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. / published_or_final_version / Pathology / Master / Master of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/180008 |
| Date | January 2011 |
| Creators | Wong, Kit-man, Sunny., 王傑民. |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Source | http://hub.hku.hk/bib/B47250665 |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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