Side population in human glioblastoma is non-tumorigenic and characterizes brain endothelial cells

Abstract

The identification and significance of cancer stem-like cells in malignant gliomas remains controversial. It has been proposed that cancer stem-like cells display increased drug resistance, through the expression of ATP-binding cassette transporters that detoxify cells by effluxing exogenous compounds. Here, we investigated the ‘side population’ phenotype based on efflux properties of ATP-binding cassette transporters in freshly isolated human glioblastoma samples and intracranial xenografts derived thereof. Using fluorescence in situ hybridization analysis on sorted cells obtained from glioblastoma biopsies, as well as human tumour xenografts developed in immunodeficient enhanced green fluorescence protein-expressing mice that allow an unequivocal tumour-stroma discrimination, we show that side population cells in human glioblastoma are non-neoplastic and exclusively stroma-derived. Tumour cells were consistently devoid of efflux properties regardless of their genetic background, tumour ploidy or stem cell associated marker expression. Using multi-parameter flow cytometry we identified the stromal side population in human glioblastoma to be brain-derived endothelial cells with a minor contribution of astrocytes. In contrast with their foetal counterpart, neural stem/progenitor cells in the adult brain did not display the side population phenotype. Of note, we show that CD133-positive cells often associated with cancer stem-like cells in glioblastoma biopsies, do not represent a homogenous cell population and include CD31-positive endothelial cells. Interestingly, treatment of brain tumours with the anti-angiogenic agent bevacizumab reduced total vessel density, but did not affect the efflux properties of endothelial cells. 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 ATP-binding cassette transporters may be a valuable therapeutic strategy to improve treatment effects in brain tumours.

Molecular crosstalk between tumour and brain parenchyma instructs histopathological features in glioblastoma

“…Using flow cytometric analysis, we found that CD31 positive cells in the tumour were GFP-positive and thus host-derived, and displayed strong efflux properties (so-called ‘side population’ (SP) phenotype), a hallmark of brain ECs (Figure 3C, Suppl. Figure 3A-3B) [16]. Although by flow cytometry a CD31 signal was occasionally (0-0.4%) detected within tumour cells (black, Figure 3C), the signal intensity was close to background and was far from the CD31 expression level (up to 100 fold lower) typically seen in human ECs or HBMVEC cells (Suppl.…”

“…We have previously shown that glioblastomas mostly comprise ECs, pericytes, microglia/macrophages and neural stem cells [16]. Because ECs constitute a major population of the glioma microenvironment and are the mediators of the angiogenic process, we focused on the interaction of tumour cells with ECs.…”

Cancer stem cells in glioblastoma

“…An alternative approach to CSC enrichment is the use of flow cytometry to isolate a side population containing CSCs, which is based on the hypothesis that stem cells contain drug efflux transporters (Yu et al 2008). While this approach has identified a population of self-renewing cells in a mouse glioma model (Bleau et al 2009), it has not been used successfully to enrich for self-renewing cells in human GBM (Broadley et al 2011;Golebiewska et al 2013), highlighting the model-and species-specific challenges of enrichment methods.…”

The role of glioma stem cells in chemotherapy resistance and glioblastoma multiforme recurrence

“…GSCs express the ATP-binding cassette (ABC) transporter channels at an increased rate compared to differentiated tumor cells, adding to the known challenges in drug delivery presented by the BBB and irregular tumor vasculature [26,70]. ABC channels actively and expeditiously transport molecules, including TMZ, both at the BBB and the cellular level to allow some cells to completely elude therapy and promote recurrence [52,71]. Expression of these proteins, with ABCG2 being the main example, is also associated with subpopulations of cells that are stem-like and multidrug resistant [26].…”