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Side population in human lung cancer cell lines and tumours is enriched with stem-like cancer cells

University of British Columbia

An emerging concept in cancer initiation and development is that cells with characteristics of the stem cell phenotype drive tumour growth and progression. Accumulating evidence reveals that solid tumours such as brain and breast cancer contain primitive cancer stem cells that have high repopulation capacity. Recent studies have demonstrated that adult stem cells can be identified by a side population (SP) phenotype. The work in this thesis was the first in investigating the existence of cancer stem cells in human lung cancer. This study used flow cytometry and the Hoechst 33342 dye efflux assay to isolate and characterise SP cells from various human lung cancer cell lines. In addition, the existence of SP cells in lung cancer tissues obtained from surgical resection was also investigated. Results indicated that all six human lung cancer cell lines contained an SP that could be reliably detected under the experimental conditions used. Evidence was found for asymmetric division by the SP to generate a population resembling the original unsorted population. SP cells and non-SP cells were characterised at the molecular level to compare mRNA expression between the two populations. SP cells from each cell line displayed elevated expression of ATP-Binding Cassette (ABC) transporters associated with multi-drug resistance. In particular, expression of ABCG2 (BRCP1) defines the SP phenotype. Human telomerase reverse transcriptase (hTERT) expression was higher in the SP cells, suggesting this fraction may represent a reservoir with high proliferative potential for generating cancer cells. mRNA levels of MCM7, a member of the mini-chromosome maintenance (MCM) family of proteins, a critical component of the DNA replication complex, was lower in SP cells suggesting that a majority of the cells from the SP fraction were in G₀ of the cell cycle. Levels of mRNA expression of BMI-1, a pathway associated with stem cell self-renewal, were higher in the SP cells compared to the non-SP cells in four of the six cell lines. The Notch-1 pathway was another self-renewal pathway that had increased mRNA expression in five of six cell lines. Functional characterisation of the SP and non-SP were investigated in both in vitro and in vivo. The Matrigel [Trademark] invasion assay demonstrated SP cells as having higher potential for invasiveness than non-SP cells, suggesting there exist a population of stem-like cells within a lung tumour that is involved in the initiation of invasion and metastasis. Non-obese diabetic/severe combined immunodeficiency (NOD/SCID) xenograft transplant experiments showed that SP cells were more tumourigenic compared to non-SP cells. Using a cell viability assay, SP cells were determined to exhibit higher resistance to drugs used to treat lung cancer, some of which are substrates for ABC transporters. Staining patterns of other putative stem cell-related surface markers such as CD24, CD34, CD44 and nestin were also examined and compared between the SP and non-SP. Using the Hoechst efflux assay, SP cells were also isolated from patient tissues, which contained a low but persistent percentage (0.025 - 1.08) of SP cells. Taken together, these studies suggested that SP is an enriched source of tumour re-initiating cells with stem cell properties and may be an important target for effective therapy and a useful tool to investigate the tumourigenic process.

Text

2011-02-25

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http://hdl.handle.net/2429/31784

Pathology

University of British Columbia

Graduate