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Squamous cell cancers contain a side population of stem-like cells that are made chemosensitive by ABC transporter blockade.

Loebinger MR, Giangreco A, Groot KR, Prichard L, Allen K, Simpson C, Bazley L, Navani N, Tibrewal S, Davies D, Janes SM - Br. J. Cancer (2008)

Bottom Line: Purified SCC-SP cells also exhibited increased clonogenic potential in secondary cultures and enhanced tumorigenicity in vivo.Despite this, SCC-SP cells remained chemotherapeutically sensitive upon ATP-dependent transporter inhibition.Future chemotherapeutic strategies should therefore consider coupling identification and targeting of this potential stem cell-like population with standard treatment methodologies.

View Article: PubMed Central - PubMed

Affiliation: Centre For Respiratory Research, Rayne Institute, University College London, 5 University Street, London WC1E 6JJ, UK.

ABSTRACT
Cancers are a heterogeneous mix of cells, some of which exhibit cancer stem cell-like characteristics including ATP-dependent drug efflux and elevated tumorigenic potential. To determine whether aerodigestive squamous cell carcinomas (SCCs) contain a subpopulation of cancer stem cell-like cells, we performed Hoechst dye efflux assays using four independent cell lines. Results revealed the presence of a rare, drug effluxing stem cell-like side population (SP) of cells within all cell lines tested (SCC-SP cells). These cells resembled previously characterised epithelial stem cells, and SCC-SP cell abundance was positively correlated with overall cellular density and individual cell quiescence. Serial SCC-SP fractionation and passaging increased their relative abundance within the total cell population. Purified SCC-SP cells also exhibited increased clonogenic potential in secondary cultures and enhanced tumorigenicity in vivo. Despite this, SCC-SP cells remained chemotherapeutically sensitive upon ATP-dependent transporter inhibition. Overall, these findings suggest that the existence of ATP transporter-dependent cancer stem-like cells may be relatively common, particularly within established tumours. Future chemotherapeutic strategies should therefore consider coupling identification and targeting of this potential stem cell-like population with standard treatment methodologies.

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Side population cells have an increased level of resistance to mitoxantrone but are sensitised by concurrent ABC transporter blockade. (A) Analysis of stem cell colony numbers in clonogenicity assays of parent, SP(1), and SP(3) cells in the absence or presence of mitoxantrone at 1 or 10 ng ml−1. Cells were grown in the drug for 3 days and grown for a further 14 days in the absence of the drug before analysis. Assays were performed in triplicate. Error bars indicate s.e.m. (B) Parental and SP(3) cells were grown in 0 or 1 ng ml−1 mitoxantrone±verapamil for 7 days and, subsequently, grown for a further 7 days in the absence of drugs and clonogenicity analysed. (C) Quantitation of large colony numbers from (B). Assays were set up in triplicate. Error bars indicate s.e.m.
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fig6: Side population cells have an increased level of resistance to mitoxantrone but are sensitised by concurrent ABC transporter blockade. (A) Analysis of stem cell colony numbers in clonogenicity assays of parent, SP(1), and SP(3) cells in the absence or presence of mitoxantrone at 1 or 10 ng ml−1. Cells were grown in the drug for 3 days and grown for a further 14 days in the absence of the drug before analysis. Assays were performed in triplicate. Error bars indicate s.e.m. (B) Parental and SP(3) cells were grown in 0 or 1 ng ml−1 mitoxantrone±verapamil for 7 days and, subsequently, grown for a further 7 days in the absence of drugs and clonogenicity analysed. (C) Quantitation of large colony numbers from (B). Assays were set up in triplicate. Error bars indicate s.e.m.

Mentions: Our observation that SCC-SP cells expressed both ABCG2 and ABCC1 multidrug transporters and exhibited elevated Hoechst 33342 efflux indicated that these cells likely possessed resistance to cytotoxic chemotherapeutic drugs including mitoxantrone. To assess SCC-SP and parent cell mitoxantrone sensitivity, 200 cells were cultured in the presence of 0, 1, and 10 ng ml−1 mitoxantrone for 3 days followed by 14 days culture in mitoxantrone-free media. Results indicated that both SP(1) and SP(3) were significantly more resistant to 10 ng ml−1 mitoxantrone treatment and capable of maintaining large colony formation in comparison with parent cells (Figure 6A). All cells appeared resistant to 1 ng ml−1 mitoxantrone in agreement with previous ABC transporter expression data (Figure 3A).


Squamous cell cancers contain a side population of stem-like cells that are made chemosensitive by ABC transporter blockade.

Loebinger MR, Giangreco A, Groot KR, Prichard L, Allen K, Simpson C, Bazley L, Navani N, Tibrewal S, Davies D, Janes SM - Br. J. Cancer (2008)

Side population cells have an increased level of resistance to mitoxantrone but are sensitised by concurrent ABC transporter blockade. (A) Analysis of stem cell colony numbers in clonogenicity assays of parent, SP(1), and SP(3) cells in the absence or presence of mitoxantrone at 1 or 10 ng ml−1. Cells were grown in the drug for 3 days and grown for a further 14 days in the absence of the drug before analysis. Assays were performed in triplicate. Error bars indicate s.e.m. (B) Parental and SP(3) cells were grown in 0 or 1 ng ml−1 mitoxantrone±verapamil for 7 days and, subsequently, grown for a further 7 days in the absence of drugs and clonogenicity analysed. (C) Quantitation of large colony numbers from (B). Assays were set up in triplicate. Error bars indicate s.e.m.
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fig6: Side population cells have an increased level of resistance to mitoxantrone but are sensitised by concurrent ABC transporter blockade. (A) Analysis of stem cell colony numbers in clonogenicity assays of parent, SP(1), and SP(3) cells in the absence or presence of mitoxantrone at 1 or 10 ng ml−1. Cells were grown in the drug for 3 days and grown for a further 14 days in the absence of the drug before analysis. Assays were performed in triplicate. Error bars indicate s.e.m. (B) Parental and SP(3) cells were grown in 0 or 1 ng ml−1 mitoxantrone±verapamil for 7 days and, subsequently, grown for a further 7 days in the absence of drugs and clonogenicity analysed. (C) Quantitation of large colony numbers from (B). Assays were set up in triplicate. Error bars indicate s.e.m.
Mentions: Our observation that SCC-SP cells expressed both ABCG2 and ABCC1 multidrug transporters and exhibited elevated Hoechst 33342 efflux indicated that these cells likely possessed resistance to cytotoxic chemotherapeutic drugs including mitoxantrone. To assess SCC-SP and parent cell mitoxantrone sensitivity, 200 cells were cultured in the presence of 0, 1, and 10 ng ml−1 mitoxantrone for 3 days followed by 14 days culture in mitoxantrone-free media. Results indicated that both SP(1) and SP(3) were significantly more resistant to 10 ng ml−1 mitoxantrone treatment and capable of maintaining large colony formation in comparison with parent cells (Figure 6A). All cells appeared resistant to 1 ng ml−1 mitoxantrone in agreement with previous ABC transporter expression data (Figure 3A).

Bottom Line: Purified SCC-SP cells also exhibited increased clonogenic potential in secondary cultures and enhanced tumorigenicity in vivo.Despite this, SCC-SP cells remained chemotherapeutically sensitive upon ATP-dependent transporter inhibition.Future chemotherapeutic strategies should therefore consider coupling identification and targeting of this potential stem cell-like population with standard treatment methodologies.

View Article: PubMed Central - PubMed

Affiliation: Centre For Respiratory Research, Rayne Institute, University College London, 5 University Street, London WC1E 6JJ, UK.

ABSTRACT
Cancers are a heterogeneous mix of cells, some of which exhibit cancer stem cell-like characteristics including ATP-dependent drug efflux and elevated tumorigenic potential. To determine whether aerodigestive squamous cell carcinomas (SCCs) contain a subpopulation of cancer stem cell-like cells, we performed Hoechst dye efflux assays using four independent cell lines. Results revealed the presence of a rare, drug effluxing stem cell-like side population (SP) of cells within all cell lines tested (SCC-SP cells). These cells resembled previously characterised epithelial stem cells, and SCC-SP cell abundance was positively correlated with overall cellular density and individual cell quiescence. Serial SCC-SP fractionation and passaging increased their relative abundance within the total cell population. Purified SCC-SP cells also exhibited increased clonogenic potential in secondary cultures and enhanced tumorigenicity in vivo. Despite this, SCC-SP cells remained chemotherapeutically sensitive upon ATP-dependent transporter inhibition. Overall, these findings suggest that the existence of ATP transporter-dependent cancer stem-like cells may be relatively common, particularly within established tumours. Future chemotherapeutic strategies should therefore consider coupling identification and targeting of this potential stem cell-like population with standard treatment methodologies.

Show MeSH
Related in: MedlinePlus