Isolation and characterization of cancer stem cells in non-small cell lung cancer

Abstract

Tumor heterogeneity has long been observed and recognized as a challenge to

cancer therapy. The cancer stem cell (CSC) model is one of the hypotheses

proposed to explain such a phenomenon. A specific cancer stem cell marker has

not been determined for non-small cell lung cancers (NSCLC), preventing the

definitive evaluation of whether the biology of NSCLC is governed by the CSC

model. This study aimed to analyze the expression of candidate CSC markers and

using the identified putative marker, to isolate CSC and determine the

applicability of the CSC model in NSCLC.

The expression or activities of four putative stem cell markers, CD24, CD44,

CD133 and aldehyde dehydrogenase 1 (ALDH1) were measured by flow

cytometry in eight NSCLC cell lines before and after chemotherapy for 24 hours.

Markers with increased expression after treatment were considered potential CSC

markers and used for isolating tumor cell subpopulations from the untreated cell

lines by fluorescence-activated cell sorting (FACS). Confirmatory analyses using

widely acceptable methodology were performed to test for CSC properties in the marker+ and marker- subpopulations. Isolated subpopulations were further

characterized by functional and phenotypic studies.

Flow cytometry showed amongst the 4 markers, only ALDH1 expression was

significantly enhanced by chemotherapeutic treatment, suggesting ALDH1 could

be a CSC marker. Untreated ALDH1+ cells formed significantly more and larger

cell spheres in non-adherent, serum-free conditions than ALDH1- cells. Likewise,

higher in vitro tumorigenic ability was observed in ALDH1+ subset using colony

formation assay. Furthermore, a higher resistance to cytotoxic drugs was observed

in ALDH1+ compared to ALDH1- cells. In vivo studies also showed ALDH1+ cells

showed higher tumorigenicity than ALDH1- cells; as few as 2,500 ALDH1+ cells

formed tumor in SCID mice which were serially transplantable to 2nd and 3rd

recipients, while no tumor was formed from ALDH- cells with even ten times the

number of cells. Also, expression analysis revealed higher expression of the

pluripotency genes, OCT4, NANOG, BMI1 and SOX9, in ALDH1+ cells. In view

of previous studies reporting CD44 as a CSC marker in lung cancer, double

marker selection of putative CSC was performed to compare ALDH1+CD44+ and

ALDH1-CD44+ subpopulations. Results of the spheroid body formation assay and

cisplatin treatment experiments revealed the ALDH1+CD44+ subpopulation

possessed higher self-renewal ability and chemo-resistance. Cell migration and

invasion assays showed differences between the ALDH1+CD44+ and ALDH1-

CD44+ subpopulations. The significance of these observations require further

investigation.

In conclusion, the result showed that ALDH1 could be a marker for NSCLC stem

cells as evidenced by enhanced self-renewal and differentiation abilities in

ALDH1+ subpopulation. Furthermore, this study observed the presence of at least

two potential stem cell subpopulations in NSCLC cells with differential selfrenewal,

chemotherapy resistance and cell mobility properties. Further

investigations are required to validate these observations and to investigate the

underlying mechanisms. Better understanding of these issues would help to solve

the challenges brought by tumor heterogeneity in lung cancer therapy.