Side Population is Not Necessary or Sufficient for a Cancer Stem Cell Phenotype in Glioblastoma Multiforme

Abstract

There is strong evidence for the existence of cancer stem cells (CSCs) in the aggressive brain tumor glioblastoma multiforme (GBM). These cells have stem-like self-renewal activity and increased tumor initiation capacity and are believed to be responsible for recurrence due to their resistance to therapy. Several techniques have been used to enrich for CSC, including growth in serum-free defined media to induce sphere formation, and isolation of a stemlike cell using exclusion of the fluorescent dye Hoechst 33342, the side population (SP). We show that sphere formation in GBM cell lines and primary GBM cells enriches for a CSC-like phenotype of increased self-renewal gene expression in vitro and increased tumor initiation in vivo. However, the SP was absent from all sphere cultures.Direct isolation of the SP from the GBM lines did not enrich for stem-like activity in vitro, and tumor-initiating activity was lower in sorted SP compared with non-SP and parental cells. Transient exposure to doxorubicin enhanced both CSC and SP frequency. However, doxorubicin treatment altered the cytometric profile and obscured the SP demonstrating the difficulty of identifying SP in cells under stress. Doxorubicin-exposed cells showed a transient increase in SP, but the doxorubicin-SP cells were still not enriched for a stem-like self-renewal phenotype. These data demonstrate that the GBM SP does not necessarily contribute to self-renewal or tumor initiation, key properties of a CSC, and we advise against using SP to enumerate or isolate CSC. STEM CELLS 2011;29:452-461 Disclosure of potential conflicts of interest is found at the end of this article.

Understanding cancer stem cell heterogeneity and plasticity

“…CSCs have been reported in brain tumors, especially in glioblastoma multiforme (GBM) [61][62][63][64][65][66][67][68][69][70][71][72][73][74]. The stemlike GBM cells have been enriched using cell surface molecules, such as CD133 [61,72], SSEA-1 (stagespecific embryonic antigen-1) [64], EGFR [67,68], and CD44 [69], or functional assays, including the SP analysis [7,63,73] and neurosphere assays [49,74].…”

“…The stemlike GBM cells have been enriched using cell surface molecules, such as CD133 [61,72], SSEA-1 (stagespecific embryonic antigen-1) [64], EGFR [67,68], and CD44 [69], or functional assays, including the SP analysis [7,63,73] and neurosphere assays [49,74]. Intriguingly, there are significant uncertainties surrounding the use of CD133 as a marker for brain tumor stem cells (BTSCs).…”

“…Although CD133 has been widely utilized to enrich BTSCs [61,72] and molecular profiling has revealed stem cell gene expression patterns associated with CD133 + GBM cells [71], tumorigenic cells are found in both CD133 + and CD133 -cells in some gliomas [62], and some CD133 + brain tumor cells may not possess high tumor-initiating capacity, and, in fact, the CD133 -cell population from some GBM may actually harbor long-term self-renewing tumor-initiating cells [66]. Similarly, the SP may or may not enrich BTSCs [63,73]. Furthermore, although it has been reported (or assumed) that there exists a lineage relationship between CD133 + and CD133 -brain tumor cells, the two populations of cells may have different cells-of-origin [70].…”

Expression of SOX2 in oral squamous cell carcinoma and the association with lymph node metastasis

“…The most frequently applied method for identifying CSCs is flow cytometry, which detects cells with the ability to excrete the vital DNA dye Hoechst 33342 (34)(35)(36)(37)(38). A distinctive small non-dyed population of cells, termed the side population (SP), has been detected in numerous tumors, and is highly tumorigenic (39)(40)(41)(42)(43)(44). SP cells express SCs markers, including cluster of differentiation (CD)44 (45), ATP-binding cassette sub-family G member 2 (44), octamer-binding transcription factor (Oct)4 (35) and B cell-specific Moloney murine leukemia virus integration site 1 proto-oncogene (45).…”

Targeted inhibition of dominant PI3-kinase catalytic isoforms increase expression of stem cell genes in glioblastoma cancer stem cell models

“…In the present study, we examined expression and signalling of class I A PI3K isoforms in two models of GBM CSC. The cell line 08/04 has high expression of embryonic and neural stem progenitor genes including SOX2, OCT4 and MSI1 and recapitulates a GBM phenotype following intra-cranial implantation (37). These cells were selected to model the effect of PI3K inhibition in maintenance of an established cancer stem cell phenotype.…”