Lung cancer contains a rare population of CD133⁺ cancer stem-like cells able to self-renew and generates an unlimited progeny of non-tumorigenic cells. The current technology allows the establishment of long-term lung cancer stem cell cultures from about one-third of the tumors.

 

1. Tumor samples were obtained.

 

2. Surgical specimens were washed several times and left overnight in culture system.
DMEM–F12 medium supplemented high doses of penicillin/streptomycin amphotericin B

 

3. Tissue dissociation was carried out by enzymatic digestion (20 μg/ml collagenase II) for 2 h at 37°C.

 

4. Recovered cells were cultured at clonal density in serum-free medium.
   50 μg/ml insulin
   100 μg/ml apo-transferrin
   10 μg/ml putrescine
   0.03 mM sodium selenite
   2 μM progesterone
   0.6% glucose
   5 mM HEPES
   0.1% sodium bicarbonate
   0.4% BSA
   glutamine and antibiotics
   20 μg/ml EGF
   10 μg/ml bFGF.

 

5. Flasks non-treated for tissue culture were used to reduce cell adherence and support growth as undifferentiated tumor spheres.

 

6. The medium was replaced or supplemented with fresh growth factors twice a week until cells started to grow forming floating aggregates.

 

7. Cultures were expanded by mechanical dissociation of spheres, followed by re-plating of both single cells and residual small aggregates in complete fresh medium.

 

8. Stem cell medium was replaced with Bronchial Epithelial Cell Growth Medium in tissue culture-treated flasks.

 

9. Identification: Antibodies used were PE-conjugated anti-CD133/1, PE-conjugated anti-CD133/2 or APC-conjugated anti-CD133/1, anti-CD56/N-CAM, FITC-conjugated anti-epithelial membrane antigen, anti-human CKs, anti-CEA, FITC-conjugated anti-CD34 and anti-CD45, anti-CD31.

 

10. Single-cell suspensions from lung cancer specimens were prepared.

 

11. After thawing, cells were double stained with PE-conjugated anti-CD133/1 antibody and FITC-conjugated anti-Ep-CAM antibody.

 

12. Purity of the CD133⁺ and CD133⁻ cell populations was evaluated.