ORIGINAL PAPER

Fabian Fehlauer Æ Martina Muench Æ Dirk Rades

Lukas J. A. Stalpers Æ Sieger LeenstraPaul van der Valk Æ Ben Slotman Æ Ernst J. Smid

Peter Sminia

Effects of irradiation and cisplatin on human glioma spheroids:inhibition of cell proliferation and cell migration

Received: 1 March 2005 / Accepted: 21 June 2005 / Published online: 12 August 2005� Springer-Verlag 2005

Abstract Purpose: Investigation of cell migration andproliferation of human glioma cell line spheroids (CLS)and evaluation of morphology, apoptosis, and immuno-histochemical expression of MIB-1, p53, and p21 of or-ganotypic muticellular spheroids (OMS) followingcisplatin (CDDP) and irradiation (RT). Material andmethods: Spheroids of the GaMg glioma cell line andOMS prepared from biopsy tissue of six glioblastomapatients were used. Radiochemosensitvity (5 lg/mlCDDP followed by RT) was determined using migrationand proliferation assays on CLS. In OMS, histology andimmunohistochemical studies of MIB-1, p53, and p21expression were examined 24 and 48 h following treat-ment. Results: Combination treatment led to a migrationinhibition of 38% (CDDP 13%; RT 27%) and specificgrowth delay of 2.6 (CDDP1.3; RT 2.1) inCLS. Cell cycleanalysis after combination treatment showed an accu-mulation of cells in the G2/M phase. In OMS, apoptosis

increased, cell proliferation decreased, and p53/p21expression increased more pronounced followingCDDP+RT. No morphological damage was observed.Conclusion: CDDP can lead to enhancement of the RTeffect in spheroids of both human glioma cell line spher-oids and biopsy spheroids from glioblastoma specimens.The exerted effect is additive rather than synergistic.

Keywords Glioma Æ Irradiation Æ Cisplatin Æ Spheroids

Introduction

Although radiotherapy following surgical resection is themost effective treatment for glioblastoma multiforme(GBM), the median survival in these patients is <1 year(Gonzalez Gonzalez and Hulshof 1993; Hulshof et al.2001). The modest increase in survival time after radio-therapy treatment has been ascribed to the high intrinsicresistance of the gliomas to X-irradiation (Taghian et al.1993, 1995). To improve survival in patients withmalignant glial tumours new treatment strategies arerequired. Further approaches include chemotherapeuticagents as an adjuvant modality or as a radiosensitiser.

Currently, several in vitro model systems to investi-gate the effects of irradiation and modulation of theradiation response of glioma are available. These includeestablished cell lines, monolayer cultures of glioma lines,both early and late passage after initial isolation, andspheroids derived from cell lines, the so-called cell linespheroids (Carlson et al. 1983; Stuschke et al. 1993;Fehlauer et al. 2000). Another culture system used tostudy gliomas in vitro are the organotypic multicellularspheroids (OMS), which resemble the original architec-ture of the in vivo tumour (Darling et al. 1983; Bjerkviget al. 1990; Fehlauer et al. 2004). It is assumed thatspheroid cultures of both, cell lines and fresh tumourspecimens, can better predict the in vivo response thanmonolayer cultures, since cell-cell contact, variation incell cycle, altered metabolism, and diffusion of nutrients,oxygen or drugs may influence the outcome (Sutherland

F. Fehlauer (&) Æ M. Muench Æ D. RadesDepartment of Radiation Oncology,Universitatsklinikum Eppendorf,University of Hamburg, Martinistr. 52,20254 Hamburg, GermanyE-mail: fehlauer@uke.uni-hamburg.deTel.: +49-40-428034031Fax: +49-40-428032846

L. J. A. Stalpers Æ F. FehlauerDepartment of Radiation Oncology,AMC University of Amsterdam, Amsterdam,The Netherlands

S. LeenstraDepartment of Neurosurgery,AMC University of Amsterdam, Amsterdam,The Netherlands

P. van der Valk Æ F. FehlauerDepartment of Pathology,VU University Medical Center, Amsterdam,The Netherlands

B. Slotman Æ E. J. Smid Æ P. SminiaDepartment of Radiation Oncology,VU University Medical Center, Amsterdam,The Netherlands

J Cancer Res Clin Oncol (2005) 131: 723–732DOI 10.1007/s00432-005-0014-3

1988; Kunz-Schunghart et al. 1998; Santini et al. 1999,2003; Walenta et al. 2000). The advantage of cell linespheroids is that they are relatively easy to obtain and tomaintain in culture. Treatment related changes of thegrowth kinetic and the outgrowth of tumour cells areestablished endpoints (Mahesparan et al. 1999; Gliem-roth et al. 2003; Kleynen et al. 2003). The advantage ofOMS over conventional spheroid culture is a remarkablestability in characteristics of the individual tumour:cellular heterogeneity (tumour cells, endothelial cell, andconnective tissue), differentiation pattern, proliferationrate and tumour cell ploidy are preserved. Individualtreatment related changes in terms of the histology andexpression of various proteins can by monitored withhistopathological and immunohistochemical techniques(Kaaijk et al. 1997).

Cisplatin (CDDP) is one of the most effective of allchemotherapeutic agents in solid tumours and hasshown to potentiate the effect of RT in various tumoursystems.

Mechanistically, CDDP molecules react with neucle-ophilic sites into DNA by forming adducts (crosslink-ing). In terms of cell cycle arrest, CDDP arrest cell cycleprogression in G1 and G2 phases, but it appears to beless active in inducing a p53 dependent G1 arrest. Cellswith disrupted p53 function tended to be less sensitive toRT (Haas-Kogan et al. 1999) and CDDP (O‘Connor1996) than cells with wt p53.

However, the combination of RT and CDDP showedunsatisfactory clinical results in malignant gliomas(Anonymus 1991), which might be due to interpatientvariation in radiochemosensitivity, intertumoural heter-ogeneity, and other physiological conditions. Localchemotherapy with CDDP-depot for GBM in combi-nation with RT was suggested to be an more effectiveapproach for those patients (Sheleg et al. 2002).

A number of in vitro studies have been publishedconcerning radiochemosensitivity of glioma cell lines byCDDP (Wilkens et al. 1996; Wolff et al. 1998, 1999;Yount et al. 1998; Fehlauer et al. 2000; Reichert et al.2002). CDDP in combination with RT exerted anadditive effect on early passage glioma cell cultures asshown by the unaltered value of the a-parameter forreproductive cell death of the linear quadratic model(Fehlauer et al. 2000).

In the present study the effect of CDDP and RT wasevaluated on spheroids from the human GaMg cell lineas well as on OMS derived from fresh tumour tissue ofnine GBM patients.

Methods

Monolayer cell culture

The GaMg cell line was obtained from a 42-years oldfemale with histologically proven GBM (Akslen et al.1988). GaMg cells were cultured as monolayer in Dul-becco’s modified Eagles medium (DMEM, Gibco BRL,

UK) supplemented with 10% fetal calf serum, 2 mML-glutamine and 50 lg/ml gentamycine (Gibco BRL) in25 cm2 culture flasks in a 37�C, 5% CO2, 95% humidi-fied air incubator.

Cell line spheroids

Spheroid cultures of GaMg were formed by liquid-overlay technique (Carlsson et al. 1983). In short,exponentially growing monolayers were trypsinized and5·106 cells were seeded in 20 ml of growth medium into80-cm2 agar-coated tissue culture flasks. After 10 days inculture, spheroids with diameter between 200 and250 lm were selected for the experiments. These spher-oids do not present central necrosis and have very fewhypoxic cells (Mueller-Klieser et al. 1986; Sutherland1986; Sminia et al. 2003).

Organotypic multicellular spheroids (OMS)

Fresh tumour tissue was obtained at surgery (openresection) from nine patients with a primary glioblas-toma, classified according to the World Health Organi-zation (WHO). The specimens were taken in tumourareas corresponding to regions with contrast enhance-ment on preoperative computerised tomography scansand transferred in Dulbecco’s modification of Eagle’smedium (DMEM; Flow Laboratories, Scotland).

For preparation of the spheroids, glioma tissue wasprocessed in the laboratory within 2 h from resection:representative tumour tissue was selected, based on botha preliminary frozen section histopathological diagnosismade by the responsible neuropathologist and on themacroscopic appearance. Blood and necrotic tissue wereremoved from tumour specimens and fragments of 0.5–1 mm3 were dissected with sterile 21G2 0.8·50 needles.48-well plates were coated with 100ll of a semisolidagarose gel, consisting of a 1:1 mixture of 1.5% agaroseand of DMEM supplemented with 4 mM L-glutamine,100 lg/ml gentamicin (all from Gibco BRL, U.K.) and20% heat-inactivated human serum (Bio-Whittaker,MD, USA). After the agarose had gelled, 300ll of cul-ture medium consisting of DMEM supplemented with2 mM L-glutamine, 100 lg/ml gentamicin and 10%heat-inactivated human serum was added to each well.

One biopsy fragment was transferred to each well ofthe 48-well plates. The spheroids were kept in a standardtissue culture incubator (98% humidity, 5% CO2, 37�C)and the culture medium was changed once a week.Cultured tissue fragments were studied twice a weekwith a phase-contrast microscope and only fragmentsthat became spherical within the first week were used forfurther study. Only tissue from patients with a fractionof fragments that formed spheroids of more than 40%were included in this study. Two weeks after onset of thecultures, the biopsy spheroids were used in the experi-ments.

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Radiation and cisplatin treatment

Irradiation set up

Speroids were single dose irradiated (OMS: 20 Gy; CLS:10 Gy) using an orthovolt X-ray generator operating at250 kV and 15 mA, with a 0.5 mm thick Cu filter and atube opening of 8·8 cm. Spheroids were irradiated inpetri dishes (60 mm), containing 3 ml of culture mediumat a temperature of 37�C. Former experiments revealedthis dose range to be effective without sterilising thespheroids (Kleynen et al. 2003; Fehlauer et al. 2004).

Cisplatin treatment set up

Cisplatin (cis-diammine dichloroplatinum, CDDP;Platosin, Pharmachemie, Haarlem, NL) concentrationused was 5lg/ml, which is known from former experi-ments to be well incorporated by spheroids withoutpermitting growth (data not shown). For incubationwith CDDP, the spheroids were transferred into a60 mm petri dish. Incubation duration was 4 h, so therewas no need to use agar coated dishes, as this is too shorta period of time for the spheroids to attach to the bot-tom. After incubation the spheroids were washed twotimes with PBS and once with DMEM. Immediatelythereafter, the spheroids were irradiated.

Treatments

Thirty-six spheroids from each tumour were treated. Anequal number of spheroids from each tumour was shamtreated: At 24 and 48 h following treatment (sham, RT,CDDP, CDDP+RT) six spheroids from each groupwere fixed in 4% neutral buffered formaldehyde solutionfollowing treatment. Subsequently, spheroids were pro-cessed and embedded in paraffin for histological andimmunohistochemical analysis.

Tumour cell migration (cell line spheroids)

Following treatment, spheroids were placed individuallyinto uncoated 16-mm multi-well dishes which were filedwith 1 ml DMEM. Each treatment group consisted ofsix spheroids. One group was used as control. Thespheroid plated within 2 h, and the cellular outgrowthfrom the spheroid was defined as a colony. Twoorthogonal colony diameters were measured daily over a4-day period (96 h). by phase contrast microscope, andthe migratory capacities of the glioma cells were thendetermined by calculation the colony areas from thediameter measurements.

Growth delay essay (cell line spheroids)

For experiments, GaMg spheroids were placed individ-ually into 16-mm multiwell dishes (Nunc), which were

base-coated with 0.5 ml 0.75% DMEM-agar and filledwith 1 ml of DMEM. Six spheroids were selected for thestudy groups (sham, CDDP, RT, CDDP+RT). Aftertreatment, the dishes were incubated at 37�C. Thediameters of the spheroids were measured two times aweek in a phase contrast microscope over a 15-dayperiod, and the spheroid volume was calculated (volumemeasurement 4/3 pr3).

Once a week the culture medium was changed. Inorder to measure growth delay, the average time re-quired to reach ten times the initial spheroid volume wasdetermined. Specific growth delay (SGD) values werecalculated by dividing growth delay of treated samplesby the corresponding doubling times of control samples.

Flow cytometric analysis of cell cycle distribution(cell line spheroids)

To analyse the cell cycle changes, cells were plated andtreated as described above. At 24 h after initial plating,10 lM bromodeoxyuridine (BrdU) was administeredfrom a 100· stock. After 2 h, the cells were harvested,fixated in 70% ethanol in phosphate buffered saline(PBS) and stored at �20�C until immunofluorescentstaining. Ethanol-fixated cells were centrifuged (1 min,2,200 rpm), resuspended in 1 ml pepsin solution(0.4 mg/ml 0.1 N HCl) and incubated for 30 min atroom temperature. Subsequently, the DNA was dena-tured by a 30 min incubation in 1 ml 2 N HCl at 37�C.After washing with PBTb (PBS, Tween-20 0.05% v/v,bovine serum albumin (Sigma) 20 mg/ml, pH 7.4, thepellet was resuspended in 0.1 ml rat anti-BrdU (HarlanSeralab, Loughborough, UK, diluted 1:100 in PBTb),and incubated at room temperature for 30 min. Afterwashing with PBTg (PBS, Tween-20 0.005 v/v, normalgoat serum (Dako, Glostrup, Denmark) 1% v/v,pH 7.4), the pellet was resuspended in 0.1 fluoresceinconjugated goat-anti-rat IgG (Jackson, nr 112–015–102,West Grove, Pa., USA, diluted 1:100 in PBTg) andincubated at room temperature in the dark for 30 min.Propidium-iodine and ethanol were added to an end-concentration of 1 lg/ml and 30% respectively. Sampleswere stored at 4�C until flow cytometric analysis. Sam-ples were syringed through a 21-gauge needle to reducecell aggregation before flow cytometry (FACScan cy-tometer, Becton Dickenson, San Jose, CA, USA). Thedistribution of cells over the cell cycle was analysed withWindows Multiple Document Interface Flow CytometryApplication (WinMDI) by placing windows around G0/G1, S and G2/M populations.

Morphology and apoptosis (organotypic multicellularspheroids)

Paraffin sections (5 lm) of spheroids were placed onorganosilan-coated (3-aminopropyltriethoxysilane; Sig-ma, USA) object slides and dried overnight at 37�C.

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Paraffin sections of the original tumour were similarlyprocessed. Histology of the spheroids was evaluated inHaematoxylin–Eosin stained sections; the same sectionswere also used for the assessment of apoptosis. Clustersof intensely basophilic nuclear fragments or an intenselybasophilic, shrunk nucleus were construed as morpho-logic evidence of the apoptotic process (Kerr et al.1972).

Immunohistochemistry (OMS)

Monoclonal antibodies were used for immunohisto-chemistry (MIB-1, p53, p21), using the Avidin-Biotin-peroxidase Complex (ABC-) technique according to themanufacturer’s protocol (MIB1, Ki67 protein, Immu-notech; DO7, wild type and mutant p53, DAKO; p21, p21 protein, PharMingen).

In brief, after sections (5lm) had undergo dewaxingand rehydration, endogenous peroxidase activity wasblocked for 30 min in methanol containing 0.3%hydrogen peroxide. Antigen retrieval was performed forall monoclonal antibodies by incubating slides in a cit-rate buffer solution (2.94 mg/ml trisodium citrate dihy-drate in distilled water; pH=6) in a microwave oven at180�C for 15 min. Thereafter, the slides were preincu-bated with normal rabbit serum in a 1:50 dilution inphosphate buffered saline with 1% bovine serum albu-mine (PBS/BSA) for 10 minutes. Subsequently, sectionswere incubated with monoclonal antibodies (overnight,4�C), and incubated with biotin-conjugated rabbit anti-mouse immunoglobulin or biotin-conjugated swine anti-rabbit immunoglobulin (all Dako, Denmark). Afterincubation with streptavidin-biotinylated horseradishperoxidase complex (dilution 1:200; Dako Denmark),peroxidase activity was developed using 3,3-diam-inobenzidine-tetrachloride (Sigma, USA) in 0.1%hydrogen peroxide as a chromogen. All sections werelightly counterstained (20 s) with haematoxylin, dehy-drated, and mounted. The signal for p21 was enhancedby the catalysed reporter deposition technique. Positivecontrol sections from known immunopositive tumourswere included for all antibodies. Negative controlsconsisted of sections of each group whereby the primaryantibody step was omitted.

Assessment and image analysis (OMS)

The evaluation of apoptosis and for all antibodies wasassessed with a conventional light microscope. Theapoptotic index was defined as percentage of apoptotictumour cells, identified as ‘apoptotic bodies’ (Kerr et al.1972). For p53, p21 and proliferation activity (MIB-1),all identifiable nuclear staining were regarded as posi-tive. The labelling index was defined as the percentage ofimmunopositive tumour cells in the spheroids. Noendothelial or haematogenous cells were included in thecounts.

The multiple factor of protein expression if treatmentgroups compared to the controls was calculated for eachtreatment group: a difference of a multiple factor >1was defined as a marked treatment effect.

The relative apoptosis count and p53, p21 and MIB-1positivity of the spheroids from each tumour was ex-pressed as % of the spheroids with the maximum posi-tivity (100%) from that tumour. Statistical comparisonof the data was performed using a unpaired sample t-testafter analysis of variance. P values <0.05 were con-sidered as significant.

Results

Cell line Spheroids

Tumour cell migration

The directorial cell migration from GaMg spheroids wasdetermined for treated and untreated spheroids (Fig. 1).,The outgrowth area was reduced by 38% (RT), 13%(CDDP), and 27% (CDDP+RT) as evaluated onday—following treatment. Combination therapy led to asignificant additional inhibition of cell migration(P<0.05).

Spheroid volume

In Fig. 2 shows the effect of irradiation alone or com-bined with cisplatin on GaMg spheroids. Combinationtreatment induced an additional growth delay. A sig-nificant reduction in spheroid growth was observed forCDDP+RT of 71% (specific growth delay, SGD 2.6),for RT alone of 42% (SGD 2.1), and for CDDP alone of32% (SGD 1.3) on day 20 (P<0.05).

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Fig. 1 Tumour cell migration for GaMg cell line spheroidsfollowing RT, CDDP, and CDDP+RT. Each treatment groupconsisted of six spheroids. Changes at day 4 were significant(P<0.05)

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Cell cycle distribution

After RT alone and in combination with CDDP, theflow cytometic DNA histograms reveals an more pro-nounced accumulation of GaMg cells in the G2/M phaseof the cell cycle (55, 59% respectively). The percentagesof the cells in G1, S, G2/M for (1) control, (2) RT, (3)CDDP, and (4) combination of both were (1) 45%,28%, 27%, (2) 27%, 18%, 55%, (3) 37%, 21%, 42%,and (4) 24%, 17%, 59%. Cell distribution of the treat-ment groups was not statistically significant.

Organotypic multicellular spheroids

Spheroid culture and volume measurements

Organotypic multicellular spheroids established from sixof the nine tumour samples could be included in thisstudy (S13, S14, S17, S18, S19, and S21). Phase contrastmicroscopy showed that approximately 70% of all tu-mour fragments prepared from fresh tissue formedspheroids within 1 week of culture. Fragments of threepatients could not be used for further experiments be-cause single cells and clumps started to be shed from thefragments without spheroid formation (S15, S16, S20).

During the culture period and following treatment,the volume of the spheroids from four tumours main-tained steady. Spheroids from two tumours showed adecrease of volume, more pronounced 48 h after com-bination treatment but not statistically significant fromcontrols.

Morphology and apoptosis

Histologically the spheroids closely resemble the originalindividual tumour. As in the tumour in vivo, OMScontained tumour cells, endothelial cells and connective

tissue. Typical features of glioblastoma were observed inuntreated and treated spheroids, including increasedcellularity, nuclear atypia, mitotic figures and non-functional capillaries, of which a few showed vascularproliferation. Neither necrosis, nor perinecrotic pseud-opalisading cellular components were observed.

Compared to the control spheroids, in none of thetreated spheroids a major morphological damage couldbe observed up to 48 h after treatment. No decrease ofcell density or changes in the glial structure was seen.Capillaries were preserved in both control and treatedspheroids of both follow-up intervals.

The number of apoptotic tumour cell increased inspheroids 24 and 48 h after treatment. Apoptosis,identified as clusters of intensely basophilic nuclearfragments were seen in both irradiated and controlspheroids from all groups.

Following CDDP and RT, individual tumoursshowed in five of six cases an maximal enhancement ofapoptotic bodies at both time points, were as a markedeffect was seen in three of six cases (S17, S18, S21) at the24 h interval, and two of six cases (S17, S18) at the 48 hinterval (Figs. 3, 4). CDDP alone was the most effectivetreatment modality in one of six cases (24 h: S13; 48 h:S19), were as the changes were only slightly more pro-nounced if compared to the other treatment groups.

Overall, a significant increase in apoptosis was de-tected following RT alone (P=0.04) and CDDP+RTtreatment (P=0.01, Table 1).

Cell proliferation

MIB-1 immunopositivity was exclusively nuclear. Con-trol spheroids showed a remarkable wide range of MIB-1 tumour cells (3–28%) which were mostly centred to-ward the peripheral zone. The mean labelling index andthe relative proliferation index was lower in spheroids oftreated groups compared to control (Fig. 3, Table 1).

Following CDDP and RT, individual tumoursshowed 24 h later in all cases an maximal decreasedproliferation labelling index, while a marked effect wasseen in two cases (S18, S21). At the 48 h interval, com-bination treatment let to decreased MIB-1 labelling in-dex in three of six cases, were as a marked effect wasonly seen in spheroids of tumour S21 (Fig. 4). CDDPalone was the most effective treatment modality in oneof six cases (48 h: S18).

Overall, CDDP+RT treated spheroids showed thehighest decrease of MIB-1 index, but changes were notsignificant.

p53 expression

In the resection material from the original glioblastomasthe overall p53 expression was less than 5%. In spher-oids established from these tumours, the absolute per-centage ranged between 2 and 8% and immunopositivitywas exclusively nuclear. A wide variety in the percentage

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Fig. 2 Volume growth of GaMg cell line spheroids following RT,CDDP, and CDDP+RT. Each treatment group consisted of sixspheroids. Changes at day 20 were significant (P<0.05)

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of p53 immunoreactive tumour cells was documentedbetween spheroids within the same group.

Following CDDP and RT, individual tumoursshowed in four of six cases an maximal enhancementof p53 expression at both time points, were as amarked effect was seen in one of six cases (S18) at the24 h interval, and two of six cases (S17, S18) at the

48 h interval (Figs. 3, 4). P53 expression was highestfollowing RT alone in one of six cases (24 h: S13;48 h: S14), were as the changes were only slightly morepronounced if compared to the other treatmentgroups.

Overall, a significant increase in p53 expression wasfound following RT alone (P=0.012), CDDP alone

Fig. 3 Apoptosis andimmunohistochemicalexpression of organotypicmulticellular spheroids (OMS)of six patients (S13, S14, S17,S18, S19, S21) as determined24 h (left column) and 48 h(right column) following RT,CDDP, and CDDP+RTcompared to the untreatedcontrols. From top, relativeapoptotic index, relativeexpression of p53, p21, andrelative cell proliferation(+, P<0.05)

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(P=0.008) and CDDP+RT treatment (P=0.008,Table 1).

p21 expression

All original tumours contained less than 5% p21immunoreactive tumour cells. In untreated spheroids,the mean p21 expression was 1–2%.

Following CDDP and RT, individual tumoursshowed in two of six cases an maximal enhancement ofp21 expression at 24 h interval (S17, S18), and three ofsix cases at the 48 h interval (S14, S18, S21). In all ofthese cases a marked effect was detected. RT alone wasthe most effective modality in one of six cases with amarked effect 24 h following treatment (S14, Fig. 3).

Overall, p21 protein expression increased significantly(P<0.05) at 24 h following RT alone and CDDP+RTtreatment (Table 1).

Discussion

The present work shows that cisplatin (CDDP) andradiotherapy (RT) exert an additive effect in humanglioma spheroids, established from the human GaMgcell line grown as cell line spheroids and from fresh tu-mour tissue of six glioblastoma patients grown as OMS.

At present, the parallel utilisation of these two assaysystems was not used in experimental neuro-oncologyfor radiochemotherapy testing. OMS derived from hu-

Fig. 4 a Haematoxylin–Eosinstained section of a organotypicmuticellular spheroid (OMS). bImmunohistochemical stainingof p53. OMS showed asignificant increased rate ofapoptosis (a) and p53 proteinexpression (b) 24 h afterirradiation and cisplatintreatment. Originalmagnification, ·40. c and dImmunohistochemical stainingof Ki-67 (MIB-1). The numberof proliferating cells was higherin control (c) compared tocorresponding singe modalitytreated spheroids or to thecorresponding spheroidstreated with irradiation andcispaltin (d). Originalmagnification, ·10

Table 1 Mean labelling index(range) of apoptosis, p53, p21and MIB-1 protein expressionin OMS (n=6) at 24 and 48 hfollowing RT, CDDP, andCDDP+RT

NS Not significant

Follow up (h) Control RT CDDP CDDP+RT

Apoptosis 24 1.3±0.4 1.8±0.7 2.3±1.7 2.9±2.0P value P=0.01 NS P=0.04

48 1.6 ± 1.0 1.8 ± 0.9 2.4 ± 1.3 2.9 ± 1.3P value NS P=0.04 P=0.022p53 24 6.8±3.6 26.4±17.8 18.6±10.8 30.0±19.0P value P=0.012 P=0.008 P=0.008

48 5.2±2.9 22.0±28.7 12.8±10.2 30.5±27.5P value NS NS NSp21 24 1.1±0.4 4.8±2.2 3.6±3.2 7.8±4.7P value P=0.001 NS P=0.006

48 1.3±0.8 9.5±7.91 2.7±1.4 11.0±14.4P value NS NS NSMIB-1 24 4.8±2.0 4.4±2.9 3.7±2.6 3.3±2.1P value NS NS NS

48 4.3±1.5 3.3±2.6 4.2±3.9 3.3±3.3P value NS NS NS

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man tumour tissue represents a methodically and bio-logically different three-dimensional culture model,which reflects in vivo situation much more reliable thancell lines or cell line spheroids do.

Spheroids derived from cell lines lack the cellularheterogeneity of OMS as they are grown from a singleimmortal cell line. However, several studies have shownthat these spheroids still have some characteristics of theoriginal tumour, which include aspects of tumour mor-phology and behaviour. In cell line spheroids, as inOMS, cell–cell contact, variation in cell cycle distribu-tion, diffusion effects, altered metabolism, and hypoxiaare presumed to influence the outcome of radiationtreatment (Olive and Durand 1994; Santini 1999; Sminiaet al. 2003). When these spheroids grow, the number ofproliferative cells decreases and the number of quiescentcells increases. Proliferation becomes limited to the outerrim of the cell line spheroids. Growth delay measure andoutgrowth assay following treatment were establishedendpoints and represent the function of surviving cells,re-growth rate, and migration property which reflectsboth radiocurability and radiosensitivity of experimentalglioma.

For human glioblastomas (GBM), the individualtreatment response of RT and radiosensitization byCDDP can be monitored excellent with OMS due to thefact, that the original tumour cell architecture, geneticprofile, extracellular matrix component, connective tis-sue, vessels and macrophages were preserved underexperimental conditions. The volume of these spheroidsmaintain stable in terms of growth, which can beattributed to a balance between cell proliferation andcell shedding. Conventional morphological studiesincluding immunohistochemical analysis of variousproteins following treatment are suitable endpoints(Bjerkvic et al. 1990; Kaaijk et al. 1995; Fehlauer et al.2004). However, cell line spheroids have the advantagethat they are relatively easy to obtain and to maintain inculture compared to OMS.

Most in vitro data on the response of GBM to RTand CDDP are based on monolayer cell cultures. In aprevious study, we evaluated the effect of RT andCDDP on primary, early passage cell cultures fromhuman GBM tumour specimens (Fehlauer et al. 2000).The survival fraction of cells following two Gray irra-diation (SF2) showed rather high values in clonogenicassay (0.61–0.72) demonstrating the high intrinsic ra-dioresistance of these cells. In combination with CDDP,an additive rather than radiosensitising effect could berecognised.

Although a CDDP-resistant human glioma cell linerequires a higher CDDP dose, the same level of sensiti-sation can be induced as in a sensitive glioma cell line,suggesting CDDP to be an effective additional cytotoxicagent to RT (Wilkens 1996).

The molecular mechanisms at the origin of theadditive effect of CDDP to RT is not yet fully under-stood. CDDP induces DNA adducts, which are gener-ally repaired by a recombination-like pathway. In

parallel, ionising radiation produces double strandbreaks, which are more likely recognised by the Kuprotein and rejoined by nonhomologous end joining,suggesting that CDDP treatment combined with radia-tion leads to the interplay of two major DNA repairpathways (Dewit 1987; Biston et al. 2004).

In our study, CDDP enhanced the effect of irradia-tion in both cell line spheroids and OMS additively. Theregrowth and migration capacity of GaMg cell linespheroids was inhibited in a additive manner whereassubpopulations of cell line spheroids survive and con-tinue to grow and migrate despite combination therapy.If compared with single mode treatment, combinationtherapy led to a reduction of the migration capacity offactor 0.8 (RT) and factor 0.7 (CDDP). A specificgrowth delay of 2.6 following combination therapycould be observed (CDDP 1.3; RT 2.1). The exertedeffect of both assays among CLS was additive ratherthan supra-additive or synergistic.

After irradiation and cisplatin, flow cytometric anal-ysis revealed only minor differences in cell cycle distri-butions if compared to single mode treatment.Radiotherapy alone and in combination with CDDPshowed an increased delay in G2 phase. The mechanismsfor the delay in the G2 phase have been demonstrated invirtually all eukaryotic cells examined in response toirradiation. After a single irradiation dose, a disturbancein DNA synthesis inflicting during late G1 or early Sphase is associated with subsequent accumulation ofcells in the G2-phase (Zatterstrom et al. 1995).

In OMS, combination treatment did not induce earlymajor histological damage or changes of the glialstructure up to 48 h post-treatment as evaluated by lightmicroscopy.

We found an remarkable increased apoptotic cell ratein spheroids of three of six patients following 20 Gyirradiation and 5 lg/ml CDDP, which was more pro-nounced 24 h post-treatment.

We also investigated the time-dependent expressionof p53 and p21, both proteins involved of control of cellcycle and apoptosis. Interestingly, we found a remark-able co-expression in spheroids S17 and S18. In thesecases, elevated p21 expression was detected following24 h following RT and CDDP, where as elevated p53expression was more pronounced at 48 h follow-up.

The evaluation of p21 expression, a downstreameffector of wt p53, has been widely used an indirect toolto assess for the functional status of p53 gene. A higherpercentage of p21 immunoreactive cells was docu-mented in p53 astrocytomas with wt p53 gene com-pared to either mutant p53 haboring p53immunonegative tumours. Thus, the expression of p21protein mediates p53-dependent cell cyclus arrest orapoptosis. It was demonstrated that the p21 gene isinduced by pathways dependent and independent fromthe functionally intact p53 tumour suppressor protein(Ono et al. 1997). The observed high variation of p53and p21 expression within the same groups underlinesthe heterogeneity of GBM. Furthermore, tumour cell

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proliferation decreased additively in the combinationgroups in both assays.

Our results suggest that the limited therapeutic gainof RT in combination with CDDP, i.e., prolongation ofdisease-free interval and survival in GBM patients, canbe achieved due to a temporary cell cycle arrest, an earlyapoptotic response, decrease in proliferating tumourcells, and reduced migration potential. Clinically, theadditive treatment effect of RT and CDDP seems to bemore pronounced, when CDDP is administered locallyin GBM patients (Sheleg et al. 2002).

Finally, the combinational use of glioma cell linespheroids and organotypic multicellular spheroids de-rived from patients’ glioblastoma tissue are useful sup-plemental 3-dimensional assays for translationalresearch in neuro-oncology to study individual treat-ment effects of irradiation in combination with experi-mental therapeutic approaches on gliomas.

Acknowledgments The authors acknowledge Dr. R. Bjerkvig(Department of Anatomy and Cell Biology, University of Bergen,Norway) for his hospitality during the laboratory visit of Dr. F.Fehlauer in Bergen and his critical comments on the manuscript.We also wish to thank the members of the European Cancer Centre(ECC) Neuro-Oncology Group for their collaborations, insightfuldiscussions and encouragement. Dr. F. Fehlauer was research fel-low and supported by a grand of the Deutsche Forschungsgeme-inschaft (Bonn, Germany) and the ECC (Amsterdam, TheNetherlands). Furthermore, we thank Dr. Dr. A.J. Terzis(Department of Neurosurgery, University of Luebeck, Germany)for providing the GaMg cell line.

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