The heterogeneity of tumour tissue, the lack of specific markers and their relative scarcity pose serious challenges to the identification of glioblastoma stem-like cells. Because of their proposed chemoresistance, we applied a detailed side population discrimination assay to identify stem-like cells in human glioblastomas. Here we show that efflux properties based on ABC transporters are absent in human glioblastoma cells, indicating that the side population phenotype cannot be used to define glioblastoma stem-like cells. Moreover, side population properties were not induced by microenvironmental stress caused by hypoxia or cytotoxic drugs. The side population phenotype, which is present in human glioblastoma tissue, was entirely stroma-derived and could be identified as endothelial cells with a small contribution of astrocytes. Interestingly the efflux properties of endothelial cells were not affected by anti-angiogenic treatment.
To our knowledge this is the first study addressing the nature of side population cells in fresh tumour tissue of human glioblastoma. Our results are in agreement with a recent in vitro
study on glioblastoma cells cultured under stem cell conditions that were found to be devoid of a side population (Broadley et al., 2011
), and resolve the controversy about the side population phenotype in human glioblastomas in vivo
by clearly demonstrating that side population cells in glioblastoma are non-neoplastic and do not identify stem-like cells. In this context it is important to emphasize that the side population discrimination assay is a highly sensitive technique that requires a stringent protocol and gating strategy in order to avoid false positive results (Golebiewska et al., 2011
). A direct data comparison is only possible if similar side population protocols and gating strategies are applied. The violet laser used in earlier studies (Bleau et al., 2009
) was not sensitive enough for an adequate discrimination of the side population in normal brain and glioblastoma xenografts. Side population cells have been found in some neoplasms including breast cancer and have been described to possess higher clonogenic and tumorigenic potential, and increased resistance to chemotherapy as compared with non-side population cells (Wu and Alman, 2008
). However, the majority of cancer cell lines and primary neoplasms including glioblastomas, appear to lack efflux properties, indicating that factors such as tumorigenicity, the stem-like phenotype and chemoresistance are not exclusively dependent on side population properties.
We further provide a detailed characterization of the stromal cells present within glioblastomas. By combining the side population assay with multicolour cell membrane phenotyping we were able to distinguish several stromal cell populations in patient biopsies, which was further confirmed and expanded upon in human glioblastoma xenografts using enhanced GFP expressing mice. In addition to endothelial cells and astrocytes, these included neural stem/progenitor cells, mesenchymal stem cells, pericytes, and microglia/macrophages. The latter four populations strongly accumulated in the glioblastomas compared with the normal brain parenchyma. The side population phenotype was clearly restricted to stromal CD31+
endothelial cells and A2B5+
astrocytes. In support of previous data on adult neural stem cells in normal brain (Mouthon et al., 2006
), we found that none of the stem/progenitor populations present within glioblastomas, e.g. neural stem cells and mesenchymal stem cells, displayed dye efflux properties. In this context it is interesting to speculate that in the adult brain, efflux properties may not be required in these cells due to an established blood–brain barrier. This may also explain the lack of side population in human glioblastoma cells, which grow in an efflux protected environment, as compared with other tumour types such as breast cancer. Importantly, we show that cancer stem-like CD133+
cells in patient biopsies are not a homogenous population and include CD31+
endothelial cells carrying efflux properties. We therefore propose the use of a detailed multicolour phenotyping assay for an adequate analysis of cancer stem-like cell populations in the brain, since many markers, including CD133 and nestin, which are typically associated with neural stem cells and cancer stem-like cells, are also detected in other cell populations such as brain endothelial cells.
The efflux properties in glioblastoma and normal brain vessels were found to rely on the ABC transporters ABCB1, ABCG2 and ABCC4, in agreement with a previous report showing a role for ABCB1 in the blood–brain barrier, while ABCC1 is more prominent in the blood–CSF barrier (Gazzin et al., 2008
). Interestingly we also found that in glioblastoma patient biopsies and in xenografts derived thereof, efflux properties in tumour vessels are fully functional, suggesting that the vessel leakiness in the tumour core may be due to mechanical abnormalities rather than a loss of functional endothelial cell properties. These data are in contrast to results from a transgenic mouse model, where the efflux properties of brain endothelial cells were reported to be impaired in glioma tissue (Bleau et al., 2009
). This discrepancy might be explained by abnormal features of the stromal compartment in RCAS PDGF-B-driven glioma models which differ from human tumours.
In clinical and preclinical studies anti-angiogenic treatment of glioblastoma has been shown to normalize blood vessels and prevent leakage of contrast agents suggesting a restoration of the blood–brain barrier (Reardon et al., 2008
; Carmeliet and Jain, 2011
; Keunen et al., 2011
). An unanswered question of major clinical interest is whether this will improve or impede drug delivery to the brain. Here we show that the efflux properties of endothelial cells are retained in glioblastoma multiformes following anti-angiogenic treatment, suggesting that drug penetration through the blood–brain barrier may still be hampered by a functional drug-effluxing endothelium. Efficient drug delivery is a major concern in glioblastoma treatment, in particular with regard to the highly invasive component of glioblastoma, which is largely shielded by an intact blood–brain barrier. Given the protective role of drug efflux transporters of endothelial cells in the brain, transiently modulating transporter function on endothelial cells may improve drug delivery to target invasive glioblastoma cells. Unfortunately, currently available inhibitors of ABC transporters have been rather unsuccessful due to their toxicity (Fletcher et al., 2010
). Therefore more potent and specific transport inhibitors will be required for application in the brain.
In conclusion our findings contribute to an unbiased identification of cancer stem-like cells and stromal cells in brain neoplasms, and provide novel insight into the complex issue of drug delivery to the brain. Since efflux properties of endothelial cells are likely to compromise drug availability, transiently targeting ABC transporters may be a valuable therapeutic strategy to improve treatment effects in brain tumours.