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B= issola=20 L
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Short = communication

Genetic alterations and in vivo tumorigenicity of neurospheres = derived=20 from an adult glioblastoma

Patrizia Tunici¹ , = Lorena Bissola¹ , = Elena Lualdi² , = Bianca Pollo³ , = Laura Cajola¹ , = Giovanni Broggi⁴ , = Gabriella Sozzi² = and=20 Gaetano Finocchiaro¹

¹Istituto=20 Nazionale Neurologico Besta, Dept. Experimental Neurology, Milano, = Italy
²Istituto Nazionale Tumori, Dept. Experimental = Oncology, Milano, Italy
³Istituto Nazionale = Neurologico=20 Besta, Dept. Clinical Neurosciences, Milano, = Italy
⁴Istituto=20 Nazionale Neurologico Besta, Dept. Neurosurgery; Milano, Italy

author email =20 corresponding author email

Molecular Cancer 2004,=20 3:25doi:10.1186/1476-4598-3-25

The electronic version of this article is the complete one and = can be=20 found online at: http://www.molecu= lar-cancer.com/content/3/1/25

Received:	30 August 2004
Accepted:	6 October 2004
Published:	6 October 2004

=C2=A9 2004 Tunici et al; licensee BioMed Central Ltd.
This = is an=20 open-access article distributed under the terms of the Creative = Commons=20 Attribution License (http://creativecommon= s.org/licenses/by/2.0),=20 which permits unrestricted use, distribution, and reproduction in = any=20 medium, provided the original work is properly cited.

Abstract

Pediatric brain tumors may originate from cells endowed with = neural=20 stem/precursor cell properties, growing in vitro as neurospheres. = We have=20 found that these cells can also be present in adult brain tumors = and form=20 highly infiltrating gliomas in the brain of immunodeficient mice.=20 Neurospheres were grown from three adult brain tumors and two = pediatric=20 gliomas. Differentiation of the neurospheres from one adult = glioblastoma=20 decreased nestin expression and increased that of glial and = neuronal=20 markers. Loss of heterozygosity of 10q and 9p was present in the = original=20 glioblastoma, in the neurospheres and in tumors grown into mice,=20 suggesting that PTEN and CDKN2A alterations are = key=20 genetic events in tumor initiating cells with neural precursor=20 properties.

Short communication

Recent data have proposed that brain tumors contain a "core" of = stem=20 cells providing them with the potential to grow aggressively, = escaping the=20 effects of radiotherapy and chemotherapy [1,2]. = These=20 cancer stem cells were isolated from medulloblastomas or gliomas = and grew=20 in vitro as neurospheres, suspended clonal aggregates containing = cells=20 with different levels of commitment [3].

Such observations, derived from pediatric tumors only, did not = include=20 data on the in vivo tumorigenicity of cancer stem cells. We have = found=20 that neurospheres from an adult glioblastoma (GBM) have the = potential to=20 express glial and/or neuronal markers and form highly infiltrating = gliomas=20 into the brain of immune-deficient mice.

The neurospheres were derived from three adult brain tumors and = two=20 pediatric malignant gliomas (BT1=E2=80=93BT5, see 1). = The=20 neurospheres of BT1, a glioblastoma multiforme (GBM) were studied = by=20 flow-cytometry and immunohistochemistry. Under differentiating = conditions=20 (EGF-bFGF-LIF withdrawal and FBS addition) nestin expression = decreased and=20 BT1 neurospheres expressed high levels of neuronal and astrocytic = markers.=20 Remarkably, most of the cells expressed both such markers, = suggesting the=20 altered function of a complete differentiation program (see 2).<= /P>

Additional=20 File 1. Additional file 1 (Tunici et al-Additional file = 1.doc)=20 contains Methods with references, comments on in vitro data and = the legend=20 to the additional file 2.

Format: DOC Size: 39KB Download=20 file

This file can be viewed with: Microsoft=20 Word Viewer

Additional=20 File 2. Additional file 2 (Tunici et al-Additional file = 2.ppt)=20 contains figures of brain tumor neurospheres, and flow cytometry = and=20 immunohistochemical data for their characterization.

Format: PPT Size: 3.8MB Download=20 file

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To test their neoplastic potential we injected BT1 and BT2 (a = central=20 neurocytoma) neurospheres into nude mice. All the mice injected=20 intracerebrally (i.c.) with BT1 neurospheres, but none of those = injected=20 subcutaneously (s.c.), developed brain tumors that were lethal = after 3, 5=20 and 6 months, respectively. After 4 months, however, none of the = mice=20 injected with BT2 neurospheres developed a tumor. Adherent cells = from the=20 same two patients were also injected i.c. and s.c. into nude mice. = Two of=20 three mice injected i.c. with BT1 adherent cells, but none of = those=20 injected with BT2 cells, developed a brain tumor that were lethal = 4 and 5=20 months after injection, respectively.

All the brain tumors in nude mice appeared as large, = infiltrating=20 gliomas (Fig. 1A-B)=20 with features of a grade II-III oligoastrocytoma (Fig 1D-E).=20 Both the primary tumor (Fig 1F)= ,=20 and the tumors in nude mice (Fig 1G-H)=20 expressed nestin.

Figure 1. Histological = analysis of=20 BT1 and BT1-derived tumors in nude mice. BT1 neurospheres = (1 =C3=97=20 10e5) were stereotactically injected into the left hemisphere of = nude mice=20 (Charles River Italia, Calco, Italy; n =3D 3) or subcutaneously (n = =3D 3).=20 Nude mice were also injected with 1 =C3=97 10e5 BT1 adherent cells = into the=20 brain (n =3D 3) or subcutaneously (n =3D 3). Cells from BT2 were = injected with=20 similar procedures into nude mice. Control mice (n =3D 3) were = injected with=20 1 =C3=97 10e5 neural stem/progenitor cells obtained from C57BL6J = mice with=20 previously described methods [11]. Fig 1A-B shows the GFAP = staining in=20 brown of coronal sections of the tumor derived from neurospheres = (1A) or=20 from adherent cells (1B). The right part on the figures correspond = to the=20 left hemisphere, were cells were injected. Fig. 1C-E show H-E = staining of=20 the primary tumor with features of a glioblastoma multiforme (1C) = and of a=20 tumor in mouse brain derived from neurospheres, showing an area = with a=20 prevailing aspect of oligodendroglioma (1D) or adherent cells, = exhibiting=20 anaplastic changes (1E). Fig. 1F-H show nestin staining of the = primary=20 tumor (1F) and of a tumor in mouse brain derived from neurospheres = (1G) or=20 adherent cells (1H).

The five chromosomal regions showing frequent allelic imbalance = in=20 gliomas (1p, 9p, 10q, 17p and 19q) were investigated on six = specimens=20 obtained from BT1 surgery. No allelic loss was detected in = specimen 1 (S1;=20 frontal area); S2 and S3 (fronto-temporal area) showed LOH on = chromosome=20 10q; S4 and S5 (temporal area) had LOH on 10q and 9p (Fig 2).= =20 Neurospheres were derived from S6 (temporal) and their analysis = showed the=20 same alterations of S5, i.e. LOH on 10q and 9p (Fig. 2).= =20 Adherent cells deriving from S6 did not show any detectable LOH = and no=20 alteration was found on 1p, 17p and 19q. In the primary tumor the = allelic=20 imbalance was partial, in neurospheres, on the contrary, it was = complete.=20 Interestingly, not only tumors deriving from BT1 neurospheres but = also the=20 tumor from adherent cells showed LOH on 9p and 10q (Fig 2).=

Figure 2. Genetic = analysis on BT1,=20 BT1-neurospheres and adherent cells and BT1-tumors in nude mice.=20 DNA was extracted from frozen tissues, cell cultures or=20 lymphocytes, using standard protocols. Primers, microsatellite = markers and=20 PCR conditions for LOH analysis were described before [12]. We = also=20 investigated markers 9S157 and 9S171 flanking the CDKN2A gene on = 9p21.=20 Before doing microsatellite analysis on mouse tumors we confirmed = that PCR=20 primers did not hybridize on mouse DNA. For cytogenetic analysis = cells=20 were harvested with 0.1 =CE=BCg/ml = Colcemid (Karyomax=20 Colcemid, Life Technologies) overnight. Hypotonic treatment, = fixation and=20 GTG banding of metaphase chromosomes were performed with standard = methods.=20 The karyotypes were described in accordance with ISCN guidelines = http://cgap.nci.nih= .gov/Chromosomes/Mitelman=20 webcite Spectral karyotyping was performed on = metaphase cells=20 according to the manufacturer's instructions (ASI, Carlsbad, CA) = and to=20 published procedures [13]. Spectral images were acquired and = analyzed with=20 an SD200 Spectral Bio-imaging System (ASI Ltd., MigdalHaemek, = Israel) and=20 a charged-coupled device camera (Hamamatsu, Bridgewater, NJ) = connected to=20 a Zeiss Axioskop 2 microscope (Carl Zeiss, Canada) and analyzed by = the use=20 of SKYVIEW (version 1.6.1; ASI) software. The upper panel = shows=20 the results of LOH analysis on 9p and 10q of the different samples = outlined on the left. The lower panel illustrates a=20 representative spectral karyotype of neurospheres obtained with = the=20 simultaneous hybridization of 24 combinatorially labeled = chromosome=20 painting probes. Karyotype display of chromosome banding (inverted = DAPI)=20 and SKY analysis (chromosomes were assigned a pseudo-color = according to=20 the measured spectrum) are shown. The number (7) next to the = marker=20 chromosome (der(3)) indicates the origin of inserted=20 material.

Cytogenetic analysis of BT1 neurospheres showed a = pseudo-diploid=20 karyotype with monosomy of chromosomes 9, 10, 18, trisomy of = chromosomes=20 19 and 20 and presence of three marker chromosomes. A = pseudo-tetraploid=20 clone was also present, resulting from duplication of the = pseudo-diploid=20 clone and with the same numerical and structural abnormalities = (Fig. 2).= =20 The G-banding karyotype of BT1 adherent cells resulted 46, XY. SKY = analysis confirmed the numerical changes (monosomies and = trisomies) shown=20 by G-banding and allowed to unravel the nature of a the marker = chromosomes=20 as a = der(3)ins(3;7)(3pter=E2=86=923q11::7q11=E2=86=927q22::3q11=E2=86=923qter)= .

Three observations are provided by the follow-up of nude mice = injected=20 with BT1 cells. First, tumors only developed into the brain and = not=20 subcutaneously. Thus, in BT1 the cancer "stem" cells required to = be in=20 their niche, i.e. the brain, to develop tumors and the evolution = of these=20 tumors resembled closely that of "real" gliomas. The phenotype of = such=20 gliomas, however, appeared less aggressive than in the original = tumor,=20 possibly because the cancer "stem" cells were conditioned by in = vitro=20 passaging and by growth in the brain of immune-deficient mice.

Second, the tumors obtained from neurospheres were completely = different=20 from those obtained from established cell lines like U87, 9L, C6 = or F98:=20 they grew slower, were highly infiltrating and showed a = morphological=20 pattern resembling that of an anaplastic, mixed glioma, but = without=20 necrotic areas and palisade cells typical of a GBM (compare Fig. = 1C = with 1D-E).=20 LOH studies demonstrated the loss of a region chromosome 10q where = PTEN is located. PTEN is a critical tumor = suppressor=20 gene in GBM but has also an important role in the regulation of = neural=20 stem cell proliferation [4-6]. Its = loss can therefore be a central event in the neoplastic = derangement of=20 brain cancer "stem" cells. We also found combined 9p LOH = associated to=20 10qLOH in S4=E2=80=935 and in the neurospheres, but not in = S2=E2=80=933, suggesting that=20 9p LOH is secondary to that on 10q. LOH on 9p suggests the = alteration of=20 the important tumor suppressor gene CDKN2A, encoding p16 = and=20 p14(ARF). p16 expression is absent or defective in glioblastomas = [7,8] = and p16=20 has an important role in the terminal differentiation of neural = precursor=20 cells [9]. = Furthermore, p16 is the main target through which Bmi1 regulates = neural=20 stem cell differentiation and self-renewal [10].

Third, LOH on 10q and 9p were present not only in the original = tumor=20 and in neurospheres but also in neurosphere-derived gliomas in = nude mice.=20 Remarkably, even if adherent cells had a normal karyotype and no = allelic=20 imbalance, the derived tumors did show 10q and 9p LOH. This = suggests that=20 few adherent cells with these genetic abnormalities escaped our = analysis=20 and underwent a positive selection in vivo. These results, = therefore,=20 point to PTEN and CDKN2A alterations as critical = events=20 in tumor initiating cells, a definition synonymous of cancer stem=20 cells.

The identification of neurospheres from adult brain tumors, and = specifically from an adult GBM, is strengthening the case for the=20 importance of cancer "stem" cells in the genesis of these = malignancies. A=20 thorough genetic dissection of such cells on a larger scale should = give=20 new insights for the therapeutic targeting of these cancer = "queen-bee"=20 cells.

Acknowledgements

We thank Ettore Salsano for collaboration with real-time PCR=20 experiments, Luigi Poliani for helpful suggestions and Francesca = Inverardi=20 for help with morphological analysis. This study was partially = supported=20 by grants to GF from the Associazione Italiana per la Ricerca sul = Cancro=20 and from the Istituto Superiore di Sanita'.

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Genetic alterations and in vivo tumorigenicity of neurospheres = derived=20 from an adult glioblastoma

Abstract

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References

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