Effects of Low Confluency, Serum Starvation and Hypoxia on the Side Population of Cancer Cell

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1 Cell Cycle ISSN: (Print) (Online) Journal homepage: Effects of Low Confluency, Serum Starvation and Hypoxia on the Side Population of Cancer Cell Lines Raluca T. Tavaluc, Lori S. Hart, David T. Dicker & Wafik S. El-Deiry To cite this article: Raluca T. Tavaluc, Lori S. Hart, David T. Dicker & Wafik S. El-Deiry (2007) Effects of Low Confluency, Serum Starvation and Hypoxia on the Side Population of Cancer Cell Lines, Cell Cycle, 6:20, , DOI: /cc To link to this article: Published online: 19 Oct Submit your article to this journal Article views: 1681 Citing articles: 38 View citing articles Full Terms & Conditions of access and use can be found at

2 [Cell Cycle 6:20, , 15 October 2007]; 2007 Landes Bioscience Report Effects of Low Confluency, Serum Starvation and Hypoxia on the Side Population of Cancer Cell Lines Raluca T. Tavaluc Lori S. Hart David T. Dicker Wafik S. El-Deiry* Laboratory of Molecular Oncology and Cell Cycle Regulation; Departments of Medicine (Hematology/Oncology), Genetics, and Pharmacology; The Institute for Translational Medicine and Therapeutics and the Abramson Comprehensive Cancer Center; University of Pennsylvania School of Medicine; Philadelphia, Pennsylvania USA *Correspondence to: Wafik S. El-Deiry, M.D. Ph.D.; University of Pennsylvania School of Medicine; 415 Curie Blvd., CRB 437A; Philadelphia, Pennsylvania USA; Tel.: ; Fax: ; upenn.edu Original manuscript submitted: 08/15/07 Manuscript accepted: 08/21/07 Previously published online as a Cell Cycle E-publication: Key words side population, cancer stem cells, starvation, hypoxia, confluency, tumor microenvironment, Hoechst 33342, cancer progression, resistance to therapy Abbreviations CSC cancer stem cell SP side population FBS fetal bovine serum FTC Fumitremorgin C Acknowledgements This work was supported by NIH grants CA75138, CA098101, CA105008, CA and the Littlefield-AACR award for research on metastatic colon cancer. LSH was supported by the Department of Pathology and Laboratory Medicine Training Grant T32CA RTT is an undergraduate student at Georgetown University who was supported by a Summer Undergraduate Research Program at the University of Pennsylvania. Abstract The cancer stem cell theory describes a small subset of cancer cells that have the ability to initiate and drive the growth of a tumor. The niche refers to the environmental factors and the surrounding cells within which the tumor develops. The exact relationship between cancer stem cells and the tumor niche is not known. However, using side population analysis by flow cytometry, it is possible to analyze the relationship between environmental stresses and putative cancer stem cells. The side population is a subpopulation of cells that efflux Hoechst and has been previously shown to be enriched for cancer stem cells. Using this technique, we characterized the response of side population cells to low confluency, serum starvation and hypoxia using three different human cancer cell lines. We found that these stresses, characteristic of the tumor niche enrich the side population of DLD1, SW480 and MCF7 cancer cell lines, thus possibly predisposing the tumor to a more malignant phenotype. Introduction Two current models explain the cellular origins of tumorigenesis. One is the stochastic model that describes cancer as a disease of the genome that occurs through stepwise accumulation of mutations. 1,2 According to this model, a cell must acquire at least six alterations in order to become malignant and drive tumorigenesis. These mutations include acquiring independence from growth, anti-growth and apoptotic signals, limited proliferation potential, ability to drive angiogenesis and the ability to metastasize. 3 The second hypothesis is the cancer stem cell theory in which only a small subpopulation of cells in a tumor has the ability to initiate and drive the growth and spread of a tumor, and also sustain the heterogeneity of the original tumor. 4,5 Cancer stem cells (CSCs) were first identified in acute and chronic myeloid leukaemias. 6,7 More recent studies have found experimental evidence for cancer stem cells in solid tumors of the breast, 8 brain 9,10 and colon. 11,12 There is much controversy regarding the origin of CSCs. Possibilities range from normal stem cells 7,13 that have become aberrant or progenitor cells that have acquired the ability to self-renew. 14,15 Despite their origin, CSCs have many properties in common with somatic stem cells. Normal stem cells are defined by the ability to differentiate, selfrenew and balance the two paths according to environmental and genetic stimuli. 16,17 CSCs are also able to self-renew and differentiate, 18 but it is not to known to what extent, if at all, CSCs respond to niche stimuli. 19 CSCs, like somatic stem cells, have increased expression of specific members ABC family of transporters such as ABCB1, ABCC1 and ABCG220 which use the energy of ATP hydrolysis to efflux drugs and fluorescent dyes from the cells. 21,22 This property is believed to contribute to chemotherapeutic resistance as many chemotherapeutic drugs are not designed to target specifically the CSCs and are effluxed out of the cells before killing them. 23 This dye-effluxing capability is also used to analyze and separate CSCs from the main cell populations by using Side Population (SP) analysis. The SP is a subpopulation of cells known to efflux the Hoechst blue fluorescent dye and to be enriched for CSCs. 24 The SP gives a characteristic profile of low Hoechst concentrations that disappears when cells are treated with verapamil, a first-generation ABC transporter inhibitor, 20 or fumitremorgin C (FTC), a specific inhibitor of ABCG2. 25 There is an extensive literature regarding the cancer stem cell theory. However, there is no clear model of the interaction between CSCs and their niche. It is known that tumors 2554 Cell Cycle 2007; Vol. 6 Issue 20

3 have regions with poor blood supply, severe hypoxia, low ph and nutrient starvation. 26,27 Also it is known that hypoxic tumors are the most resistant to anticancer drugs and hence have poor prognosis. 28,29 We hypothesized that the niche may have a major influence on the regulation of tumor progression. Performing side population analysis with flow cytometry, we analyzed the effects of low confluency, serum starvation and hypoxia, which are all important characteristics of the niche, on the side population of human cancer cell lines to ultimately begin to learn about the interplay between the niche and cancer stem cells. Our results suggest that CSCs are affected by and respond to changes within their microenvironment. MATERIALS AND METHODS Cell culture. Tumor cell lines SW480, DLD1 and MCF7 were obtained from the American Type Culture Collection (Manassas, VA). All cells were grown in DMEM (Gibco) with 10% FBS and 1% Penicillin/Streptomycin at 37 C in 5% CO 2. Flow cytometry. Flow cytometry was performed using an Elite ESP flow cytometer (Beckman-Coulter, Miami, FL). To analyze the SP population, a solid state 355 nm UV laser (Lightwave Electronics, Mt. View, CA) was used to excite the Hoechst and the dual emission was capture with a 450/20BP (Hoechst Blue) and a 675/40BP (Hoechst Red) separated with a 550LP dichroic. Forward scatter and PI (675/40BP) were captured from a 488 nm argon laser 40υsec upstream of the UV laser. Cells were harvested using trypsin (Gibco). Cells (1 x 10 6 /ml) were incubated in HBSS (Gibco) containing Hoechst (Invitrogen,) for hours at 37 C, vortexing gently every 30 min. A negative control sample was treated with verapamil (Sigma) or Fumitremorgin C (Sigma) for 15 min at room temperature prior to the addition of Hoechst Dead cells were gated out based on positive staining with propidium iodide (2 µg/ml). Concentrations used for specific cell line: SW480 (5 µg/ml Hoechst 33342, 50 µm verapamil), DLD1 (6 µg/ml Hoechst 33342, 50 µm verapamil) and MCF7 (9 µg/ml Hoechst 33342, 10 µm FTC). Confluency. For each cell line, 1, 2, 4 and 6 million cells were plated on day 1. At 48 hours later, photomicrographs were taken using the Axiovert 100 microscope, (Carl Zeiss MicroImaging, Inc, Thornwood, NY) and the cells were harvested for flow cytometry. Serum starvation. For each cell line, 5x100 cm2 flasks were plated with approximately 2 million cells on the evening of day 1 in DMEM media. On the morning of day 2, the media was changed with the appropriate Fetal Bovine Serum (FBS) concentration. The control flask had 10% FBS. The starvation levels had 5%, 2%, 1% and 0% FBS. The cells were harvested for flow cytometry after 48 hours of incubation at different serum levels. Hypoxia. 3 flasks (control, cisplatin and hypoxia) with approximately 2 million cells were plated on the evening of day 1. In the morning of day 2, plates were moved into the hypoxia chamber (Invivo2-200, Ruskinn, Leeds, UK) at the specific hypoxia level. On day 2, cells under normoxia or hypoxia were treated with cisplatin (6 µl/ml). On day 3, the cells were harvested for flow cytometry. Figure 1. Harvesting density is inversely proportional to SP percentage in DLD1, SW480 and MCF7. The DLD1 cell line had the least significant response to varying harvesting densities. Higher harvesting densities had a more significant impact on the SP percent of SW480 and MCF7. The harvesting density range of million cells/cm 2 resulted in the highest side population percentage in all three cell lines. RESULTS Confluency (harvesting density) correlates with the percent of side population. In three different cell lines (DLD1, SW480 and MCF7), we found that cells plated on day 1 at a density of 1 million cells per plate, which correlated 48 hours later with a harvesting density of about 0.05 million cells/cm2, had the highest SP percent. Also, as the harvesting density increased, the SP percent had a downward trend (Fig. 1). In particular for the DLD1 cell line, the flasks plated with 1 and 2 million cells resulted in similar harvesting densities of and million cells/cm 2, respectively, and consequently had SP percentages close in value. The flasks plated with 4 and 6 million cells, after 48 hours, had much higher resulting densities and SP percentages about 20 30% lower. In the SW480 colon cancer cell line, the effect of confluency was analyzed at two different time points, 24 and 48 hours. In both trials, the highest SP percent was found at the same harvesting density of million cells/cm2, which represented the flask plated with 2 million cells at 24 h (Fig. 2A) and 1 million cells at 48 h (Fig. 2B). Higher harvesting densities resulted in a decrease of SP percent of up to 80 90%. Similarly, for the MCF7 breast cancer cell line, the side population percentage was highest for the flask plated with 1 million cells or with the harvesting density of million cells/cm 2. As the harvesting density increased, the percent of SP clearly decreased in an inversely proportional relationship. The inverse relationship between percent of confluency (harvesting density) and side population percentage was demonstrated in the three different cell lines. The harvesting density in the range of 0.04 to 0.06 million cells/cm 2 appears to be an optimal harvesting density at which the SP is enriched. Serum starvation enriches the side population in three different cell lines. After starving three different cell lines at various levels for 48 hours, we discovered that serum starvation enriches the side population in DLD1, SW480 and MCF7 cancer cell lines (Fig. 3). The DLD1 cell line appeared to be most independent of nutrients and growth factors found in the media because upon starvation, the Cell Cycle 2555

4 Figure 2. SW480 Hoechst profiles at 24 or 48 hours after plating. (A) At 24 hours after plating, SW480 cells had the highest side population at the 2 million cells plate. The SP of 1.86% was by far the highest as compared to the other side populations. (B) At 48 hours after plating, SW480 cells had the highest side population at 1 million cells plate. This SP was 1.55%. SP increased, making the population more robust (Figs. 3 and 4). Starving the cells to half the usual amount of nutrients and growth factors (5% serum) did not affect the cells as much, producing only a 10% SP increase. However, 2% starvation appeared to induce a high stress on the cells thereby prompting a three fold increase in SP. At 1% starvation there was enrichment of the SP by 40% as compared to the 10% FBS control. Complete starvation was toxic for the DLD1 cell line thereby decreasing the SP to 60%. Similarly, the SW480 colon cancer cells were responsive to serum starvation by increasing the SP at 1%, 2% and 5% serum. However, 2556 Cell Cycle 2007; Vol. 6 Issue 18

5 Figure 3. A total of 48 hours serum starvation enriched the side population of DLD1, SW480 and MCF7. For all three cell lines, 0% serum decreased the side population. For the DLD1 and SW480 cell lines, 1%, 2% and 5% serum enriched the SP as compared to the control of 10% FBS. The SP of the MCF7 cell line was only enriched at 5% starvation. the enrichment was not as high or as varied as in DLD1. The highest enrichment was observed at 5% starvation to twice the control value. The 1% and 2% serum treatments produced 75% enrichment, while complete starvation decreased the SP proving again to be toxic to SW480 cells. The MCF7 breast cancer cells appeared to respond differently to serum starvation. Only at 5% serum was the side population enriched by 20%. The harsher levels of starvation of 2%, 1% and 0% serum were toxic and the SP decreased up to 2.5 times in value. The trend evident in the three cell lines showed that the SP can be modulated by serum starvation to either increase or decrease in percent. Complete nutrient and growth factor removal proved to be toxic for all three human cancer cell lines and resulted in decreased percent of SP cells. Severe hypoxia induces the enrichment of SP in DLD1 and SW480 colon cancer cell lines. The side population of cells, treated for 24 hours with cisplatin or various hypoxic exposures, was compared to the side population of cells grown under normoxic conditions. Cisplatin, a chemotherapeutic drug, shown to enrich the side population (Sussman et al., Cancer Biology and Therapy, in press, 2007) was used as a positive control for enrichment. Analysis of the effects of hypoxic stress was performed in two colon cancer cell lines DLD1 and SW480. After multiple experiments and various low hypoxia levels, the side population of the hypoxia treated plates was enriched as compared to the control, suggesting that hypoxic stress stimulates an increase in SP and consequently the increase in cancer stem cell numbers (Table 1). The response of the DLD1 cancer cell line at different hypoxia levels is shown in Figure 5A. Treating the cells with three different Figure 4. DLD1 serum starvation. The SP profiles show that 1%, 2% and 5% FBS enriched the SP compared to the 10% FBS control. The 0% starvation had the lowest SP percent. Cell Cycle 2557

6 Table 1 Hypoxia enriches the SP compared with normoxia Figure 5. Hypoxia enriched the side population in DLD1 and SW480 cells. (A) Hypoxia treatment of the DLD1 cell line enriched the side population. 0.2%, 1.0% and 2.0% hypoxia enriched the SP as compared to normoxic conditions, while 0.5% oxygen maintained the SP at the same percent as in the normoxic plate. (B) Hypoxia treatment of SW480 cell line enriched the side population at 0.2%, 0.5% and 1.0% oxygen. The % oxygen levels refer only to the hypoxia and cisplatin experiments. The cisplatin dose was as described in Sussman et al., Cancer Biology and Therapy 2007; In press. levels of hypoxia 0.2% (Fig. 6A), 1.0% (Fig. 6C) and 2.0% (Fig. 6D) increased the side population by 1.5- to 2.0-fold. The hypoxia level of 0.5% maintained the side population of the cells at a level equal to normoxia. The cisplatin positive control enriched the side population of the 0.2% and 0.5% experiments, while for the 1.0% and 2.0% experiments it decreased the side population. The side population percentages of treated cells were validated through use of a verapamil-treated negative control. The DLD1 cell line therefore proved to be sensitive to hypoxic treatments. Similarly, the SW480 colon cancer cell line was sensitive to hypoxic levels (Fig. 5B). The experiment analyzing the effect of 0.2% hypoxia was repeated four times resulting in the same trend the hypoxia plate had a side population percentage higher than the normoxia plate. The exact fold changes were calculated and are shown in Table 1. The hypoxia level of 0.5% was tested two times and in both experiments the hypoxic plate was enriched in SP cells as compared to the control plate at 21% oxygen (Fig. 6B). 1.0% oxygen was analyzed in one experiment with the same result of enrichment in SP. We could therefore conclude that hypoxic conditions enrich the SP in different cancer cell lines. DISCUSSION There is a delicate balance between self-renewal and differentiation of cancer stem cells. It appears that under stressful conditions the CSCs self-renew more than differentiate into progenitor cells, as observed by the increase side population percentages after induction of stress by low confluency, serum starvation and hypoxia. Cell contact is part of the regulatory mechanism that drives the proliferation of SP cells. In the DLD1 cell line, the different harvesting densities did not appear to have a high impact on the SP. The proliferation pattern of the DLD1 cells could provide an explanation for this decreased effect. There is a large difference in the Hypoxia Trial # Cisplatin Hypoxia Level Fold Change Fold Change SW % % % DLD1 0.20% % % % In this list, we show the fold change increases of the side populations treated with cisplatin and different hypoxia levels. density of the different plates; however, there is not a large difference in the growing pattern of the DLD1 cells because patches of cells are characteristic of all the densities (Fig. 7A). Even the lowest density had cells growing in patches. The direct contact resulting from this growth pattern might explain why the SP percent did not vary much with increasing harvesting densities. In contrast, in the SW480 and MCF7 cell lines, the different harvesting densities appeared to have a more significant impact on the SP. These cells grew separated from each other, rather than in patches, resulting in diminished direct contact at lower densities (Fig. 7B). The absence of direct contact between the cells at the low confluency might explain why the SP is more greatly affected at low densities as compared to high densities. Two hypotheses explain the inverse relationship between percent of confluency and SP percentage. Malignant cells might interpret low confluency and density as a stressful condition that triggers an increase in proliferation of cancer stem cells the support basis of the tumor. Unique CSC signals dependent on the number and closeness of the cells could drive the balance between self-renewal and differentiation. At low densities, the number of CSCs is low, thus the aggregate of signals is also lower, triggering self-renewal rather than differentiation or triggering dedifferentiation, both mechanisms resulting in an increase of the SP. At higher confluency, the number of CSCs is higher and their contact signals are also increased thus preventing the aforementioned mechanism and rather directing differentiation into progenitor cells and consequently increasing the non-sp or by association decreasing the SP percentage. Alternatively, higher confluency and density may downregulate the expression of the ABC family of transporters and consequently decrease the amount of fluorescent dye that is effluxed out of the cell. If at higher densities, the ABC transporters pump out less Hoechst 33342, then flow cytometry would reveal a decrease in the percent of SP, as we observed. Problems and questions raised by the confluency study. The confluency study is difficult to apply in vivo because it is assumed 2558 Cell Cycle 2007; Vol. 6 Issue 18

7 Figure 6A B. SP profiles of hypoxia treatment show how in hypoxic conditions the side population is enriched as compared to the side population of normoxic conditions. (A) DLD1 cells at 0.2% oxygen have a 2-fold SP increase as compared to the control. (B) SW480 at 0.5% oxygen have a 3.2-fold SP increase as compared to normoxia. (C) DLD1 cells at 1.0% oxygen have a 1.54-fold SP increase over the control. (D) DLD1 cells at 2.0% oxygen have a 2-fold SP enrichment as compared to 21% oxygen. that in the body all the cells are attached and in direct 3-dimensional contact with each other as opposed to in a plate where the cells are spread throughout the area of the dish in a relatively 2-dimensional manner. If this is the case, the confluency study is useful when used together with other experiments such as applying other treatments or stresses to these cells. Side population analysis by flow cytometry of the cells plated at the optimal density would remove the confluency variable from the experiment, helping to ensure that the resulting data is not confounding. However, using the hypothesis that CSCs release specific contact signals only in response to each other, and not to non-cscs or non-cancer cells, the confluency study can be applied to in vivo studies. Cell Cycle 2559

8 Figure 6C D. SP profiles of hypoxia treatment show how in hypoxic conditions the side population is enriched as compared to the side population of normoxic conditions. (A) DLD1 cells at 0.2% oxygen have a 2-fold SP increase as compared to the control. (B) SW480 at 0.5% oxygen have a 3.2-fold SP increase as compared to normoxia. (C) DLD1 cells at 1.0% oxygen have a 1.54-fold SP increase over the control. (D) DLD1 cells at 2.0% oxygen have a 2-fold SP enrichment as compared to 21% oxygen. To strengthen the results of this experiment, a wider range of harvesting densities with their specific SP percentages would give a better correlation of the relationship between SP and confluency. Also, it would be interesting to determine if repeating this study in other cancer cell lines known to have an SP results in the same relationship. If the same results are found, the conclusions and the implications drawn in this study would be further validated. Manipulating the stress induced by serum starvation may have therapeutic potential. The three cell lines studied had different levels of dependence upon outside nutrients and growth factors. The DLD1 and SW480 colon cancer cell lines appeared to respond most robustly to withdrawal of nutrients and growth factors found in FBS, while the MCF7 breast cancer cell line was found to be most dependent on these factors. Depriving the colon cancer cell lines of serum triggered the increase in percentage of SP. This can result from either death of the non-sp cells or an increase in the SP cells due to the induced stress. The question that arises is whether this effect can be repeated and can 2560 Cell Cycle 2007; Vol. 6 Issue 18

9 Figure 7. At 48 hours after plating, the density of the DLD1 and SW480 cell lines vary. (A) DLD1 cells grew in patches at all ranges of densities. (B) SW480 cells grew as separate cells at low densities and connected into patches at higher densities. be shown to occur in other colon cancer cell lines. If serum starvation does enrich the SP in other cell lines, colon cancer might prove to acquire its malignancy from this possible increase in CSCs. In contrast, the MCF7 cell line was vulnerable to lower levels of serum starvation showing that it is dependent to a greater degree on the nutrients and growth factors found in the environment. Also, it would be interesting to determine if other breast cancer cell lines respond in similar ways to serum starvation. Both effects of increase and decrease of SP due to starvation can be used in future experiments. For example, manipulating cell lines to increase their SP by serum starvation would provide a good model of the nutrient deprived tumor and a good control for malignant cells believed to be independent of outside growth factors. Lower levels of starvation in MCF7 and complete starvation in all three cell lines used in this study with a decrease in the SP demonstrated that serum starvation can also have toxic effects on the cell lines and their SP s. Comparing the differences in molecular mechanisms stimulated in complete starvation (that induced a decrease in SP) versus lower levels of starvation (that induced an increase in SP) could potentially show how CSCs are vulnerable and therefore could potentially allow therapeutic targeting of these areas of vulnerability. The increased SP in hypoxic conditions can explain the worse prognosis associated with hypoxic tumors. Hypoxic tumors have been shown to be associated with worse prognosis. 28 Our results show that hypoxic conditions enrich the SP of all three human cancer cell lines used in this study. The hypoxic stress on a tumor appears to increase the number of SP cells (or decrease drastically the number non-sp cells). If it is the number of SP cells that is increased then this could be an explanation as to why hypoxic tumors are more aggressive. If it is the number of non-sp cells that is decreased, then this correlates with current findings of radiation therapy that are successful in reducing the size of the tumor, but not degenerating the tumor. Another explanation could be that hypoxia helps stabilize the hypoxia inducible factors (HIFs) which target, among others, the human gene that encodes for the ABC transporters, increasing their expression and consequently the amount of drug that is effluxed out of the cell. 30,31 It is known that HIF is stabilized in hypoxic conditions and that it helps drive the malignancy of the tumor. However, we have showed here that it is the increase in SP cells, which alongside with HIF stabilization, is what might confer the malignant phenotype to hypoxic tumors. Future directions in the study of cancer stem cells. Problems that arise with SP analysis stem from the definition of the SP. The side population is only enriched for CSCs and does not include all the CSCs present. Because SP analysis does not provide definite conclusions since it does not explain the behavior of all the CSCs, other analyses methods must be used to confirm the trends discovered. 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