The role of CD133 in the identification and characterisation of tumour-initiating cells in non-small-cell lung cancer.
OBJECTIVE
Emerging evidence suggests that specific sub-populations of cancer cells with stem cell characteristics within the bulk of tumours are implicated in the pathogenesis of heterogeneous malignant tumours. The cells that drive tumour growth have been denoted cancer-initiating cells or cancer stem cells (hereafter CSCs). CSCs have been isolated initially from leukaemias and subsequently from several solid tumours including brain, breast, prostate, colon and lung cancer. This study aimed at isolating and characterising the population of tumour-initiating cells in non-small-cell lung cancer (NSCLC).


METHODS
Specimens of NSCLC obtained from 89 patients undergoing tumour resection at the Cancer National Institute of Naples were analysed. Three methods to isolate the tumour-initiating cells were used: (1) flow cytometry analysis for identification of positive cells for surface markers such as CD24, CD29, CD31, CD34, CD44, CD133 and CD326; (2) Hoechst 33342 dye exclusion test for the identification of a side-population characteristic for the presence of stem cells; (3) non-adherent culture condition able to form spheres with stem cell-like characteristics. Definition of the tumourigenic potential of the cells through soft agar assay and injection into NOD/SCID mice were used to functionally define (in vitro and in vivo) putative CSCs isolated from NSCLC samples.


RESULTS
Upon flow cytometry analysis of NSCLC samples, CD133-positive cells were found in 72% of 89 fresh specimens analysed and, on average, represented 6% of the total cells. Moreover, the number of CD133-positive cells increased markedly when the cells, isolated from NSCLC specimens, were grown as spheres in non-adherent culture conditions. Cells from NSCLC, grown as spheres, when assayed in soft agar, give rise to a 3.8-fold larger number of colonies in culture and are more tumourigenic in non-obese diabetic (NOD)/severe combined immunodeficiency (SCID) mice compared with the corresponding adherent cells.


CONCLUSIONS
We have isolated and characterised a population of CD133-positive cells from NSCLC that is able to give rise to spheres and can act as tumour-initiating cells.
Tirino
Virginia
V
Department of Experimental Oncology, National Cancer Institute, Naples, Italy.
Camerlingo
Rosa
R
Franco
Renato
R
Malanga
Donatella
D
La Rocca
Antonello
A
Viglietto
Giuseppe
G
Rocco
Gaetano
G
Pirozzi
Giuseppe
G
D000071916:AC133 Antigen; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000032:analysis*; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism / Q000473:pathology*; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000378:metabolism; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism / Q000473:pathology*; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000378:metabolism; D009368:Neoplasm Transplantation; D014411:Neoplastic Stem Cells / Q000378:metabolism / Q000473:pathology*; D010455:Peptides / Q000378:metabolism*; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
D000071916:AC133 Antigen; D015703:Antigens, CD; D014408:Biomarkers, Tumor; D006023:Glycoproteins; D009363:Neoplasm Proteins; C000606023:PROM1 protein, human; D010455:Peptides; C000606024:Prom1 protein, mouse