Glioblastoma-derived spheroid cultures as an experimental model for analysis of EGFR anomalies - PubMed (original) (raw)

. 2011 May;102(3):395-407.

doi: 10.1007/s11060-010-0352-0. Epub 2010 Aug 29.

Piotr Rieske, Krystyna Hułas-Bigoszewska, Magdalena Zakrzewska, Robert Stawski, Dominika Kulczycka-Wojdala, Michał Bieńkowski, Ewelina Stoczyńska-Fidelus, Sylwia M Grešner, Sylwester Piaskowski, Dariusz J Jaskólski, Wielisław Papierz, Krzysztof Zakrzewski, Maciej Kolasa, James W Ironside, Paweł P Liberski

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Glioblastoma-derived spheroid cultures as an experimental model for analysis of EGFR anomalies

Monika Witusik-Perkowska et al. J Neurooncol. 2011 May.

Abstract

Glioblastoma cell cultures in vitro are frequently used for investigations on the biology of tumors or new therapeutic approaches. Recent reports have emphasized the importance of cell culture type for maintenance of tumor original features. Nevertheless, the ability of GBM cells to preserve EGFR overdosage in vitro remains controversial. Our experimental approach was based on quantitative analysis of EGFR gene dosage in vitro both at DNA and mRNA level. Real-time PCR data were verified with a FISH method allowing for a distinction between EGFR amplification and polysomy 7. We demonstrated that EGFR amplification accompanied by EGFRwt overexpression was maintained in spheroids, but these phenomena were gradually lost in adherent culture. We noticed a rapid decrease of EGFR overdosage already at the initial stage of cell culture establishment. In contrast to EGFR amplification, the maintenance of polysomy 7 resulted in EGFR locus gain and stabilization even in long-term adherent culture in serum presence. Surprisingly, the EGFRwt expression pattern did not reflect the latter phenomenon and we observed no overexpression of the tested gene. Moreover, quantitative analysis demonstrated that expression of the truncated variant of receptor-EGFRvIII was preserved in GBM-derived spheroids at a level comparable to the initial tumor tissue. Our findings are especially important in the light of research using glioblastoma culture as the experimental model for testing novel EGFR-targeted therapeutics in vitro, with special emphasis on the most common mutated form of receptor-EGFRvIII.

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Figures

Fig. 1

Fig. 1

Initial evaluation of EGFR gene dosage in glioblastoma tumors. Results of real-time PCR (means ± SD) allowed the identification of GBMs exhibiting EGFR overdosage (values describing relative EGFR dosage above 2)

Fig. 2

Fig. 2

Spheroid and adherent cultures of GBM-derived cells. Light photomicrographs presenting the cells derived from the selected tumors with EGFR overdosage cultured as spherical aggregates and monolayer

Fig. 3

Fig. 3

Proliferation ability of GBM-derived culture in vitro assessed with BrdU. a The spherical aggregates obtained from tumors showing EGFR amplification demonstrated maintained proliferation ability during the whole period of culture (up to 8–12 weeks). b The short-term adherent cultures derived from GBMs with EGFR amplification showed gradual decrease of proliferative activity to a very low level at the final stage of culture (8–12 weeks). c The proliferation ability was sustained even in long-term adherent cultures derived from tumors showing polysomy 7 (up to 20–24 weeks)

Fig. 4

Fig. 4

Proliferative and differentiation potential of GBM cells in vitro evaluated with SOX2 expression. Immunocytochemistry data demonstrated a ability of spheroids to maintain SOX2 expression, and b gradual loss of this neural stem cell marker in adherent culture

Fig. 5

Fig. 5

Verification of EGFR gene dosage status with the use of FISH method. FISH with an EGFR probe (red signals) and 7q control probe (green signals) confirmed EGFR overdosage in selected GBMs and allowed a distinction between amplification of the tested gene and polysomy 7. G1, G32, and G48 were recognized as samples showing EGFR amplification (accompanied by low polysomy 7 in single cells). G16 and G33 were assessed as GBMs with polysomy 7. EGFR amplification was observed as homogenous or clustered distribution of EGFR signals in cells with EGFR/CEP7 ratio > 2 (representative cells in circles). Polysomy 7 was detected as the nuclei contained three or more signals specific for CEP 7 (representative cells in rectangles). EGFR gene amplification and polysomy 7 was scored as negative if only 2 red signals (EGFR)/2 control green signals (CEP7) were observed

Fig. 6

Fig. 6

Quantitative analysis of EGFR gene dosage at DNA level. a The short-term monolayer cultures derived from the tumors presenting EGFR amplification (G1, G32, G48) showed gradual decrease of EGFR gene dosage, in contrast to spherical aggregates able to maintain EGFR amplification at a level higher than the adherent cells. b The long-term cultures derived from GBMs with polysomy 7 (G16, G33) showed a gain in EGFR dosage at higher passages. c The initial culture demonstrated a rapid decrease of EGFR amplification level in comparison to the original tumor tissue. Real-time PCR data were presented as means ± SD; P < 0.05 was considered significant

Fig. 7

Fig. 7

Quantitative analysis of EGFRwt expression at mRNA level. a The tumors with EGFR amplification (G1, G48) showed EGFRwt overexpression, followed by downregulation of the tested gene in adherent culture and maintenance of high level of EGFR mRNA in spheroids. b The cell cultures derived from tumors showing polysomy 7 showed EGFRwt expression at a level similar to control (NHA) in long-term culture (G16), or underexpression of EGFRwt irrespective of the culture length (G33). Real-time PCR data were presented as means ± SD; P < 0.05 was considered significant

Fig. 8

Fig. 8

EGFRvIII expression pattern in G48-derived cell culture. a RT-PCR results carried out with two pair of primers (P1 amplicon size: 131 bp; P2 amplicon size: 90 bp), singled out G48 as _EGFRvIII_-positive sample; C(−) negative control; MW molecular weight marker. b Quantitative analysis demonstrated a rapid decrease of EGFRvIII mRNA level in adherent culture at passages 5 and 10, in contrast to spherical aggregates showing EGFRvIII expression at a level similar to the tumor tissue and passage 0. Real-time PCR data were presented as means ± SD; P < 0.05 was considered significant

Fig. 9

Fig. 9

Invasion ability of GBM spheroids and adherent cells in vitro. a, b The results of comparative invasion assay for spheroid and monolayer cultures established from the tumor tissue originally showing EGFR amplification with the use of Matrigel invasion chambers. a Quantitative data demonstrated decreased invasiveness of GBM cells in monolayer in comparison to spheroids (P = 0.03). b The cells invaded through the Matrigel-coated filters were fluorescently stained with DAPI. c The immunocytochemistry data demonstrated downregulation of EGFR protein level in adherent cells in comparison to spheroids, a phenomenon parallel to the decrease of invasiveness. d Visualization of three-dimensional invasion assay for spheroids. Photomicrogarphs of spheroids implanted into Matrigel at day 1 and day 4 of the culture presented the cells invaded radially from the spheroid core

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