We observed novel features in a sub-population of SCLC SP fraction cells that have cancer stem cell-like characteristics. Previous studies using SP fractions from many tumours showed relative enrichment in tumour-initiating cells, but several hundred or thousand cells were required for tumour reconstitution (Al Hajj et al, 2003
; Collins et al, 2005
; Ponti et al, 2005
). In studies of non-small-cell lung cancer, 1–5 × 103
SP cells (Ho et al, 2007
) or 1 × 104
cells that express CD133 markers (Eramo et al, 2008
) were needed for tumour reconstitution. In contrast, we found that as few as 50–100
SP cells from SCLC lines were sufficient to reconstitute tumours in NOD/SCID mice, whereas at least 500 non-SP fraction cells were required to form tumours under the same conditions, although few of those tumours reached 300–400
after an additional 30 days of observation. Implantation of 50–100 non-SP cells resulted in either no tumour or formation of barely palpable tumours that did not progress during the additional 30 days of observation. A recent report showed that as little as 10 cells co-implanted with one million unselected immune cells reconstitute haematopoietic tumours in syngeneic recipients (Kelly et al, 2007
). The differences with reports from solid tumours were attributed to use of a syngeneic model. However, studies comparing tumour growth rates of cells from transgenic Wnt1 mouse mammary tumours implanted into syngeneic, nude or SCID mice did not show appreciative differences (Varticovski et al, 2007
; Svirshchevskaya et al, 2008
). Thus, the high capacity of tumour reconstitution using haematopoietic tumour cells may be an intrinsic feature of haematopoietic malignancies.
In addition to a faster growth rate, the tumours that arose from SCLC SP cells showed a significant degree of neo-angiogenesis. These tumours, the corresponding SP fraction cells and tumours that arose from these cells showed upregulation of VEGF, a feature not previously reported for these cells. Side population-sorted cells also reconstituted the entire cell population within 2 week in culture, a feature consistent with efficient self-renewal, which is an essential characteristic of cancer stem cell-like cells. In addition, the SP cells overexpressed NANOG and SOX2, genes associated with self-renewal process.
As non-SP fraction cells also formed tumours, although at a significantly reduced rate and with delayed tumour growth rates, it is possible that sorting SP cells, as defined by Verapamil gating, do not permit absolute selection, and a few cells in non-SP fraction have the capacity of dye efflux. This was confirmed in the self-renewal experiments. Cells sorted from the non-SP fraction after 2 weeks in culture acquired 0.6–0.8% of cells that were able to efflux the dye. Thus, it is not possible to achieve complete separation of dye by excluding cells in a single sorting passage by flow cytometry, and the non-SP fraction is likely to contain a small percentage of cells that can also efflux the dye. Thus, the SP fraction analysis needs to be compared with other methods, including staining for ALDH. Alternatively, efflux of Hoechst dye is only one of the many functional characteristics of tumor-initiating cells. Previous reports indicate that haematopoietic stem cells are also present in the non-SP compartment (Morita et al, 2006
), and mammary repopulating cells have also been found in the non-SP fraction. Thus, these compartments are not strictly defined in normal organs, and some overlap is expected.
These considerations prompted us to undertake an extensive search for additional cell surface and genetic markers to define further the cancer stem-like cell population in the SP fraction. In spite of the extensive search, we could not identify any common surface markers that would be enriched in the SP fraction of all SCLC cell lines we examined. However, we observed a consistent decrease in SP cells in two markers, CD56 and CD90. These cell markers represent features associated with neuronal differentiation of SCLC and other organs, and their low expression is consistent with the primitive nature of SP cells. CD56 (N-CAM, neural cell adhesion molecule 1 isoform) is a hallmark of SCLC. CD90 (Thy-1) is a neuronal and mesenchymal stem cell marker that also defines the neuronal differentiation of these neuroendocrine tumours. In contrast to our data using SCLC, cell surface expression of Thy-1 was reported to be enriched in CD133+ tumour stem cells from brain tumours (Liu et al, 2006
; Mizrak et al, 2008
) characterised by self-renewal, high proliferative capacity in vitro
and tumour reconstitution in vivo
(Al Hajj et al, 2003
; Ponti et al, 2005
; Sales et al, 2007
; Eramo et al, 2008
). However, we found no change in frequency or enrichment in any of the previously identified cell surface markers, including CD133, in the SP fraction cells in SCLC cells. This may reflect high expression of CD133 in H146 and H82 cells (20% and 90%, respectively). Thus, stem-like cells from SCLC have features similar to the haematopoietic malignancies, with enrichment in SP fraction, rather than expression of cell surface markers described for other solid tumours.
ABCG2 transporter expression is associated with drug resistance and Hoechst dye efflux (Farnie and Clarke, 2007
; Ho et al, 2007
). We did not detect an increase in ABCG2 transporter protein on cell surface. However, gene expression analysis showed upregulation of mRNA for the ABCG2 transporter. These changes were subsequently confirmed by independent RT–PCR. The previously reported rapid membrane turnover of ABC transporters may be responsible for this phenomenon (Robey et al, 2007
Of significance, SP fraction cells showed upregulation of genes that werepreviously associated with normal stem cell biology and pluripotency. Similar to our observations, transcriptional profiling of SP cells from several non-malignant tissues showed that more genes were upregulated than downregulated and included genes associated with multi-drug resistance, regulation of transcription, cell signalling, Notch and Wnt pathways (Liadaki et al, 2005
; Behbod et al, 2006
; Larderet et al, 2006
). Side population fraction cells examined in our studies have upregulated genes that are involved in pathways modulating stemness, including MYC, FGF1, OCT4, KLF4, NOTCH2
. These data confirm that SCLC cell lines contain a population of highly undifferentiated cells with stem cell-like characteristics.
Development of agents that target the Hedgehog pathway, which is re-activated in bronchial mucosa following epithelial injury and in progenitor cells (Watkins et al, 2003
; Vestergaard et al, 2006
), renewed the interest in using these agents in treatment of SCLC. Recent studies showed that stem-like cells in brain tumours are selectively vulnerable to agents inhibiting the Notch pathway (Fan et al, 2006
). The feasibility of similar approaches in SCLC remains to be established.
In summary, our findings identified a small population of SCLC cells that resides in the SP fraction and has functional and molecular features consistent with cancer stem cells. Further characterisation of their genetic signature, epigenetics and metabolic features could have direct therapeutic implications.