¡CANCERRESEARCH 50. 7444-7449. December I, 1990|

Glycosphingolipids of Human Gliomas1

Richard Jennemann, Appletree Rodden, Bernhard L. Bauer, Hans-Dieter Mennel, and Herbert WiegandtZentrum fur Operative Meili:in, Abtl. Neurochirurgie [R. J., A. R., B. L. B.J, Zentrum fur Pathologie, Abtl. Neuropathologie ¡H-D.M.], anäPhysiologisch-ChemischesInstitut ¡H.W.j, Philipps-Universitat Marburg, Federal Republic of Germany

ABSTRACT

Histologically characterized human gliomas of various grades of malignancy obtained during surgery were extracted, and their glycolipidswere isolated and partially identified. Among the gliomas analyzed, threetypes of glycolipid component distribution could be identified. The gly-cosphingolipid (CSL) type I pattern correlated closely with that of themost malignant gliomas (Grade IV). Its neutral GSLs consisted ofglucosyl- and, as a major component, dihexosylceramide, in addition toglobo- and neolactotetraosylceramide. Galactosylceramide and sulfatidewere absent. The gangliosides of GSL type I were almost exclusively ofthe GI „,family, aside from small amounts of neolacto-series-derivedspecies. The neutral components of GSL type II were similar to those ofGSL type I. The acidic compounds of GSL type II were gangliosides ofthe <•„,family and trace amounts of neolacto-series sialoglycolipids, inaddition to G, „,I and <., ,„2. GSL type II contained no (>,,.,gangliosidesand no sulfatide. The GSL type III pattern was that of the most benigngliomas, with all glycolipids present that are found in normal brain and,in addition, those of the GSL type II.

INTRODUCTION

Aside from palliative surgical debulking and, in some cases,radiation, the treatment of most brain tumors remains ineffective. For the rational development of new concepts in therapy,it would be well to know more of the proliferative properties ofsuch tumors, in particular of the important group of thegliomas. The most common of these, glioblastoma multiforme,presents particular difficulties because of its notorious heterogeneity (1). Traditionally, the gliomas are categorized in gradesfrom I to IV according to their clinical malignancy (2-4).Methods of describing include the analysis of marker substancesknown to be correlated with cellular proliferation, differentiation, or dedifferentiation (5, 6). Ideally, such molecular markersof cell differentiation/dedifferentiation would be located on thecell surface so that they might be used as tumor-specific targetsfor diagnosis and therapy. GSLs2 might conceivably satisfy

these criteria (7). Within a given animal species, the GSLcomponent pattern reflects the histológica! cell type, as well asthe state of cellular growth, maturation, and differentiation (8).Cellular dedifferentiation usually is accompanied by a reductionin the structural complexity of the GSL components of a givencarbohydrate series, accumulation of "precursor" GSLs, and in

some cases the additional appearance of neo-GSLs that are notpresent in the differentiated cell.

The present investigation was initiated in order to determine

Received 11/14/89; accepted 7/25/90.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was generously supported by grants from the Ursula Kulemann-Stiftung and Deutsche Forschungsgemeinschaft (WÌ222/27).

2The abbreviations used are: GSL. glycosphingolipid; BSA, bovine serumalbumin; PBS. phosphate-buffered saline; CDH. ceramide dihexoside; CMH,ceramide monohexoside; CTetH, ceramide tetrahexoside; CTH, ceramidc tri-hexoside; Gangliosides (G) of the sialoganglio-serics are given by the short abbreviations described earlier (10): a suffix to G designates the neutral carbohydrateportion, e.g., GU^ (lactose). Glri (gangliotriaose. Ggj). or G,„(gangliotetraose.Gg«).The number of sialic acid residues is given in arabic numerals, positionalsialo-isomers by the addition of a. b, c; GSLs are designated according to thesuggestions of the IUPAC-IUB Commission on the Nomenclature of Glycocon-jugates; TLC. thin-layer chromatography; HPTLC, high-performance TLC.

the range of the GSL component variability among differenthuman gliomas and to explore the possibility of improvingdiagnostic validity on the basis of GSL composition. It has nowbeen recognized that basically three types of GSL componentpatterns of gliomas exist. Tumors of the respective types weredesignated glioma GSL types I, II, and III. Several humangliomas revealed GSL component profiles that represent stagesof transition between the basic GSL types, thereby possiblyreflecting subtle differences in tumor growth and its state ofcell dedifferentiation.

MATERIALS AND METHODS

Tissue Samples. Tumors were obtained after surgical removal in ourown neurosurgical unit or were kindly provided by Professor K. Roosen(Department of Neurosurgery, University of Giessen, Giessen, FederalRepublic of Germany) or by Professor R. Lorenz (Department ofNeurosurgery, University of Frankfurt, Frankfurt, Federal Republic ofGermany). Tumors from our own neurosurgical unit were processedduring operation, and immediately thereafter (intraoperatively) cyto-logical analysis by smear preparation (9) and rapid diagnosis withfrozen sections were carried out. Postoperatively, routine histologyfrom paraffin sections was determined. These tests were augmentedwith immunohistochemical determinations using a panel of antibodiesagainst intermediary filament proteins and other neural differentiationmarkers (9). Finally, tumor samples were examined electromicroscop-ically. In tumors from outside departments, routine histology andimmunohistochemical determinations were performed. The diagnosisand gradings were made in all cases by one of the authors (H. D. M.)according to the criteria of the World Health Organization—Histológica! Typing of Central Nervous System Tumors (1979). Some tumorsunderwent analysis in several portions. In these instances, a "partial"

diagnosis of the tumor portion can be at variance with the tumordiagnosis and grading as a whole.

Tumors analyzed were gliomas of Grades II, III, and IV (glioblasto-mas).

Immunoreagents. Monoclonal antibodies against lactoneotetraose(1B2) and globotetraose (SSEA-3) were gifts from Professor S. Hako-mori (University of Washington, Seattle, WA). Monoclonal antibodiesagainst G, ac2(R24) and Glri2/Gu.,2b (BW 625/445), as well as choleratoxin and anti-cholera toxin, were provided by Dr. W. Dippold (University of Mainz) and Dr. K. Bosslet and Dr. J. Stärk(Behring Werke,Marburg, Federal Republic of Germany), respectively. Alkaline phos-phatasc conjugated with anti-mouse immunoglobulin antibody waspurchased from Dakopatts (Hamburg, Federal Republic of Germany).

Extraction and Purification of GSL. Lyophilized tumor tissue (5-25mg dry weight) was first freed from the bulk of nonpolar componentsby acetone extraction (2 x 2.5 ml) followed by low-speed centrifugation.The first extraction lasted for 10 h and the second for 30 min. In afurther extraction, the samples were sonified in the presence of 2.5 mlof chloroform:methanol:water (10:10:1, v:v:v) for 15 min, incubated at50°Cfor 15 min, separated by centrifugation, and air dried. This

procedure was repeated one time. For saponification of acylglyceroli-pids and separation of neutral from acidic GSLs, the extracted glycolipid fraction was suspended in 1 ml of methanol to which 100 ^1 of 3M KOH was added. Saponification was carried out at 50°Cfor 5 h. The

resultant solutions were dialyzed against 3x5 liters of distilled waterfor 24 h and then dried in a stream of air. The acidic glycolipids wereseparated from the neutral GSL components on DEAE-Sephadex A25(Pharmacia Fine Chemicals, Uppsala, Sweden) by the method of Le-deen et al.(\\ ).

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For purification of the neutral GSLs, the fraction of the neutralGSLs was evaporated to dryness in a vacuum desiccator over P2O5,redissolved in 400 n\ of chloroform, 100 ¿ilof acetic anhydride, and100 M!of 1% dimethylaminopyridine (in chloroform) and incubated at37'C for 1 h. One ml of toluene was added to the now completely

peracetylated GSLs before drying them in a stream of nitrogen.The samples were dissolved in dichloroethane-n-hexane (4:1, v:v),

once in 2 ml and then 2 x I ml, and layered onto small columns Tilledwith Florisil (60-100 mesh; Merck AC, Darmstadt, West Germany)which had been suspended in and then washed with 20 ml of an identicalsolvent mixture. An additional 20 ml were run through the loadedcolumn and then discarded. The neutral GSLs were eluted with dichlo-roethane:acetone (1:1, v:v), dried, and transferred to Pyrex vials via 3x 1 ml of chloroform:methanol (1:1, v:v). The samples were deacety-lated by the addition of SO¿ilof l M sodium methylate in methanol.

Interfering salts were then removed by running the solution throughSep Pac RP-18 cartridges (Waters Associates Inc., Milford, MA) whichhad been prewashed with 20 ml of methanol, then 10 ml of metha-nohwater (1:1, v:v), and finally 25 ml of water. Samples were appliedto the cartridges in 10 ml of water. The vials were rinsed twice with 2ml and then washed out with 25 ml of water. The vials were rinsedagain with 2 ml of methanohwater (1:1, v:v) and the columns washedwith an additional 10 ml of the same solution. The desalted GSLs wereeluted with 20 ml of methanol.

TLC of Neutral Glycosphingolipids and Gangliosides. TLC of thesamples was carried out on HPTLC-Siw plates (Merck AC). For theneutral GSLs, a solution of chloroform:methanol:water (65:25:4, v:v:v)was used for separation. The glycolipids were visualized by sprayingthe plates evenly with 2% orcinol in 2 N sulfuric acid and heating at110°Cfor 30 min. The acid fraction glycolipids were separated using a

mixture of chloroform>thanol;2% aqueous CaCI2 (60:35:8, v:v:v). Forthe detection of sialic acid-containing compounds, they were stainedwith resorcinol-HCl reagent by heating at 95°Cfor 30 min. With this

reagent, sulfatide forms a grayish color.The GSL patterns were quantitated densitometrically using a Shi-

madsu dual wavelength TLC scanner CS 910 at a wavelength of 440and 580 nm for orcinol-H2SO4- and resorcinol-HCI-stained plates,respectively.

Borate Separation of Glucosyl- and Galactosylceramides. The neutralGSLs were applied to an HPTLC silica aluminum foil plate (MerckAG). After thorough spraying with 1.5% aqueous sodium tetraboratesolution, the plates were dried in a desiccator over P2O5. A solution ofchloroform:methanol:water:25% aqueous ammonium hydroxide(60:25:4:0.5, v:v:v:v) was used to affect separation of the gluco- andgalactocerebrosides.

Immunostaining. For immunostaining, two aliquots of the neutralGSLs from the tumors were applied to HPTLC silica plates. Afterchromatography, the plates were broken in half by using a glass cutter.On one-half, the neutral GSLs were visualized by treatment with orcinolreagent.

On the other half, immunostaining was carried out. These plateswere first mechanically stabilized by a 2-min bath in 2% Plexigum 28(Rohm and Haas, Darmstadt, Federal Republic of Germany) in n-hexane. n-Hexane was allowed to evaporate for 5 min and the plateswere then incubated for 10 min in a 2% solution of BSA in 10 mMPBS, pH 7.2, with 0.05% Tween 20. The plates were further incubatedfor 2 h at room temperature with the murine first monoclonal antibody.Plates were then washed five times with 20 ml of the PBS/Tweensolution (without BSA). A second incubation with PBS/Tween/BSA asdescribed above was then carried out. This was followed by another 2-h incubation at room temperature with alkaline phosphatase-conju-gated anti-mouse immunoglobulin antibody, diluted 1:100 with PBS/Tween/BSA, in preparation for visualization of the positive TLC bands.

After this incubation with the second antibody, the plate was washed5 times with 20 ml of PBS/Tween. Finally, substrate for the phospha-tase [1 mg of 5-bromo,4-chloro,3-indolylphosphate, /Moluidine salt(Sigma) in 0.2 ml A'.A'-dimethylformamide] in 10 ml of 10 mM Tris-

buffered saline, pH 8.5, which contained 2 mM MgCl2 and 0.2 mM

ZnCI2 was layered on the plate. Positive bands were visible after 5-30min.

Hydrolysis Using Sialidase. The solvent-free ganglioside samplesfrom the tumors were dissolved in 0.5 ml of "neuraminidase buffer"

(0.2 Msodium acetate and 2 mM CaCI2 in 0.5% acetic acid, pH adjustedto 5.2 with dilute NaOH). To this solution, 3 x 50 ^1 of neuraminidasefrom Vibrio cholerae (Sigma), activity 0.01 unit/50 p\ of suspensionwas added over the course of 24 h. Incubation was at 37°C.After the

incubation, reaction products were separated from the buffer salts bypassing the solution through Sep Pak RP-18 cartridges by the methodalready described above. After desalting, the resulting compounds wereseparated on DEAE-Sephadex into neutral and acidic components.

RESULTS

The qualitative and quantitative analysis of the GSL distribution of the various human gliomas investigated revealed thatthey could be classified into three basic types of GSL componentpatterns, the GSL types I, II, and III (Figs. 1 and 2; Table 1).

GSL Type I

Neutral GSL. Whereas normal brain contains galactosylcer-ebroside with only trace amounts of lactosylceramide as thesole neutral glycolipid constituents, the latter CDH forms thepredominant component of the neutral fraction GSL of gliomasGSL type I. In addition, higher neutral GSLs are present inmajor proportions (Fig. 1, Lanes a and c). The CMH of gliomaGSL type I was characterized as predominantly glucosylcer-amide (data not shown).

The CTetH components consist of glycolipid migrating onHPTLC as globoside (Gb4Cer) reacting immunologically withSSEA-3 antibody and paragloboside (nLc4Cer). The latter wasidentified on HPTLC using 1B2 antibody (data not shown).

Acidic GSL. Whereas sulfatide constitutes one of the majoracidic GSLs of normal glial tissue, the glycolipid componentsof GSL type I pattern are conspicuously devoid of this sulfato-glycolipid. The gangliosides consist predominantly of the lactose sialoderivatives, i.e., Gi,acl and GLac2, in addition to traceamounts of immunologically identified gangliosides of the nLcseries. Most typically, no or at most minor amounts of G,ricomponents are found, whereas G,c, gangliosides are alwayscompletely absent (Fig. 1, a and c. Table I).

GSL Type II

Neutral GSL. The component distribution of GSL type IIneutral glycolipids is similar to the glioma GSL type I (Fig. 1,Lanes </-/; Table 1). It consists mainly of CDH, CTH, andCTetH, in addition to low proportions of CMH in the form ofglucosylceramide. CDH and/or CTetH is the predominantneutral GSL. The CTetH of GSL type II consists of globoside(positive with SSEA-3 antibody) and paragloboside (positivewith 1B2 antibody) (data not shown).

Acidic GSL. In possible correlation with the absence ofgalactosylcerebroside, GSL type II also does not contain anysulfatide. The gangliosides consist of those derived from lactoseand gangliotriaose, i.e., GLacl< GLaC2,G,ril, and G,r¡2.Gg4-sialoderivatives were not, or only in traces, present in theganglioside fraction. Paragloboside that could be identifiedamong the products of sialidase degradation of the GSL typeII acidic fraction glycolipids revealed the presence of nLc4-derived gangliosides (data not shown).

GSL Type III

The GSL component profile is seen in general as an overlapof the components present in normal brain tissue and those of

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Neutral GSL

GlcGal

CDHr

CTH-Gtucer—nLcícer—

Fig. 1. HPTLC of the neutral and theacidic glycosphingolipids of human gliomas..Vi, standard bovine brain gangliosides; No,normal human brain gray matter gangliosides.Nonganglioside impurities marked by X. Lanesa, b, and c, GSL type I pattern; Lanes d, e, and/ GSL type II pattern; Lanes g, h, i, and j,GSL type III pattern. For running solvent andstaining conditions, see "Materials and Methods." For description of deviation of pattern

in Lane b from GSL type I, see text.

St a b f g h i j No

(Suif)—

Glac 1—

Glac 2—Gtet 2a—Gtri 2-^

Gtet 2b-~Gtet 3b—

Ganaliosides

Stabcdefghi

Fig. 2. Scanning profiles of neutral andacidic GSL of human tumors representative ofGSL types I (Fig. I, Lane a). II (Fig. 1, Laned), and III (Fig. 1, Lane i) and normal braingray matter (Fig. 1, Lane No). For runningsolvents and staining conditions, see "Materials and Methods."

Normal

cote

CTH-

Type Type I Type I

Neutral GSL

GSL type II (Fig. 1, Lanes h-j; Table 1).

Neutral GSL. The major GSL of the GSL type III pattern isCMH, as galactosylcerebroside. CDH, CTH, and CTetH occurin lesser proportions. The CTetH consists of globoside and noparagloboside could be detected.

Acidic GSL. In contrast to the GSL types I and II, the GSLtype III contains sulfatide as a major component. The sialo-glycolipids of this type consist of those found in GSL type II,i.e., the Lac- and the Gg.i-derived gangliosides GLacl and 2 andGlril and 2. In addition to these, the major Gg4-derived brain

Gangliosides

gangliosides G,cl, 2a, 2b, and 3b are also present (Fig. 1, LanesA-;; Table 1).

The major characteristics of the glycolipid component distribution of glioma GSL types I, II, and III may therefore besummarized as follows. GSL type I contains neither GalCernor its sulfatation product, sulfatide. Instead, CDH, CTH, andCTetH, the latter consisting of Gb4Cer and nLc4Cer, are themajor neutral GSL components. The major gangliosides areLac-sialoderivatives. nLc4-derived gangliosides are present inlow amounts. GSL type II is similar to the former GSL type.

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Table 1 Quantitation of glioma GSL distribution as shown in Fig. I

Lane

°Values are percentage of total neutral GSL or total gangliosides. respectively.*Traces of <2% of the total.

No

Neutral GSLGlcCerGalCerCDHCTHGb4CernLc4CerGangliosidesCL«!G,rilG„,lG^2G„,2aG,ri2G,„2bG,„3b25.4°64.75.32.53.051.648.4bbb77.410.77.72.51.745.854.21bb16.0b75.43.81.33.452.048.0bbb42.450.24.21.91.338.525.126.210.2bb16.262.310.45.95.130.832.128.88.3bh13.932.130.57.214.61.634.524.828.412.3bb89.08.21.01.60.213.08.26.642.8i27.4b2.095.43.60.40.66.55.421.424.813.315.07.75.991.18.60.312.49.528.412.613.013.45.65.099.70.39.48.318.823.812.815.36.84.898.51.54.63.227.25.022.24.819.213.5

The gangliosides, however, consist of the sialoderivatives oflactose and gangliotriaose. Trace amounts of nLc4-derived gangliosides are also present. GSL type III is clearly distinguishedby the presence of GalCer and sulfatide, as well as the ganglio-tetraose-derived major brain gangliosides.

Within the three defined GSL type patterns of humangliomas, considerable variations among the ganglioside compositions were observed with regard to the degree of sialylationof the neutral carbohydrate backbone, i.e., the relative proportions between GLacl and GLat2, G,r¡land G,r¡2,and G,cll, GIC,2a,G,c,2b, and G,CI3b.

From the above observations, it could be recognized that themajor difference in the ganglioside component distributionbetween the human glioma GSL types is the varying lengths oftheir neutral carbohydrate backbone. In order to investigate thevalidity of this notion, an "abbreviated" form of ganglioside

identification was considered. Hereby, the total acidic GSLfraction of a respective tumor sample was treated with V.cholera neuraminidase. The molar ratios of the products thatwere formed, i.e., lactosylceramide representing the Lac-gan-gliosides, and the gangliosides Glril and G,,,l for all Gg.,- andGg4-derived sialoglycolipids, were then estimated (Table 2).The observed molar ratios of the GLac,Glri, and G,«sialoglycolipids of the respective GSL type glioma indeed confirmedour above conclusions.

Of nine cases of glioblastoma multiforme of malignancyGrade (WHO) IV diagnosed on the basis histology, five showedthe GSL type I pattern and three were of the GSL type II. Oneglioblastoma Grade IV did not conform with either GSL typeI or II (Table 3). Even though this tumor presented a gangliosidedistribution of GSL type I, the neutral GSLs were atypical ofthe latter GSL type, containing galactosylcerebroside and onlya comparably low relative proportion of CDH (Fig. 1, Lane b).

Table 2 Relative molar distribution of gangliosides G^, G,„,and G,,, in gliomasof GSL Type I, II, and III as calculated from the products of treatment of total

gangliosides with V. cholerae neuraminidase

Table 3 GSL type, histodiagnosis, and malignancy grading of humansupratentorial gliomas

GNormalbrain white matter"

Normal brain graymatter"Normal

brain gray matterGSL type IIIGSL type IIGSL type IL«

GW1.48

0.430.48

0.78 ±0.32s0.57 ±0.27*0.07 ±0.07*G,«,8.1

5.27.3

1.34 ±0.40*

<0.03

Code"abc91/8816/8838/88def45/8833/8874/88gh,ji73/8848/88GSLtype|(I)*IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIHistodiagnosisGlioblastomaGlioblastomaGlioblastomaGlioblastomaGlioblastomaGlioblastomaGlioblastomaAstrocytomaGlioblastomaGlioblastomaAstrocytomaAstrocytomaAstrocytomaAstrocytomaAstrocytomaAstrocytomaAstrocytomaGradingIVIVIVIVIVIVIVIl/IliIVIVIIIIIIII11IIIIIIPatient'ssex/age(yr)F/52M/50F/64M/56M/57F/55M/66M/67F/60F/69M/74F/35F/59M/60F/22F/49M/66Samples22212112413122411

" Calculated from the ganglioside component distribution reported by Eto and

Shinoda(17).* Deviation of three tumor samples of the same GSL type. Mean ±SD.

" GSL patterns of tumor samples (a-j) are given in Fig. I.4 For description of this tumor type, see text.

In contrast, five astrocytoma samples diagnosed as Grade IIshowed the GSL type III. Two probes of astrocytoma GradeIII and one of Grade II/III each revealed a GSL type II pattern.Thus, >60% of malignant gliomas (glioblastoma IV) displayedthe "malignant" GSL component profile I. All of the comparably benign astrocytomas (astrocytoma II) had the "benign"

pattern of GSL type III. No constant correlation of tumor typeand grade was observed for the type II GSL component distribution present in either astrocytoma Grade III or glioblastomaGrade IV.

DISCUSSION

Glycosphingolipids occur predominantly as constituents ofthe outer surface plasma membrane of eukaryotic cells. Theirstructure, in particular that of their complex carbohydratemoiety, is characteristic for the state of proliferation and thedegree of cell differentiation in a given animal (9). GSL analysisis therefore uniquely suited to yield information concerning thestate of the cell. Since, except for the galactosylcerebroside ofthe glial cells, neutral GSL are present in the central nervoussystem only in very minor quantities, earlier reports of humanbrain tumor glycolipids were generally restricted to the analysisof gangliosides.

In 1965, Seifert and Uhlenbruck (12) were the first to discoverin human brain tumors the accumulation of the less complex,

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and for the normal brain atypical, ganglioside GLacl (13).In 1977, Kostic and Buchheit (14) again analyzed brain

tumors and considered the presence or absence of abnormalgangliosides that could possibly be correlated to the degree ofmalignancy.

In 1979, Yates et al. (15) reported that all brain tumorsinvestigated revealed structurally less complex gangliosides. In1980, Traylor and Hogan (16) were able to identify a componentfrom human gliomas as the ganglioside GLac2. In a more detailed study of the GSL of human brain tumors, Eto andShinoda in 1982 (17) also showed that, in gliomas of differentdegrees of malignancy, the relative proportion of the disialo-gangliosides Gi^c2 and G,r¡2is increased. Three years later,Berràet al. (18) recognized a correlation between malignancyand the concentration of ganglioside GLac2 in surgically removed astrocytomas. The occurrence of the ganglioside GLac2as the major GSL constituent is the more noteworthy since thisglycolipid is present in only minor proportions in isolated glialcells or astrocytes (19-22).

In 1986, Mânssonet al. (23) reported GSL analyses of humanglioma cells cultured in mice. Under these cell culture conditions, three GSLs were characterized that were typical for theglioma, i.e., ganglioside of the lacto-series, globoside and lac-totetraosylceramide. That these GSLs also occur in humanbrain gliomas was shown in 1988 by Fredman et al. (24). Theseauthors reported that the ganglioside IV'NeuAc-Lc^er is typ

ical for gliomas of malignancy Grade IV. In addition, theycould identify the presence of gangliosides GLacl, Glril,IV3NeuAc-nLcjCer, G,et1, and higher gangliosides derived fromlinear and branched chain oligosaccharides of the (neo)lacto-

series (24).In view of the characteristics in the tumor-associated ganglio

side composition that was in principle reported by all previousauthors, it seemed desirable to establish criteria that wouldallow for a clear distinction among different glial tumors byanalyzing their GSL component distribution.

The glial tumors that showed the least deviation from thenormal GSL distribution of normal brain tissue were designatedto be of GSL type III. The characteristics of these tumors ascompared to normal brain tissue consisted of the appearanceof higher neutral GSL components with Gb4Cer as the CTetHconstituent. The pattern of the gangliosides of GSL type IIIgliomas was characterized by a more or less drastic increase ofthe Lac and the Gg., gangliosides relative to the normal majorGg4 brain gangliosides.

What possibly reflects a further dedifferentiation of the GSLtype III gliomas to those of GSL types II and I is the shorteningof the neutral carbohydrate backbone of the gangliosides fromgangliotetraose to gangliotriaose and lactose. Thereby, the gangliosides of the most dedifferentiated GSL type I gliomasdisplay only Lac derivatives. There is, however, an increase inthe proportion of the nLc4 gangliosides in GSL type I gliomasas compared to the GSL type II.

A particularly conspicuous difference that distinguishes manyGSL type I and II gliomas from normal glial tissue is theabsence of galactosylcerebroside and sulfatide. Instead, gluco-sylcerebroside, lactosylceramide, and higher neutral GSLs arefound. Among the latter, GSL types I and II express as CTetHcomponents globoside and neolactotetraosylceramide (para-globoside), whereby the more malignant GSL type I gliomas ofmalignancy Grade IV show a relatively increased proportion ofthe latter neutral GSL.

Since there is an increase in ganglioside complexity depend

ing on phylogenetic and/or ontogenetic differentiation, thesesubstances have often been considered as differentiation markers, i.e., to be expressed in "lower" forms in tumors of increasing

anaplasia. For gliomas, most investigators have found changesin sphingolipid composition corresponding to degrees of malignancy (for review, see Ref. 25). Some authors, however, failedto distinguish between tumors of different grades (24) or couldsee clear distinctions only between tumors of Grades I-II andGrade IV (16).

Our findings tend to confirm both sides. From solely biochemical observations, it is possible to establish three patternsof glycosphingolipids, i.e., Ill, II, and I, with increasingly simplified ganglioside compositions. These seem to indicate anincreasing degree of malignancy in the respective tumors. Twoof these patterns, the most benign and the most malignant,were clearly associated with corresponding histológica! types.There was, however, no correspondence of intermediate malignant GSL pattern to an intermediate malignant histology. Ithas not been established whether or not the GSL compositiondepends upon the tumor as a whole or on the cellular make upof the particular sample under direct investigation. If the latteris true, then the tissue sampling is critical since an admixtureof nontumorous compartments cannot be completely avoided.

At present, there is no conclusive information available concerning the nature of the gangliosides in the normal astrocytein vivo. Several authors have shown that isolated postembryonicneuronal perikarya and glial cells share a very similar complement of cerebroside, sulfatide, and gangliosides including GLac,G,r¡,and Glc, glycolipids (19-21). Ganglioside G(;ail, interestingly, was not found as a constituent of astrocytes (22). Culturedembryonic astrocytes, on the other hand, contain, in additionto gangliosides, Gi.acl and GLac2, no galactosylceramide, andno G,cl glycolipids (26). Since the gangliosides Gi.acl and Gi,ac2were also reported to constitute the major glycolipids of ratastroblasts (21) and of reactive glial cells of mutant mousecerebellum (27), it was suggested that an increased quantity ofthese GLac gangliosides were characteristic of active as contrasted with resting astrocytes.

It remains to be established whether the GSL profiles of thevarious gliomas compared reflect tumor derivation from comparable parental glial cells. If this indeed were the case, theGSL types III, II, and I gliomas might represent stages ofcellular dedifferentiation. Successive loss of differentiationwould then be associated with a reduction of the chain lengthof the neutral part of the sialoganglioseries carbohydrate backbone. The degree of sialylation of these sugar backbones maybe a reflection of the proliferative state of the neoplastic cells.Second to the loss of complexity within these gangliosides, atcertain stages during dedifferentiation, neobiosynthesis of GSL,of one or later of two GSL carbohydrate series, the globo- andthe lacto-series, neither of which is typically expressed by fullydifferentiated neuroectodermal cells of the brain, would beinitiated. By characterizing multiple, serial specimens of intra-cranial tumors obtained by stereotactical biopsy, the study ofGSL profiles may yield information concerning the biologicaldynamics of these malignancies.

REFERENCES

1. McComb, R. D.. and Bigner, D. D. The biology of malignant gliomas—acomprehensive survey. Clin. Neuropalhol., 3: 93-106, 1984.

2. Mennel, H. D. Classification of supratentorial gliomas. In: C. Voth and P.Krauseneck (eds.). Chemotherapy of gliomas, pp. 305-309. Berlin. WestGermany: Walter de Grüyter,1985.

7448

Research. on September 5, 2020. © 1990 American Association for Cancercancerres.aacrjournals.org Downloaded from

GLYCOSPHINGOLIPIDS OF HUMAN GLIOMAS

3. Hildebrand, J. Chemotherapy of malignant supratentorial gliomas in adults:a ten year experience of the E.O.R.T.C, brain tumor group. In: D. Voth andP. Krauseneck (eds.). Chemotherapy of gliomas, pp 317-320. Berlin, WestGermany: Walter de Grüyter,1985.

4. Krauseneck, P. Der Stand der Chemotherapie bei malignen Gliomen desErwachsenenalters. In: E. H. Graul. S. Pütter,and D. Low (eds.), MedicenaleXVIII Iserlohn, pp. 329-343. Iserlohn, West Germany: Medice, 1985.

5. Gerdes, J., Schwab, U., Lemke, H., and Stein, H. Production fo a mousemonoclonal antibody reactive with a human nuclear antigen associated withcell proliferation. Int. J. Cancer, 31: 13-20. 1983.

6. Burger, P. C., Shibata, T., and Kleihues. P. The use of the monoclonalantibody Ki-67 in the identification of proliferating cells: application tosurgical neuropathology. Am. J. Surg. Pathol.. 10: 611-617, 1986.

7. Hakomori. S. Aberrant glycosylation in cancer cell membranes as focused onglycolipids: overview and perspectives. Cancer Res.. 45: 2405-2414, 1985.

8. Hakomori. S. Glycosphingolipids in cellular interaction, differentiation andoncogenesis. Annu. Rev. Biochem., 50: 733-764, 1981.

9. Mennel H.-D., Rossberg, C., Lorenz, H., Schneider, H., and Hellwig, D.Reliability of simple cytological methods in brain tumor biopsy diagnosis.Neurochiurgia, 32: 129-134, 1989.

10. Wiegandt, H. The gangliosides. In: A. Neuberger and L. L. M. van Deenen(eds.), Glycolipids New Comprehensive Biochemistry, Vol. 10, pp. 199-260.Amsterdam. The Netherlands: Elsevier. 1985.

11. Ledeen, R. W., Yu, R. K.. and Eng, L. F. Gangliosides of human myelin:sialylgalactosylceramide (G7) as a major component. J. Neurochem.. 21:829-839, 1973.

12. Seifert, H.. and Uhlenbruck, G. ÃœberGanglioside von Hirntumoren. Naturwissenschaften, 52: 110, 1965.

13. Seifert, H. Überein weiteres hirntumorcharakteristisches Gangliosid. Klin.Wochenschr.. ¥4:469-470. 1966.

14. Kostic. D., and Buchheit, F. Gangliosides in human brain tumors. Life Sci..9:589-596, 1977.

15. Yates, F. A. J., Thompson. D. K.. Boesel, C. P., Albrightson, C., and Hart,R. W. Lipid composition of human brain tumors. J. Lipid Res., 20: 428-436, 1979.

16. Traylor, T. D.. and Hogan. E. L. Gangliosides of human cerebral astrocyto-

mas. J. Neurochem.. 34: 126-131, 1980.17. Eto, Y., and Shinoda, S. Gangliosides and neutral glycosphingolipids in

human brain tumors: specificity and their significance. Adv. Exp. Med. Biol.,752:279-290, 1982.

18. Berra. B., Gaini, S.. and Riboni, L. Correlation between ganglioside distribution and histological grading of human astrocytomas. Int. J. Cancer, ifi:363-366, 1985.

19. Hamberger, A., and Svennerholm. L. Composition of gangliosides and phos-pholipids of neuronal and glial cell enriched fractions. J. Neurochem., 18:1821-1829, 1971.

20. Abe, T., and Norton, W. T. The characterisation of sphingolipids fromneurons and astroglia of immature rat brain. J. Neurochem., 23:1025-1036,1974.

21. Robert. J., Freysz, L., Sensenbrenner. M., Mandel. P., and Rebel, G. Gangliosides of glial cells: a comparative study of normal astroblasts in tissueculture and glial cells isolated on sucrose-Ficoll gradients. FEBS Lett., 50:144-146, 1975.

22. Byrne, M. C., Farooq, M., Sbaschnig-Agler, M., Norton, W. T., and Ledeen,R. W. Ganglioside content of astroglia and neurons isolated from maturingrat brain: consideration of the source of astroglial gangliosides. Brain Res.,461: 87-97, 1988.

23. Mânsson. J.-E., Fredman, P.. Bigner. J. J.. Molin. K., Rosengren, B.,Friedman, H. S., and Svennerholm, L. Characterisation of new gangliosidesof the lactotetraose series of murine xenografts of a human glioma cell line.FEBS Lett.. 201: 109-113, 1986.

24. Fredman, P., van Hoist, H., Collins, V. P., Granholm, L., and Svennerholm,L. Sialolactotetraosylceramide, a ganglioside marker for human malignantgliomas. J. Neurochem., 50: 912-919, 1988.

25. Yates. F. A. J. 1988. Glycolipids and gliomas. Neurochem. Pathol.. S: 157-180. 1988.

26. Sbaschnig-Agler, M, Dreyfus, H., Norton, W. T., Sensenbrenner, M., Farooq,M., Byrne, M. C., and Ledeen, R. W. Gangliosides of cultured astroglia.Brain Res., 461: 98-106, 1988.

27. Seyfried, T. N.. Yu, R. K., and Miyazawa, N. Differential cellular enrichmentof gangliosides in the mouse cerebellum: analysis using neurological mutants.J. Neurochem., 38: 551 -559, 1982.

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1990;50:7444-7449. Cancer Res   Richard Jennemann, Appletree Rodden, Bernhard L. Bauer, et al.   Glycosphingolipids of Human Gliomas

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