Adenocarcinoma Engraftment: A Method to Implant Cancer Cells into the Murine Lungs

Protocollo

All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Bleomycin Treatment

1. Plan to inject mice intratracheally with bleomycin 14 days prior to tumor cell engraftment.
2. Thaw bleomycin stock on ice 2 h prior to injection.
3. Once thawed, dilute bleomycin to the desired working concentration (0.02 U/50 µL or 0.005 U/50 µL), and keep it on ice.
   NOTE: Concentration of bleomycin depends on the mouse strain used for the experiments (Table 1). Titration of bleomycin in mice should be performed to determine optimal dose.
4. Prepare anesthesia by diluting ketamine and xylazine to a final concentration of 10 mg/mL and 1 mg/mL, respectively, Using a 1 mL syringe and 27 G needle, anesthetize each mouse by injecting intraperitoneally ketamine/xylazine solution at 100/10 mg/kg respectively.
5. Monitor the breathing of mice and employ a toe pinch. Confirm proper anesthetization when mouse does not respond to toe pinch. Apply vet ointment on eyes to prevent dryness while under anesthesia.
6. Place 1 mouse at a time on an intubation platform by hanging its front teeth with its back against the platform.
7. Illuminate the upper chest using a fiber-optic illuminator to help with visualization of the trachea. Open the mouth of the mouse and pull the tongue out gently with sterile flat forceps.
8. Use an intravenous catheter without the needle to avoid blocking breathing. Position the catheter over the white light emitted from the opening of the trachea.
9. Insert the catheter into the trachea until the top of the catheter reaches the front teeth. Confirm proper placement of the catheter in the trachea by visualizing the white light shining through the opening of the catheter in the mouth.
10.  Using a pipette, dispense 50 µL of bleomycin or vehicle (PBS) directly into the catheter to ensure that the entire volume is inhaled. Perform this step under sterile conditions.
11. If the catheter is correctly inserted, the mouse will immediately inhale the contents of the catheter. Wait a few seconds until the entire volume travels down the catheter. Then remove the catheter from the trachea and dispose in 10% bleach solution.
    NOTE: The average time per mouse of steps 1.7-1.11 is around 5 min.
12. If the mouse is not inhaling the liquid, carefully monitor breathing and adjust the catheter position. If the mouse stops breathing, remove the catheter immediately and allow the mouse to resume breathing normally before re-inserting the catheter.
13. Place the injected mouse on an IACUC approved heating pad on their back on a flat surface for recovery. The whole procedure will take 10 min per mouse. Do not return an animal that has undergone injection to the company of other animals until fully recovered.
14. Place a "Hazardous Chemical in Use" Card on the cage for 24 h.

2. Engraftment of Lung Adenocarcinoma Cell Lines

NOTE: Perform engraftment of cells 14 days after the injection of bleomycin (step 1.1)

1. Allow cells to grow in 75 cm² flasks undisturbed for 3 days prior to the day of injection with corresponding media as stated in step 1.3.
   NOTE: They should be 80% confluent at the day of injection (which corresponds to 2-3 x 10⁶ in each flask depending on the cell line).
2. Wash the cells growing on 75 cm² flasks with 5 mL of PBS at room temperature.
3. Aspirate PBS and add 1.5 mL of trypsin to the cells and incubate the cells at 37 °C until the cells detach (typically 2-5 min).
   NOTE: We recommend checking every 2 min for cell detachment from the plastic by simple visualization of the bottom of the flask or under a light microscope. Do not exceed 5 min of incubation with trypsin.
4. Neutralize trypsin by adding 4 mL of media containing 10% FBS (same media as step 2.1). Collect the cells into a 15 mL tube and centrifuge it at 200 x g for 3 min at room temperature.
5. Aspirate the media and resuspend the cell pellet in 5 mL of PBS to wash cells. Centrifuge the cells at 200 x g for 3 min at room temperature to pellet cells. Repeat this 1 time.
6. Aspirate the PBS and resuspend the pellet in 1.5 mL of PBS per flask.
7. Mix 50 µL of cell mixture with 50 µL of trypan blue and count the cells using a Cell Counter/Hemocytometer chamber.
8. Prepare cell suspension by diluting the cells in PBS to inject 1 x 10⁵ cells (or other amount indicated) in 50 µL per mouse. Keep the cells on ice prior to injection.
   NOTE: Prepare at least two times more cells for injection to avoid running out of cell suspension.
9. Prepare anesthesia by diluting ketamine and xylazine to a final concentration of 10 mg/mL and 1 mg/mL, respectively, Using a 1 mL syringe and 27 G needle, anesthetize each mouse by injecting intraperitoneally ketamine/xylazine solution at 100/10 mg/kg respectively.
10. Monitor the breathing of mice and employ a toe pinch. Confirm proper anesthetization when mouse does not respond to toe pinch. Apply vet ointment on eyes to prevent dryness while under anesthesia.
11.  Place 1 mouse at a time on an intubation platform by hanging its front teeth with its back against the platform.
12. Illuminate the upper chest using a fiber-optic illuminator to help with visualization of the trachea.
13.  Resuspend the cells gently using a p200 pipette to make sure they do not form clumps. Open the mouth of the mouse and pull the tongue out gently with sterile flat forceps.
14. Use an intravenous catheter with the needle removed to avoid blocking breathing. Position the catheter over the white light emitted from the opening of the trachea.
15.  Insert the catheter into the trachea until the top of the catheter reaches the front teeth. Confirm proper placement of the catheter in the trachea by visualizing the white light shining through the opening of the catheter in the mouth.
16. Using a pipette, dispense 50 µL of cells directly into the catheter to ensure that the entire volume is inhaled. Perform this step under sterile conditions.
    NOTE: If the catheter is correctly inserted, the mouse will immediately inhale the contents of the catheter.
17. Wait a few seconds until the entire volume travels down the catheter, and then remove the catheter from the trachea and dispose in 10% bleach solution.
18.  If the mouse is not inhaling the liquid, carefully monitor breathing and adjust the catheter position. If the mouse stops breathing, remove the catheter immediately and allow the mouse to resume breathing normally before re-inserting the catheter.
    NOTE: The average time per mouse of steps 2.11-2.18 is around 5 min.
19. Place the injected mouse on an IACUC approved heating pad on their back on a flat surface for recovery.
    NOTE: The whole procedure will take 10 min per mouse. Do not return an animal that has undergone injection to the company of other animals until fully recovered.
20. Shave the entire rib area both ventrally and dorsally of B6129SF1/J and other mouse strains with dark hair using an electric shaver prior to imaging.
21.  2-3 min after cell injection, inject 100 µL of luciferin retro-orbitally (15 mg/mL) using an insulin needle. Alternate the eye used for injection every imaging session.
22.  After at least 2 min, place up to 5 mice in an animal bioluminescence imager in the dorsal recumbency position and acquire a ventral picture using luminescence settings .
23. Under the control panel adjust the resolution and sensitivity settings to measure luminescence of a given cell line. For most applications, start with 3 min (or "Auto" if saturated), binning=Medium (4), F/Stop=1, Field of View=D, Subject Height=1.50.
    NOTE: If the injection is successful, luminescence signal is detected in the upper chest area, in one lung or both lungs.
24. Flip mice onto sternal recumbency position and acquire a dorsal picture as above.
    NOTE: If cell injection is not performed properly, cells may cluster at the entrance of the trachea or in the neck.
25. Inject ketoprofen 5 mg/kg intraperitoneally to alleviate pain.
26. Move mice back to their cage and place a "BSL-2 Agent in Use" Card on the cage.
27. Monitor mice for respiratory distress immediately after intubation, and then every 15 min until mice wake up from anesthesia. Do not leave mice unattended until they have regained sufficient consciousness to maintain sternal recumbency.
28. Examine the mice 24 h post intubation and three times a week for any signs of morbidity.
29. Keep record in the logbook of the date of procedure, animal identification, type of procedure, type of anesthetic and post-operative monitoring observations and times.
30. Monitor mice for weight loss, respiratory distress, behavioral abnormalities, and a body condition score <2 (segmentation of vertebral column evident/dorsal pelvic bones are palpable) bi-weekly following inoculation of bleomycin.
    NOTE: Mice with lung tumors may exhibit respiratory irritation, weight loss, cachexia, and/or death.
31. Euthanize mice under respiratory distress or mice that have experienced 15% loss of body mass by carbon dioxide or by ketamine/xylazine followed by cervical dislocation if collecting tissues. Confirm euthanasia by conducting palpation for respiratory activity.

3. Monitoring of Tumor Growth by Bioluminescence Imaging

1. Repeat imaging of the mice at day 3 post-engraftment and weekly thereafter.
2. Anesthetize mice by injecting ketamine/xylazine (as described in steps 2.4-2.5) or by inhaled isoflurane (2%). Monitor breathing of mice and employ a toe pinch to confirm proper anesthetization. Apply vet ointment on eyes to prevent dryness while under anesthesia.
3. Shave the entire rib area both ventrally and dorsally of B6129SF1/J and other mouse strains with dark hair using an electric shaver prior to imaging.
4. Once the mice are under anesthesia, inject 100 µL of luciferin retro-orbitally (15 mg/mL) using an insulin needle. Wait 2 min. Alternate the eye used for injection every imaging session.
5. Place mice in the imager and acquire dorsal and ventral images as described in steps 2.22-2.24.
6. Place mice back into the cage after imaging, on their back on a flat surface for recovery.
   NOTE: The whole procedure will take 5 min per group of 5 mice. Do not leave mice unattended until they have regained sufficient consciousness to maintain sternal recumbency.
7. Analyze the images using the imager/bioluminescence analysis software.

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Risultati

Table 1: List of recommended doses of bleomycin for mouse strains tested.

Mouse Strain	Cell Line	Number of cells injected	% engraftment		p value
			Vehicle

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Materiali

Name	Company	Catalog Number	Comments
Rodent Intubation stand	Braintree Scientific	RIS-100	Recommended stand for intratracheal injection
Bleomycin	Sigma	B5507-15UN	CAUTION Health hazard GHS08
Exel Catheter 24G	Fisher	1484121	Remove needle. For intratracheal injection
Ketamine (Ketaset inl 100 mg/mL C3N 10 mL)	Butler Schein	56344	To anesthetize mice
Xylazine	Butler Schein	33198	To anesthetize mice
Syringe 1ml	BD / Fisher	14-823-434	For intraperitoneal injections
Puralube Veterinary Ophthalmic Ointment	BUTLER ANIMAL HEALTH COMPANY LLC	8897	To prevent eye dryness while under anesthesia
MI-150 ILLUMINATOR 150W MI-150	DOLAN-JENNER INDUSTRIES	MI-150 / EEG2823M	To illuminate and visualize trachea
Graefe Forceps, 2.75 (7 cm) long serrat	Roboz	RS-5111	For intratracheal injection
Flask T/C 75cm sq canted neck, blue cap	Fisher / Corning	353135
Phosphate Buffered Saline	Life Technologies	14190-144
0.25% Trypsin-EDTA	Life Technologies	25200-056
Pipet-Lite Pipette, Unv. SL-200XLS+	METTLER-TOLEDO INTERNATIONAL	17014411
Countess Automated Cell Counter	Life Technologies	AMQAX1000
D-Luciferin powder	Perkin Elmer Health Sciences Inc	122799	For luminescent imaging. Reconstitute powder with PBS for a working concentration of 15mg/ mL. Protect from Light
IVIS Spectrum Xenogen Bioluminiscence	Perkin Elmer Health Sciences Inc	124262	For in vivo bioluminescence imaging
B6129SF1/J mice	Jackson Laboratories	101043
NIH-H2030 cells	ATCC	CRL-5914
PC9 cells	Nguyen DX et al. Cell. 2009;138:51–62
Living image software	Perkin Elmer Health Sciences Inc	128113	For in vivo bioluminescence analysis