Fluorescence-guided surgical sampling of glioblastoma identifies phenotypically distinct tumour-initiating cell populations in the tumour mass and margin

Abstract

Background: Acquiring clinically annotated, spatially stratified tissue samples from human glioblastoma (GBM) is compromised by haemorrhage, brain shift and subjective identification of 'normal' brain. We tested the use of 5-aminolevulinic acid (5-ALA) fluorescence to objective tissue sampling and to derive tumour-initiating cells (TICs) from mass and margin.

Methods: The 5-ALA was administered to 30 GBM patients. Samples were taken from the non-fluorescent necrotic core, fluorescent tumour mass and non-fluorescent margin. We compared the efficiency of isolating TICs from these areas in 5-ALA versus control patients. HRMAS (1)H NMR was used to reveal metabolic alterations due to 5-ALA. We then characterised TICs for self-renewal in vitro and tumorigenicity in vivo.

Results: The derivation of TICs was not compromised by 5-ALA and the metabolic profile was similar between tumours from 5-ALA patients and controls. The TICs from the fluorescent mass were self-renewing in vitro and tumour-forming in vivo, whereas TICs from non-fluorescent margin did not self-renew in vitro but did form tumours in vivo.

Conclusion: Our data show that 5-ALA does not compromise the derivation of TICs. It also reveals that the margin contains TICs, which are phenotypically different from those isolated from the corresponding mass.

Figure 1

The TICs can be isolated using GBM samples obtained from 5-ALA-administered patients. (A) Human brain under the light (left) and the fluorescence microscope (right) during resection of GBM. The 5-ALA technology allows us to discriminate between three areas of disease in human GBM: fluorescent tumour mass (T), non-fluorescent necrotic area (N) and non-fluorescent margin (M). (B) In vitro, cells isolated from T of 5-ALA+ve GBMs (left) and cultured in SF media grow as neurospheres (right) as previously reported (Piccirillo et al, 2009b; Fael Al-Mayhani et al, 2009) (Magnification × 100; scale bar, 50 μm). (C) Cells isolated from T of 5-ALA+ve GBMs are long-term expanding (left) and clonogenic as the cells isolated from T in 5-ALA−ve samples (right). Note that SP6 and SP18 TICs are derived from 5-ALA−ve GBMs and SP10 and SP20 TICs are established from 5-ALA+ve GBMs. (D) Upon in vitro differentiation, T cells from 5-ALA+ve GBMs resemble the phenotypes of normal neural stem cells by expressing astroglial and neuronal markers (GFAP, green and Tuj1, fuchsia; left) and oligodendroglial-markers (O4, red; right) (Magnification × 400; scale bar, 20 μm). (E) Representative HRMAS spectra of 5-ALA+ve GBM (top) and 5-ALA−ve GBM (bottom). Clearly visible peaks are annotated in both spectra. One example is shown as representative of 10 5-ALA+ve GBMs and 10 5-ALA−ve GBMs giving similar results. (F) Xenograft obtained upon injection of T cells of a 5-ALA+ve GBM. The H&E staining reveals the presence of tumour (left) and GFAP immunoreactivity (green; right) confirms the glial nature of the tumour, counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (Magnification × 200; scale bar, 100 μm). Abbreviations: TM=tumour mass and C=core.

Figure 2

Margin area in GBM contains a distinct population of TICs. (A) In vitro, primary cells isolated from N (left) and M (right) of 5-ALA+ve GBMs do not grow in SF media (Magnification × 100; scale bar, 20 μm). (B) Growth-curve analysis show that only cells derived from the T are capable of long-term expansion. On the contrary, N cells die in a few passages. M cells are not capable of long-term proliferation in these culture conditions. This analysis has been repeated on 20 sets of samples (T, M and N) giving similar results. (C) RT– PCR shows that the proliferation marker Ki67 is significantly less expressed in M cells in comparison with T cells (n=3) in accordance to the growth-curve analysis shown in B. The number of data points included in each sample is 3. (D) M cells show a poor ‘stem-cell molecular signature’ in comparison with their corresponding T cells. Gene expression of Ki67, Nestin, Gfap, Sox2 and Notch2 has been evaluated using 18S as housekeeping (n=3). The number of data points included in each sample is 3. (E) Table summarising the tumorigenic ability of M versus T cells in four samples. Cells were injected uncultured (directly from patients) or briefly cultured in vitro for two passages in SF media before injection into the brain of Nod/Scid mice.