3.1 Generation of metastasis
Multiple methods can be applied to generate metastasis in the lung. These methods have been reviewed elsewhere(30). Methods can vary from genetically engineered mouse models of metastasis, to fat pad injected cell lines that spontaneously develop metastasis, to experimental metastasis (intra-venous injected cell lines). The time for development of metastasis depends heavily on cell line, route of administration, injected cell number, and background of mice/cell lines and must be determined empirically for each model. The method presented here covers both the spontaneous (orthotopic injection followed by resection) and experimental metastasis (tail vein injection) methods (20) using MMTV-PyMT derived cells containing the fluorescent protein Cerulean (Cer2-MMTV PyMT).
Preparation of cells for injection
1. Culture Cer2-MMTV PyMT cells with complete DMEM in a T75cm2 flask with filter cap to between 30-70% confluency. Passage cells on the day prior to injection to reach about 70% confluency on the day of injection (see Note 3).
2. To detach Cer2-MMTV PyMT cells flask, remove complete DMEM and rinse flask once with PBS to remove remaining protein and cell media.
3. Add 2mL of Trypsin-EDTA per flask and ensure that liquid is covering cells. Incubate cells for about 10 min at 37C, 5% CO2 in incubator to detach cells. Observe detachment of cells under a widefield microscope.
4. Neutralize Trypsin-EDTA with 10mL complete DMEM and transfer solution into 50 mL Falcon tube.
5. Centrifuge cells for 5 min at 300g, room temperature.
6. Discard supernatant and vigorously resuspend cells in 1 mL ice-cold PBS, top up to 10 mL with ice-cold PBS and repeat centrifugation.
7. Resuspend cell pellet in 1 mL ice-cold PBS and pass through 30µm cell strainer to remove and dissociate cell clusters.
8. Count cells using hemocytometer or cell counter (CellDrop FL, Denovix).
9. Adjust cell density to 5x106 cells per mL in PBS for tail vein injections (1x106 cells per mouse) and 5x106 in 1:1 PBS:Matrigel (2.5x105 cells per mouse) for fat pad injection. Ensure that for both injections cell suspension is kept continuously on ice.
Spontaneous metastasis model
1. The day prior to injection, shave mice from fourth and fifth nipple to midline and weigh mice to monitor weight changes after injection.
2. Autoclave a pair of tweezers and place an aliquot of matrigel on ice at 4°C to thaw overnight.
3. On the day of injection, anesthetize the mice with 5% isoflurane for 1 min, then decrease dose of isoflurane to 2%.
4. Confirm anesthesia by toe pinch and wait until there is no reflex observed.
5. Apply ophthalmic ointment to eyes of mice to prevent drying.
6. Clean shaved area with a chlorhexidine soaked cotton swab using circular movement away from the incision site.
7. Using the tweezers, gently lift tissue at fifth nipple and insert the needle bezel up, gently moving in a diagonal line to the fourth nipple, while keeping the needle close to skin.
8. While holding the needle horizontally, inject 50µL of cell suspension into the mammary fat pad around fourth nipple. A weal should be observed where the cell suspension was injected.
9. Slowly withdraw needle to avoid leakage from injection site.
10. Return mice to a heated cage (using a heat lamp or heat pad). Monitor mice for 15 minutes after they regain consciousness.
11. Administer 0.1 µg/mL antibiotic (baytril) into the drinking water to minimize the risk of infection.
12. Resect primary tumor when it reaches 5mmx5mm and macro-metastases can be observed: around four months after orthotopic injections.
Experimental metastasis model
1. Pre-heat water bath to 37°C.
2. Prepare 200µL cell suspensions per 29G ½ inch syringe per mouse: remove air bubbles and position on ice until use.
3. Position mouse in a restrainer to avoid sudden movements of the mouse.
4. To ensure dilation of veins, warm the mouse tail by dipping the tail into the water bath (about 1-2min, avoid restraining the mice for an extended duration to minimize stress).
5. If mouse has turned within the restrainer, confirm the orientation and location of lateral vein before carrying out injections.
6. Insert the needle with bevel up into the vein towards the direction of the head and keep the syringe parallel to the tail. There is no need for deep insertion of needle into the tail vein as the veins are superficially located. Proper placement can be verified when there is no resistance to injection and the needle advances slowly into the vein. (see Note 4)
7. Slowly inject the cell suspension into the vein, if the needle is inserted correctly, then the vein will appear to blanch during the injection.
8. Remove the needle and apply gentle compression to the tail to prevent bleeding.
9. Return mouse to cage and monitor for 15min to observe any adverse effects.
10. Macrometastases can be observed around 4 weeks after tail vein injection.
3.2 Generation of Precision-Cut Lung Slices
Steps:
1. Sterilize forceps, scissors, and disposable razor blades.
2. Cool PBS and complete DMEM (4°C)
3. Pre-cool metallic buffer holder and disposable blades for 1h at -20°C prior to slicing.
4. Prepare 24-well plate by pipetting 1 mL ice-cold complete DMEM into each well and transfer plate on ice until slicing.
5. Prepare two 50mL aliquots of complete DMEM and PBS and store at 4°C for after lung dissection.
6. Pre-heat 2% LMP Agarose and transfer at 37°C until lung inflation (see Note 5)
Dissection and inflation of lungs
7. Humanely euthanize C57BL/6 mouse with a fatal dose of ketamine (see Note 6).
8. Sterilize mouse chest and tracheal area using 70% Ethanol spray.
9. Make a lateral incision from the lower end of the rib cage to the upper end of the throat.
10. Open chest cavity along the midline from abdomen to chin and remove front ribs to generate sufficient space for lung inflation.
11. Cut artery between lungs and liver for exsanguination and to confirm death of mice.
12. Perfuse lungs through the left/right cardiac ventricle with 10mL ice cold PBS using 10mL syringe fitted with 25G needle.
13. Expose trachea by carefully removing surrounding muscle tissue.
14. Gently lift trachea with forceps and make a loose knot around it.
15. Laterally insert 19-21G needle into trachea using forceps to help guide the needle and secure needle by tightening the knot.
16. Fill 2mL syringe with 2% pre-heated LMP Agarose and connect to 19-21G syringe.
17. Inflate lungs with about 1 mL LMP Agarose (see Notes 7 & 8). Optimal inflation of lungs can be observed when tips of lung lobes stiffen. Ensure that lung lobes are not over-inflated (see Note 9).
18. Leave needle and syringe attached until solidification of agarose (2-3min).
19. Cover lung with sterile parafilm and cover with ice-cold PBS or ice cubes to facilitate solidification.
20. Remove needle and tighten knot, cut trachea above knot, and, holding trachea, excise lung and move to 10mL dish with ice-cold PBS.
21. Place dish on ice until further processing.
Preparation of precision cut lung slices
22. Turn on hood around vibratome and spray area and blade holder with 70% Ethanol. Remove metallic buffer holder from the freezer and sterilize with 70% Ethanol, connect to vibratome.
23. Take a disposable blade from –20°C and screw into blade holder.
24. Transfer lung slices into a fresh 10cm dish with forceps and use dissection scissors to separate individual lobes.
25. Glue one lung lobe into the middle of the specimen holder with cyanoacarylate glue and place into buffer holder.
26. Fill buffer holder with PBS/1%BSA until lung lobe is immersed in buffer.
27. Lower blade to about 1mm above specimen and set parameters for cutting (thickness: 300µm, cutting speed: 7, cutting oscillation: 5).
28. Use single slice mode and select start position for the blade about 2mm behind the tissue and select the end position about 2mm from back of the tissue.
29. Switch to continuous slicing mode and proceed with slicing of the tissue.
30. Use a plastic loop to gently transfer lung slices into a 24 well plate prepared on ice.
31. To cut additional lobes, remove buffer using 50mL pipette and dry before addition of new specimen.
32. Repeat step 25-31.
33. Incubate lung lobes at 37°C, 5% CO2 overnight before preparation of imaging.
3.3 Labelling of immune cells
Novel development of therapies for metastatic cancer focus on modulating the environment and immune responses at the metastatic site to increase susceptibility of cancer cells to treatment. To assess whether therapies change the microenvironment, dynamic changes between immune cells and cancer cells and the behavior of cells upon treatment have to be examined. In order to evaluate these interactions ex vivo and in real-time, other cells of interest have to be labelled using fluorescent tags (see Note 10). Celltracker dye forms succinimdyl esters with free amine groups of cellular proteins.
1. To label cells using commercially available Celltrackers, centrifuge cells of interest (Note 11) at 300g, 5 min, room temperature, discard supernatant and resuspend in 1 mL PBS.
2. Count cells using hemocytometer or cell counter (CellDrop FL, Denovix).
3. Aliquot number of cells needed, centrifuge at 300g, 5 min, room temperature and resuspend at 1x106 cells per mL in PBS.
4. Add 0.5 µM CelltrackerTM Green CMFDA and incubate for 25min on the rotary shaker at room temperature.
5. Centrifuge cells at 300g, 5 min, room temperature and resuspend in complete phenol red free media at 106 cells per mL.
3.4 Culture of slices and preparation for imaging
1. Preheat complete Phenol-red free DMEM and 2% LMP Agarose at 37°C in the water bath
2. Autoclave custom-made stainless-steel window
3. Transfer one lung slice per well into 24 well glass bottom plate and secure lung tissue with custom-made stainless-steel window frame (allowing media to access the lung tissue while securing the tissue edges for imaging).
4. Put four drops of LMP Agarose around stainless-steel window to avoid movement of window and allow micro-cartography(31).
5. If investigating immune cell and cancer cell interactions, add 100µL of immune cells on top of metastatic lung slices.
6. Incubate for 15 min at 37°C to support migration of cells into the tissue
7. Add 900µL of phenol-red free complete DMEM as well as additional treatments of interest (see Note 12).
8. Plates can be imaged immediately or incubated at 37°C, 5% CO2 until imaging experiment.
4. Image acquisition
1. Turn on heater for imaging stage at 37°C and, for longitudinal live imaging, use an imaging stage with built-in incubator with 37°C and 5% CO2 (see Note 13).
2. Turn on epifluorescent lamp and lasers on the multiphoton or confocal microscope.
3. Change microscope stage insert to one that accepts a multiwell plate and position plate on microscope within incubator.
4. For imaging experiments, use of a 25x1.05 NA water immersion lens is preferable. Add one drop of MilliQ water on top of lens and focus on lung tissue using epifluorescence light, observing the autofluorescence of tissue (green or blue) or the fluorescent cell labels (Note 14).
Microcartography:
To follow the effect of a therapy over multiple days, microcartography can be used to repeated relocalize multiple fields of view over many imaging sessions (Figure 2). This is accomplished by recording and comparing the coordinates of etched grooves in the custom-made stainless-steel window frame at each imaging session, as previously described(31).
5. When focused on tissue, set stage coordinates to x,y,z = 0.
6. Using brightfield imaging, find the etched grooves on the top left window. Record the xy location for each of the three grooves and save their xy coordinates.
7. Return back to the tissue and find positions of interest, recording their xy locations.
8. When relocalization is needed for next imaging session, repeat step 1 to 6 and use mathematic prediction software(31) to predict the new coordinates of the positions of interest.
Labeling of nucleus and apoptosis:
9. For visualization of all cells and to measure apoptosis induction upon therapy, add 1 uM NucSpot Live 650 (nucleus) and 150 nM Apotrackertm Green (Figure 3).
10. Z-Stack for time lapse imaging is set by focusing about 25μm below top (closest to coverslip) of tissue in 3-5μm steps for 50μm (see Note 15).
11. Duration of time lapse imaging depends on kinetics of interactions as well as frequency of events.
5. Image analysis and quantification
Dynamics of immune cells and cancer cells can be studied using the ROI-Tracker plugin and associated motility parameters Excel spreadsheet calculator as published by our group in 2011(32) (Figure 4). The method for use of ROI_Tracker for ImageJ is as followed:
1. Open timelapse of interest and separate individual channels.
2. Threshold image to mark the cells of interest.
3. Open ROI Manager and draw a region of interest around the area the cell of interest is moving in.
4. Use the Analyze Particles feature and add the resultant positions to ROI Manager.
5. Export and save positions to a file.
6. Open ROI_Tracker and add positions saved in ROI Manager.
7. To track movement path, select animate and path.
8. Export parameters calculated by ROI Tracker.
9. Open ROI_Tracker in the analysis Excel sheet and insert calculate parameters.
10. Velocity, Average Speed, and Directionality are calculated automatically by the spreadsheet.
6. Immunofluorescent staining of slices after real-time imaging
To fully understand the therapeutic responses observed through intravital imaging, lung slices can be fixed and further analyzed for markers of the microenvironment or diverse immune cells. Here we describe an example staining protocol to analyze proliferation and leukocyte numbers within the lung slices.
1. Wash lung slices twice in PBS supplemented with 2 mM EDTA for 10min at room temperature.
2. Fix slices for 2h in 1% paraformaldehyde at room temperature in the dark.
3. Wash sections for about one hour in PBS-T, changing PBS-T three times every 20 min at room temperature.
4. Remove excess PBS-T and block slices in blocking buffer for 2h at room temperature.
5. Aspirate blocking buffer and add primary antibodies for CD45 (1:500) and Ki67 (1:100) in blocking buffer
6. Cover slices and incubate overnight in a humidified environment at 4°C.
7. Remove primary antibody and wash three times in PBS-T for 15 min.
8. Dilute secondary antibodies 1:1000 in blocking buffer and incubate for 2h at room temperature in the dark.
9. Wash three times in PBS-T for 15min and incubate with 3ug/mL Hoechst 33342 (in PBS) for 30min at room temperature in the dark.
10. Image immediately on the confocal or two-photon microscope (see Figure 5).