7. 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.

Protocol

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

1. 4T1 and 4T1-GFP Cell Culture
   NOTE: Perform all steps using sterile solutions in a laminar airflow (LAF) bio-safety cabinet unless specified otherwise.
   1. Maintain 4T1 and 4T1-GFP cells in RPMI medium supplemented with 10% heat inactivated fetal bovine serum and 100 U/ml penicillin G and 100 μg/ml streptomycin sulfate.
      NOTE: Determine a passage number by counting every trypsinization and plating of cells. It is important to use cells with the same passage number to all mouse experiments, as the number of passages affects cell tumorigenicity and metastatic potential.
   2. Prior to cell injections remove the T75 cm² flask, containing tumor cells at 80 % confluency, from an incubator and aspirate medium. Add 10 ml of PBS and rotate the flask gently to wash out remaining medium, and then aspirate PBS.
   3. Add 2 ml of 0.25 % trypsin with EDTA to the flask and tilt it to distribute the solution uniformly on the surface and incubate in a humidified incubator for 3 min at 37 ˚C with 5 % CO₂.
   4. Tap the flask to facilitate detachment of cells and add 8 ml of fresh medium. Pipette up and down several times to disrupt clumps and get a single cell suspension.
   5. Centrifuge cells for 5 min. at 500 x g at RT. Aspirate the supernatant and wash cells in 10 ml of PBS.
   6. Pipette up and down several times to disrupt clumps and get a single cell suspension. Take 100 µl of a cell suspension and count the cells using the cell viability analyzer as per manufacturer's instructions.
   7. Calculate number of cells required for all injections using formula: 10⁵ cells per mouse x # of mice used in an experiment (always prepare extra cells to be on a safe side).
   8. Centrifuge cells as in 1.1.5. Remove the supernatant through aspiration and resuspend cells in the amount of fresh PBS required to obtain a final cell concentration of 1 x 10⁶ cells/ml. Keep the cell suspension on ice until ready for injection.
2. 4T1 or 4T1-GFP Cell Injection-mouse Procedures
   1. The day before tumor cell injection, anesthetize mice in an induction chamber with 3 % isoflurane. Once mice are asleep and breathe regularly, place the mouse on a surgical pad with nose inside the nose cone, and connect it to the isoflurane vaporizer. Maintain anesthesia with 2.5 % isoflurane. Pinch the mouse toe to ensure that the mouse is anesthetized.
   2. Apply hair removal cream using a cotton swab to the injection site (right pectoral mammary fat pad) and wait for 2 min. Clean the site using a wet paper towel, place the mouse back into the cage and monitor an animal until it regains sufficient consciousness to maintain the sternal recumbency.
   3. On the day of injection, anesthetize the mice as in 1.2.2 and place on the surgical pad.
   4. Vortex the tube containing tumor cells to get uniform cell suspension. Aspirate 100 μl of a cell suspension, containing 1 x 10⁵ tumor cells, into a 0.5 ml insulin syringe (29 G, 12.7 mm-needle length).
   5. Lift the skin using the thumb and finger index near the 2^nd and 3^rd nipple, insert the needle of the syringe into the mammary fat pad just below the third nipple under the skin between the fingers and inject cells slowly to form a bubble (subcutaneous injection).
   6. Place the mouse back into the cage after injection and monitor as in 1.2.2.
3. Monitoring Tumor Growth
   1. Caliper Measurements¹⁴
      NOTE: Injected 4T1 or 4T1-GFP cells form aggressive breast tumor. Usually the palpable tumors appear ~ at day 4 - 5 after cell inoculation. 4T1-GFP cells form tumors that grow slower and give metastases later. Measure the tumor two to three times a week. If clinical status of animals deteriorates, animals lose more than 10 % body weight, tumors exceed 10 % of body weight or become ulcerated, euthanize mice immediately.
      1. Anesthetize a mouse as in 1.2.1., weigh, place on the surgical pad and wet the area with 70 % ethanol.
      2. Palpate tumor in the site of injection (4 - 5 days after cell inoculation).
      3. Measure the largest diameter (D) and smaller diameter (d1) with a caliper.
      4. Calculate tumor volume using formula Volume= (D X d1 X d1)/2 mm³. Monitor mouse recovery from anesthesia as in 1.2.2.
   2. Imaging (Only for 4T1 GFP Cells)
      1. Anesthetize the mouse as in 1.2.1. Place the mouse on a movable stage inside the imaging instrument. Maintain anesthesia through the nose cone.
      2. Turn on the imaging instrument and the light source as per manufacturer's instructions.
      3. Under the tab "Acquisition" go to "Lighting" and switch to the empty position (no filter) for white light. Ensure that the light engine is ON and select 'Trans' in the light table.
      4. Under the tab "Microscope", select "Clear" emission filter and 0.5X magnification. In the "Camera" tab, make sure that binning for capture is '1 x 1' and preview '4 x 4'.
      5. Click on preview to locate the tumor in white light, focus to get clear image, and click capture.
      6. Go to "Acquisition" tab and change lighting to filter 1 (filter for GFP as per manufacturer's instruction). In the "Microscope" tab, change emission filter to "530/20 GF". Preview and capture image in a green channel. Adjust exposure time to get the appropriate brightness.
      7. Apply pseudocolor to the image and save in appropriate folder (Figure 1).

2. Intranasal Administration of Liposomes¹⁰

NOTE: Perform all steps using sterile solutions in a laminar airflow (LAF) Bio-safety cabinet unless specified otherwise.

1. On day 6 after tumor cell injection anesthetize mice as in 1.2.1.
2. Vortex the clodronate or control (PBS) liposome suspension obtained from the manufacturer. Take 60 µl liposome suspension using sterile pipette tip.
3. Slowly release liposome solution (5 µl each time) near the nostrils allowing the mouse to breathe in the solution, repeat until the entire dose of 60 µl is administered.
4. Let the mouse regain consciousness before placing it back in the cage.
5. Repeat liposome administration every 3 days until the mice are sacrificed. Do not administer liposomes on a day of sacrifice.

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.

1. On day 22 (for 4T1 cells) or day 26 (for 4T1-GFP cells) after tumor cell injection anesthetize the mouse as in 1.2.1. and place on the surgical pad with a nose cone connected to vaporizer, maintain anesthesia as in 1.2.1. Pinch one of the toes to ensure that the mouse is unconscious.
2. Pin the toes using needles to the dissection board. Spray ethanol on the mouse skin.
3. Lift the skin using forceps and make an incision with the surgical scissors. Slowly expose the peritoneum and continue cutting skin through the thorax until the neck.
4. Using the surgical scissors make an incision in the peritoneum and expose carefully the organs with cotton tips avoiding damage to the blood vessels.
5. Move the organs to one side and expose the inferior vena cava. Puncture the vein and collect blood with the 29 G insulin syringe. Place collected blood into the tube and leave on ice for further processing.
6. Cut the diaphragm to expose the rib cage, lungs and heart. Slowly cut the rib cage on both sides using the bone cutter and lift the top of ribcage. Grab the heart with forceps and cut the connective tissue connecting the heart and lungs to the chest cavity.
7. Place the lungs in small amount of PBS in the 60 mm Petri dish on ice until ready for imaging and further processing to frozen sections for immunofluorescent microscopy or routine histology [including hematoxylin and eosin (H & E) staining].

4. Lung Metastases Evaluation

1. Counting and Scoring Surface Metastases
   1. Place the Petri dish with the lungs under the dissection microscope. Focus to get a clear view of the lung surface.
   2. Using a dissection microscope observe the lung surface. Count metastases on the anterior and posterior sides of both lungs.
      NOTE: The normal lung is whitish or pinkish and porous and has a sponge like structure. 4T1 metastases are solid and nonporous, sometimes appear to be similar to a drop of liquid (Figure 2A).
2. Lung imaging
   1. Place the Petri dish with lungs on the movable stage inside the imaging instrument.
   2. Turn on the instrument and the biolite multispectral source. Open the software. Under the tab, "Acquisition", go to "Lighting", and switch to empty position (no filter) for white light. Light Engine--> ON, Light table--> Epi. Under the tab "Microscope" Select 'Clear' emission filter and 1.66X magnification. In the "Camera" tab, make sure that binning for capture is '1 x 1' and preview '4 x 4'.
   3. Click on preview and focus to get a clear picture of lungs in white light and click capture.
   4. Go to "Acquisition" tab and change to the filter 1 (filter for GFP as per manufacturer's instruction). In the "Microscope" tab, change emission filter to "530/20 GF". Preview and capture image in a green channel. Adjust exposure time to get the appropriate brightness.
   5. Apply pseudocolor to the image and save in appropriate folder.
   6. Flip the lungs to get image of the other side in a similar way. This procedure generates images of GFP-positive metastases that can be further quantified using appropriate software (Figure 2B)¹⁴.
3. Immunofluorescent Microscopy
   1. Fix the lungs in 4% paraformaldehyde, at 4 °C for 4 hr in dark.
   2. Wash the tissue twice with 10 ml PBS for 5 min to completely remove the fixative. Transfer to 18 % sucrose in PBS and incubate O/N at 4 °C.
   3. Remove the tissue from the sucrose and dab off any excess.
   4. Take a cryomold, cover the bottom layer with OCT medium. Place the tissue on OCT medium. Fill the mold with OCT medium to completely cover the tissue and place on dry ice.
   5. Store block at -80 °C until sectioning.
   6. Prepare 5 µm thick sections with a cryostat and place on charged slides.
   7. Store unused slides in -80 °C until further usage.
   8. Air dry the slides for 7 min. and wash with PBS to remove OCT. Wipe the slide with tissue or paper towel to remove excess of water, mount cover slips with mounting medium containing DAPI and visualize with GFP filter under microscope.
   9. Quantify GFP metastases (Figure 3A) with the use of appropriate software¹⁴.
4. Histology and Digital Pathology
   1. Click the manual load button under the start tab and wait for the slide holding rack to eject from the scanner.
   2. Take the H&E stained tissue section, clean it with tissue to remove any dirt, and place it securely in the slide holding rack.
   3. Enter the slide description in the popped-up window.
   4. Select the Scan Area tab on the SS imaging console window and adjust the green square to define the region of interest (ROI) to be scanned.
   5. Drag the blue calibration diamond to a clear portion of the slide, where tissue is absent, however, within the ROI.
   6. Next, select the Focus Points tab and click the auto select button to obtain several focus points (yellow plus signs) on the tissue in the ROI.
   7. If necessary, place additional focus points manually by double-clicking within the ROI.
   8. Proceed to the calibrate tab and select the calibrate button to begin slide calibration at the blue calibration diamond point. After the calibration is completed the image from the calibration point will be displayed.
   9. Inspect this image to ensure that it is clear and free of dirt or artifacts.
   10. Proceed to the Scan tab and select Start Scan to begin scanning of the ROI. Scanned histological section is converted to a digital slide (Figure 3B and 4A)
   11. Quantify metastatic burden as previously described (Figure 4B)¹⁴.

5. Flow Cytometry (FACS) Analysis of Alveolar Macrophages in the Lungs

1. Preparation of Single Cell Suspension
   1. After imaging and counting of metastases, wash the lungs with sterile PBS and place to the Petri dish. Make 1 - 2 mm pieces of the lungs using surgical scissors and forceps.
   2. Transfer the lung pieces to the 15 ml conical tube with 3 ml of the digestion buffer containing 1 mg/ml collagenase P, 0.04mg/ml DNase I, and 10 µg/ml trypsin inhibitor dissolved RPMI medium (sterile).
   3. Incubate the conical tube on a rotating shaker in the incubator at 37 °C for 40 min.
   4. Remove the tube conical from the incubator and pipette the solution up and down to dissociate the tissue.
   5. Pass the solution through the 40 µm strainer to avoid clumps and get a single cell suspension. If needed, to disintegrate better the digested tissue press tissue against the cell strainer with the help of a syringe plunger.
   6. When a single cell suspension has been achieved, collect the cells by centrifugation and lyse red blood cells with 2 ml of ACK buffer for 10 min at RT. Then, wash a pellet two times in 8 ml of RPMI.
   7. Resuspend cells in 3 ml of the complete RPMI medium and proceed to cell counting using the cell viability analyzer as per manufacturer's instructions.
2. Staining of Lung Cells for Surface Markers
   NOTE: Perform all incubations at 4 °C. Centrifuge plate at 500 x g and 4 °C.
   1. Pipette 1 x 10⁶ cells in 100 µl of FACS buffer (1% FBS in PBS) to individual wells of a V-bottom 96-well plate. Using a V-bottom 96 well plate facilitates handing of multiple samples.
   2. Pre-incubate the cell suspension with 1 µg Mouse Fc Block purified anti-mouse CD16/CD32 in 100 µl of FACS buffer for 15 min. Centrifuge the V-bottom 96 well plate to pellet the cells and remove the supernatant by flipping the plate and letting the solution drain.
   3. For surface staining, in advance, dilute the antibodies to murine antigens in one tube (master mix): BV605 CD45 (30-F11), PE CD11b (M1/70), PE/Cy7 F4/80 (BM8), APC/Cy7 CD11c (N418), PerCPCy5.5 I^A/I^E (MHCII) (M5/114.152), PE CD80 (16-10A1), AF 647 CD86 (GL-1) to a final concentration of 2.5 µg/ml in FACS buffer containing 10 µg/ml of Fc block CD16/CD32 antibody.
      NOTE: This set of antibodies is useful for the identification and the evaluation of a functional status of alveolar macrophages and dendritic cells.
   4. Add 100 µl of diluted antibodies (master mix) to the wells of the V-bottom 96 well plate and incubate at 4 ˚C for 30 min.
   5. Centrifuge the plate to pellet cells and remove supernatant as in 5.2.2. Wash cells with 200 µl of FACS buffer, centrifuge at 500 g for 5 min. Remove the supernatant as in 5.2.2. and resuspend cells in 200 µl of PBS.
   6. Centrifuge the plate to pellet cells and add viability dye diluted in PBS (1:1,000). Incubate for 20 min at 4 ˚C.
   7. Centrifuge the plate to pellet cells and remove the supernatant as in 5.2.2. Wash with 200 µl of PBS and centrifuge the plate again.
   8. Resuspend cells in 100 µl of 1% paraformaldehyde and store at 4 °C until the acquisition by FACS instrument. Prior to acquisition transfer cell suspensions to the polypropylene FACS tubes.

6. Analysis of FACS Data: Gating Strategies and the Identification of Cell Subpopulations

1. Perform FACS analysis as previously reported¹⁰. Gate acquired cells/events based on forward-(FSC) and side-scatter (SSC); and then gate live and CD45⁺ cells. For alveolar macrophage identification, gate CD11b-negative and then CD11c⁺F4/80⁺ cells. (Figure 5A).

Results

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 metastati...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
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
15 ml conical tube	BD falcon, Franklin Lakes, NJ, USA	352096	Tissue culture supplies
50 ml conical tube	BD falcon, Franklin Lakes, NJ, USA	352098	Tissue culture supplies
60 mm2 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