Source: Stevens, L.E. et al, Pre-Conditioning the Airways of Mice with Bleomycin Increases the Efficiency of Orthotopic Lung Cancer Cell Engraftment. J. Vis. Exp. (2018).
This video describes a method to engraft lung adenocarcinoma cells in murine lungs by preconditioning the airways with fibrosis-inducing agent–bleomycin. This method significantly enhances orthotopic engraftment of lung cancer cells into murine lungs
All procedures involving animals have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Bleomycin Treatment
2. Engraftment of Lung Adenocarcinoma Cell Lines
NOTE: Perform engraftment of cells 14 days after the injection of bleomycin (step 1.1)
3. Monitoring of Tumor Growth by Bioluminescence Imaging
Table 1: List of recommended doses of bleomycin for mouse strains tested.
Mouse Strain | Cell Line | Number of cells injected | % engraftment | p value | |
Vehicle | Bleomycin | ||||
Athymic | H2030 | 5×105 | 0% (0/7) | 100% (6/6) | 0.0006 |
Athymic | H2030 | 5×104 | n.d. | 85.7% (6/7) | n.a. |
Athymic | H2030 | 5×103 | n.d. | 85.7% (6/7) | n.a. |
Athymic | PC-9 | 1×105 | 0% (0/7) | 85.7% (6/7) | 0.0023 |
NSG | H2030-BrM3 | 1×105 | 16.7% (1/6) | 100% (3/3)* | 0.0476 |
B6129SF1/J | 368T1 | 1×105 | 0% (0/7) | 71.4% (5/7)* | 0.0105 |
The authors have nothing to disclose.
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 |