Glycolipid GD3 and GD3 synthase are key drivers forglioblastoma stem cells and tumorigenicityShih-Chi Yeha,b,1, Pao-Yuan Wangb,c,d,1, Yi-Wei Loub, Kay-Hooi Khooe, Michael Hsiaob, Tsui-Ling Hsub,and Chi-Huey Wongb,2

aInstitute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan; bGenomics Research Center, Academia Sinica, Taipei115, Taiwan; cChemical Biology and Molecular Biophysics, Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan; dInstitute ofBiochemical Sciences, National Taiwan University, Taipei 106, Taiwan; and eInstitute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan

Contributed by Chi-Huey Wong, March 30, 2016 (sent for review January 30, 2016; reviewed by Xuefei Huang and Valentin Wittmann)

The cancer stem cells (CSCs) of glioblastoma multiforme (GBM), agrade IV astrocytoma, have been enriched by the expressed markerCD133. However, recent studies have shown that CD133− cells alsopossess tumor-initiating potential. By analysis of gangliosides onvarious cells, we show that ganglioside D3 (GD3) is overexpressedon eight neurospheres and tumor cells; in combination with CD133,the sorted cells exhibit a higher expression of stemness genes andself-renewal potential; and as few as six cells will form neuro-spheres and 20–30 cells will grow tumor in mice. Furthermore,GD3 synthase (GD3S) is increased in neurospheres and humanGBM tissues, but not in normal brain tissues, and suppression ofGD3S results in decreased GBM stem cell (GSC)-associated proper-ties. In addition, a GD3 antibody is shown to induce complement-dependent cytotoxicity against cells expressing GD3 and inhibitionof GBM tumor growth in vivo. Our results demonstrate that GD3and GD3S are highly expressed in GSCs, play a key role in glio-blastoma tumorigenicity, and are potential therapeutic targetsagainst GBM.

GBM | ST8SIA1 | gangliosides | glycosphingolipids | cancer stem cells

Glioblastoma multiforme (GBM) is extremely infiltrative anddifficult to treat, and most patients develop recurrence after

therapy. Over the past decade, many studies have suggested thatbulk GBM tumors harbor cancer stem cells (CSCs) (1, 2), adistinct subpopulation of cancer cells that are able to initiate newtumors efficiently, have long-term self-renewal capacity, andsurvive better against chemo- or radiotherapy (2–4). CD133 hasbecome a widely used marker for the enrichment of GBM CSCs(GSCs) and other tumor types (5–10). However, recent studieshave shown that CD133 is not specific for GSCs because CD133−

cells also possess tumor-initiating potential (11–13), indicatingthe need to identify more specific and exclusive markers forGSCs to facilitate our understanding of GSCs and therapeu-tic development against GBM. Several reports have proposedL1CAM, A2B5, integrin α6, MET, and CD15 as markers forGSCs (14–18). However, none of these protein markers could beused specifically to identify GSCs, and no study was reportedwith respect to glycans as potential markers, although glycanbiosynthesis involves multiple genes and it is possible to createdifferent structures in cancer progression. It is noted that gan-glioside D2 (GD2) and ganglioside D3 (GD3) were found on thesurface of neural stem cells (NSCs) and that stage-specific embryonicantigen 3 (SSEA3) and SSEA4 were found on embryonic stem cellsand cancer cells (19–21), but there is no glycan marker found on thesurface of GSCs.Gangliosides are sialic acid-containing glycosphingolipids

(GSLs) that are most abundant in the nervous system (22). Theexpression levels and patterns of gangliosides during brain devel-opment shift from simple gangliosides, such as GM3 and GD3, tocomplex gangliosides, such as GM1, GD1a, GD1b, and GT1b (23,24). Moreover, several unique ganglioside markers, includingSSEA3, SSEA4, GD2, and GD3, have been identified in stem cells(19). GD3, a b-series ganglioside containing two sialic acids, is

highly expressed in mouse and human embryonic NSCs (20, 25).In cancers, GD3 is highly accumulated in human primary mela-noma tissues as well as in established melanoma cell lines (26),whereas human normal melanocytes express no or minimal levelsof GD3 (27). Moreover, malignant gliomas contain higher levelsof GD3, and its expression correlates with the degree of malig-nancy (28). GD3 is produced from the precursor GM3 by theactivity of GD3 synthase (GD3S), which mediates the propertiesof CSCs through the c-MET signaling pathway and correlates withpoor prognosis in triple-negative human breast tumors (29). Thesefindings suggest that GD3 may play an important role in thetransformation of normal cells into tumors, and imply that GD3could be a cell surface marker for GSCs.This study was designed to identify glycan markers for the en-

richment of GBM stem cells and then uses these enriched GBMstem cells to characterize tumorigenicity, their association withclinical GBM specimens, and their regulation in tumor progres-sion. The results showed that GD2 and GD3 were positivelystained on GBM neurospheres. We found that cells with highGD3 expression display functional characteristics of GSCs. Sup-pression of GD3S, a critical enzyme for GD3 synthesis, impededneurosphere formation and tumor initiation. The expression ofGD3S correlated with the grades of astrocytomas and mediatedself-renewal through c-Met activation. Furthermore, a GD3 an-tibody was found to eliminate the GD3+ cells through comple-ment-dependent cytotoxicity (CDC) in vitro and to suppresstumor growth in mice. These results suggest that GD3 could be asignificant biomarker for GSCs, that CD3 could be combinedwith CD133 for the enrichment of GSCs, and that both GD3 and

Significance

Glioblastoma multiforme (GBM) is the most malignant brain tu-mor. The recurrence or chemoresistance of GBM is attributed tothe presence of cancer stem cells (CSCs). Although many studiesindicate that CD133 protein is a biomarker for GBM CSC (GSC)enrichment, CD133 is not specific for GSCs and is also present oncancer cells. In this study, we report that ganglioside D3 (GD3) isnot only an alternative marker but also an additional marker toCD133 for the identification and enrichment of GSCs. We furtherprove that the properties of CSCs in GBM are suppressed whenGD3 synthase is inhibited, supporting GD3 as a GBM stem cellmarker and a promising therapeutic target for GBM treatment.

Author contributions: S.-C.Y., Y.-W.L., M.H., T.-L.H., and C.-H.W. designed research; S.-C.Y.,P.-Y.W., and Y.-W.L. performed research; S.-C.Y., P.-Y.W., Y.-W.L., K.-H.K., and T.-L.H. ana-lyzed data; S.-C.Y., T.-L.H., and C.-H.W. wrote the paper.

Reviewers: X.H., Michigan State University; and V.W., University of Konstanz.

The authors declare no conflict of interest.1S.-C.Y. and P.-Y.W. contributed equally to this work.2To whom correspondence should be addressed. Email: chwong@gate.sinica.edu.tw.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1604721113/-/DCSupplemental.

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GD3S could be targets for the development of new therapiesagainst GBM.

ResultsExpression Levels of Glycan Epitopes Are Evaluated on GBM Cells andNeurospheres. We enriched GBM stem-like cells in a serum-freemedium containing EGF and basic FGF (30) to generate neu-rospheres in four human GBM cell lines: LN18, U251, DBTRG,and LN229 (Fig. 1A and Fig. S1A). All GBM neurospheres wereexamined for GSC characteristics. The mRNA expression lev-els of stemness genes (SOX2, Oct4, NANOG, and NES) and the

reported marker CD133 were dramatically increased in GBMneurospheres (Fig. S1B). The protein expression levels of Oct4,NES, and CD133 were also highly increased in GBM neurospherescompared with parental cells (Fig. S1C). Moreover, the cells dis-sociated fromDBTRG neurospheres showed an increased numberof neurospheres compared with parental cells (Fig. S1D) andpowerful tumor growth and tumor initiation ability in immuno-deficient mice (three tumors generated in three mice) (Fig. S1E).These results suggested that the established culture system for en-riched stem-like cells showed GSC properties in vitro and in vivo.

GD2 and GD3 Are Expressed at High Levels in Various GBM Neurospheres.After the neurosphere system was established, we profiled theglycan-related molecules by flow cytometry and MALDI-MS (Fig.1A). To check the expression levels of glycan epitopes, antibodystaining was conducted and analyzed by flow cytometry on fiveGBM cell lines: LN18, U138MG, U251, DBTRG, and G5T. Theglycan epitopes stained were gangliosides (GM3, GM2, GM1,GD1a, GD3, GD2, GT1b, and A2B5), Lewis antigens [Lex, sialylLex (sLex), and Ley], globoseries GSLs (SSEA-3, SSEA-4, andGlobo H), O-linked glycans [Thomsen–Friedenreich antigen (TF),Tn, and sialyl Tn (sTn)], and stem cell-associated glycoproteins(Table S1). High levels of SSEA4 were observed on most GBMparental cells, but decreased on GBM neurospheres. SSEA3staining was positive on LN18, U138MG, and G5T parental cells,but weak on their neurospheres, and VK9 (anti-Globo H) stainingwas negative on GBM cells. Both GBM parental cells and neu-rospheres showed high levels of TF, Tn, Lex, and Ley; a low levelof sLex; and no sTn. The levels of CD44 and CD90 were abundanton both GBM cells and neurospheres, but CD24 was low onneurospheres and only DBTRG neurospheres displayed CD133staining. With respect to gangliosides, GBM parental cells andneurospheres showed high levels of GM2, GM1, GD1a, and GT1band a low level of GM3 and A2B5. Moreover, GD3 and GD2staining intensity was low on GBM parental cells, but high on GBMneurospheres (Table S1).Because antibodies were not available for every ganglioside

staining, we analyzed the ganglioside profile from DBTRG cellsand DBTRG neurospheres by MALDI-MS (Fig. 1B). Twoceramide isoforms with differences in the sphingosine moiety(C16:0 and C24:0) were commonly detected in human tissues.The respective ganglioside profiles were thus assigned basedon the m/z values of the major molecular ions, adjusted withthe permethylation of hexose (Hex), N-acetylhexosamine, orN-acetylneuraminic acid residues. The MS profiles showed thatthe major species of gangliosides on DBTRG cells was GM2,whereas the most predominant complex gangliosides on DBTRGneurospheres were GM3, GM2, GM1, GD1, GD3, and GD2 (Fig.1B). The result was consistent with the expression profile of var-ious gangliosides determined by flow cytometry using differentantiganglioside antibodies (Table S1). Based on these findings, wefurther examined the levels of gangliosides on additional GBMparental cells and neurospheres by flow cytometry, and found thatGD3 and GD2 expression levels were negative or low on all GBMparental cells, but were relatively high on most of the GBMneurospheres (Fig. S2 A and B).

Isomeric Structures of GM1 and Their Distribution. Of the ganglio-sides examined, GM1 exists as a mixture of structural isomers andcould not be distinguished by MALDI-MS or reverse-phase liquidchromatography (LC)-MS alone and by existing antibodies. Wetherefore developed a method based on porous graphitized car-bon LC-MS (31) to separate the isomers in positive ion mode,which were structurally confirmed and quantified by collision-induced dissociation (CID) MS/MS fragmentation and peak area.Fig. 1C shows a representative demonstration of this method usedin the identification of GM1 isomers in DBTRG cells. The GM1isomers of DBTRG cells are composed of mostly 2-3 sialyl

Fig. 1. Profiling and discovery of glycan markers for GBM stem cells.(A) Glycan-related molecules specifically expressed in GBM neurospheres(stem-like cells) were screened and verified by flow cytometry and MS, re-spectively. The cells carrying these specific glycan markers were enriched fromGBM xenograft tumors and further examined for their abilities of self-renewaland tumorigenicity in vivo. (B) Extracted gangliosides from DBTRG cells andneurospheres were permethylated and analyzed by MALDI-MS. The majorganglioside in DBTRG cells was GM2 (m/z = 1,617.0), whereas the most pre-dominant complex gangliosides in DBTRG neurospheres were GM3 (m/z =1,371.9), GM2 (m/z = 1,617.1), GM1 (m/z = 1,821.2), GD1 (m/z = 2,294.5), GD3(m/z = 1,733.2), and GD2 (m/z = 2,090.4). Gangliosides with the same glycanmoiety but with different fatty acyl contents are bracketed. (C) Isomericstructures of GM1 in DBTRG cells were separated by a porous graphitizedcarbon LC-MS–based platform. The major gangliosides include 2-3 sialyl lac-totetraose (Lc4) (21.4%), 2-3 sialyl neolactotetraose (nLc4) (70.6%), and a smallamount of sialyl-lacto-N-tetraose b (LSTb) (4%) and GM1a (4%). Mono-saccharide symbols were used as follows: yellow circle, galactose; blue circle,glucose; yellow square, N-acetylgalactosamine; blue square, N-acetylglucos-amine; purple diamond, N-acetylneuraminic acid.

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lactotetraose (Lc4) (21.4%), 2-3 sialyl neolactotetraose (nLc4)(70.6%), and small quantity of sialyl-lacto-N-tetraose b (LSTb)(4%) and GM1a (4%). With this method established, it was foundthat the ratio of GM1 isomers differs from one cell to another andcould serve as a characteristic fingerprint of individual cell types.On the contrary, GD3 and GD2 have no isomers found, and theirstructures could be unambiguously confirmed by both MS andexisting antibodies. This platform was also applied to other GBMcells (Fig. S2 C–E).

GD3 and CD133 as Markers for the Enrichment of GSCs. We dissoci-ated the cells with GD3 and related markers from DBTRG xe-nograft tumors that were inoculated in immunodeficient mice. Toinvestigate the correlation between GD3 (or GD2) and CD133expression, we first analyzed the expression levels of CD133 inGD3+ or GD2+ tumor cells. More than 57% of GD3+ tumor cellsdisplayed high CD133 signals, and less than 10% of GD3− tumorcells showed low CD133 signals, whereas 53% of GD2+ tumor cellsand 30% of GD2− tumor cells were positive for CD133, suggestingthat GD3 expression positively correlated with CD133 expression(Fig. 2A). Based on the expression levels of the isotype control, wegated and separated the tumor cells into GD3lo, GD3hi, GD2lo, andGD2hi cells (Fig. S3A), and it was found that GD3hi cells alsoexpressed high levels of the stemness genes SOX2 and NES, andgenerated more neurospheres than GD2hi cells (Fig. 2 B and C).We further investigated the population of GD3hi cells vs. thepopulation of CD133hi or GD3hiCD133hi cells with regard to thecontent of GSCs. GD3hi or CD133hi cells had higher expressionlevels of SOX2 and NES than GD3lo or CD133lo cells, and the cellswith GD3hiCD133hi expression exhibited higher expression levels ofstemness genes than GD3hi or CD133hi cells (Fig. 2D). In addition,GD3hi or CD133hi cells formed spheres more efficiently than thecorresponding GD3lo or CD133lo cells, and more neurospheresformed from GD3hi cells than from CD133hi cells (Fig. 2E). Wealso found that an increased number of neurospheres was observedin wells from CD133hiGD3hi cells, but not from CD133loGD3lo

cells, and that CD133hiGD3hi cells showed a higher potential forself-renewal than GD3hi or CD133hi cells (Fig. 2E). To estimate thefrequency of stem cells in vitro, a limiting dilution assay for neu-rosphere formation was also conducted and computed using theextreme limiting dilution analysis (ELDA) algorithm as describedin Materials and Methods, and it was found that GD3hi or CD133hi

cells required more than 10 cells to form one neurosphere, whereasonly six CD133hiGD3hi cells were enough to form one neurosphere(Table S2). Taken together, these results demonstrated thatGD3hiCD133hi cells are a better representative of GSCs becausethis population has self-renewal ability with expression of stemnessgenes and a higher propensity to form neurospheres.To evaluate the tumorigenicity of glycan molecule-enriched

cells, we dissociated cells from tumors that were inoculated inimmunodeficient mice, and then recapitulated the selected cellsinto immunodeficient mice for evaluation. By monitoring the lu-ciferase activity, we analyzed the potential of tumor initiation andtumor growth. Using 1,000 cells per site, however, there were nosignificant differences in the tumor initiation ability among thesesix populations: GD3hi and CD133hi cells generated tumors fasterthan GD3lo and CD133lo cells, respectively, and CD133hiGD3hi

cells showed the most effective tumor growth among these sixpopulations (Fig. 2F and Fig. S3B). At the level of 50 cells per site,GD3hi, CD133hi, and CD133hiGD3hi cells generated more tumorsat a higher frequency than GD3lo, GD3lo, and CD133loGD3lo

cells, respectively, and CD133hiGD3hi cells generated tumors in allmice (five of five mice). Moreover, at the lower level of 20 cellsper site, there were fewer CD133hiGD3hi cells required for tumorinitiation (two of five mice bearing tumors) compared with theGD3hi or CD133hi population (none of five mice bearing tumors)(Fig. 2F). These data firmly demonstrated that the cells enrichedby CD133 plus GD3 were capable of tumor initiation at a higher

frequency than the cells with GD3 or CD133 only, suggesting thatGD3 is a major determinant of GSCs.Because SSEA3 and SSEA4 were specifically found on em-

bryonic stem cells (19) and breast CSCs (32, 33), as well as on 15different types of cancer cells, including GBM (21), but not onnormal cells, we hypothesized that SSEA3 and SSEA4 would behighly expressed in our defined CD133hiGD3hi GSCs. We ana-lyzed the expression of SSEA3 and SSEA4 in the sorted cellpopulation using CD133 and GD3 as markers, and found thatmore than 31.3% of CD133hiGD3hi cells were SSEA3+ and59.4% were SSEA4+, whereas less than 10% of CD133loGD3lo

cells were positive for SSEA3 and SSEA4 (Fig. S3C). Moreover,SSEA3 or SSEA4 was highly expressed in CD133hiGD3hi cellscompared with other divided cell populations.

GD3S Mediates GSC Characteristics. To understand the relationshipbetween glycosyltransferases and GD3 expression, we analyzedthe mRNA levels of GM3 synthase (GM3S, ST3GAL5), GD3S(ST8SIA1), and GM2/GD2 synthase (GD2S, B4GALNT1)

Fig. 2. GD3 identifies GSCs in GBM tumors. (A) DBTRG tumor cells werecostained with anti-GD2, anti-GD3, and anti-CD133 antibodies. We calculatedthe percentage of cells expressing CD133 in GD2+, GD2−, GD3+, or GD3− cells.(B) Expression levels of SOX2 and NES were examined in sorted cells byquantitative PCR (Q-PCR). Results are shown as mean ± SD (n = 3). (C) Sphereformation assays were performed in sorted cells. The mean ± SD for eachgroup (n = 10) is shown. (D) Q-PCR analyses of stemness genes were performedin tumor cells sorted by the indicated molecules. Results are shown as mean ±SD (n = 3). (E) Self-renewal potential of each subpopulation from DBTRG tu-mors was evaluated in neurosphere formation assays. Cells seeded at a densityof 100 cells per well were represented, and data are shown as mean ± SD (n =10). (F) In vivo limiting dilution assay of separated subpopulations derivedfrom xenograft tumors was conducted in mice. The numbers of tumor cell-injected mice and tumor-bearing mice are shown (20–1,000 cells per mouse,n = 4 or n = 5 mice per group). The P value was determined by an unpairedStudent’s t test between groups (B and C) and by one-way ANOVA for mul-tiple comparisons (D and E). *P < 0.05; **P < 0.01.

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individually in the parental cells and neurospheres. First, it wasfound that GM3S and GD3S, the enzymes involved in the con-version of lactosylceramide to GM3 and GM3 to GD3, respectively(Fig. S4A), were significantly up-regulated in neurospheres (Fig. 3Aand Fig. S4B). In particular, GD3S was significantly up-regulatedwhen GBM cells were cultivated into neurospheres, as shown bythe cell lines LN18, LN229, U251, and DBTRG.GD2S was slightlyincreased in LN18 and LN229 neurospheres, whereas no changesin U251 and DBTRG neurospheres were observed (Fig. S4C).Interestingly, GD3hi cells exhibited the most abundant expressionof GM3S and GD3S in fractionated GD3hi cells from DBTRGtumors (Fig. S4D). To investigate the functional role of GD3 inGSCs, we suppressed the expression ofGD3S in DBTRG cells witha lentiviral shRNA expression vector or enhanced the expression ofGD3S using a pcDNA3 expression vector. As expected, the GD3Sknockdown (KD) showed no effect on parental cells with no de-tectable GD3, whereas the expression ofGD3S and the percentageof GD3+ cells were significantly reduced from 63.9 to 9.06% inDBTRG neurospheres (Fig. S4 E and F). GD3S overexpression(O/E) cells displayed strong intensities of GD3S and GD3, andwere further enhanced in neurospheres (Fig. S4 G and H). Theseresults suggested that the expression level of GD3 is regulated byGD3S in GSCs. Interestingly, both GD3S KD and GD3S O/E

caused the down- or up-regulated expression of stemness genes inneurospheres in comparison to parental cells (Fig. 4 B and C). Inaddition, suppression of GD3S inhibited neurosphere formation,but had no effect on parental cell growth (Fig. 3D and Fig. S4I).Adversely, O/E of GD3S increased neurosphere formation andpromoted cell growth (Fig. 3D and Fig. S4J). Moreover, micebearing GD3S shRNA cells showed significantly reduced tumorgrowth (Fig. 3E) and tumor formation (Table S3). Even after 20wk, 10,000 GD3S shRNA cells had no tumor formation, whereasthe control shRNA cells generated tumors in two of four mice.Adversely, mice bearing GD3S O/E plasmid showed increasedtumor size and tumor initiation compared with the control on theindicated days (Fig. 3E and Table S3). Taken together, thesefindings demonstrated that GD3S is necessary for GSCs in vitroand in vivo.

GD3S Is Highly Expressed in GBM Specimens.GD3S has been reportedto be a potential therapeutic target for inhibiting breast cancerinitiation (34), but the expression status of GD3S in GBM tissueswas unclear. To investigate whether GD3S was overexpressed inclinical GBM specimens, we analyzed the expression of GD3S ingrade I–IV astrocytomas and in normal brain tissues by screeninghuman tissue arrays using immunohistochemistry. The resultsshowed that most normal brain tissues were GD3S− (Fig. 4A), andGD3S was strongly located in the cytoplasm of GBM cells (Fig. 4B).Furthermore, the statistical results indicated that 38 of 46 GBMtissues (84.8%) were GD3S+ and more than half of the GBM spec-imens (60.8%) were intensely stained, with a score of 2+ or higher(Fig. 4C). On the contrary, 57.1% of low-grade astrocytoma speci-mens were weakly stained (scored as 1+) by GD3S antibody, andthe score of GD3S intensity was positively correlated with thegrades of astrocytomas. These results demonstrated that GD3S ishighly expressed in GBM tumors.

GD3S Regulates Sphere Formation Through Activation of c-Met.Previous studies showed that GD3S enhances the proliferationand tumor growth of MDA-MB-231 cells through c-Met signal-ing (35); hence, we analyzed the expression of phosphorylatedc-Met (p-c-Met) in neurospheres, GD3S O/E, and GD3S KDcells. Interestingly, we observed no change in total c-Met andincreased p-c-Met in neurospheres compared with their parentalcells, and decreased p-c-Met in neurospheres expressing GD3SKD relative to their KD control counterparts (Fig. 5A). More-over, the p-c-Met was strongly expressed in the GD3S O/E cellscompared with O/E control cells (Fig. 5B), suggesting that GD3Sregulates only the function of c-Met and not its expression. To

Fig. 3. Manipulation of GD3S mediates stemness genes, sphere forma-tion, and tumor initiation. (A) The expression level of GD3S in DBTRGparental cells and neurospheres was measured by Q-PCR. (B and C) Theexpression levels of stemness genes were determined in neurospheres thatexpressed vector control, GD3S KD (B), or GD3S O/E (C ) plasmid. Results areshown as mean ± SD (n = 3). (D) Neurosphere formation assays usingvector control, GD3S KD, or GD3S O/E cells were performed in 96 wells. Themean ± SD for each group (n = 10) is shown. (E ) Tumor growth generatedfrom 105 vector control, GD3S KD, or GD3S O/E cells was monitored using acaliper to measure the tumor size every 4 d between 8 and 12 wk (n = 4mice per group). (Right) Gross view of isolated tumors. (Scale bars, 1 cm.)The P value between groups was determined by an unpaired Student’s ttest. *P < 0.05; **P < 0.01.

Fig. 4. Expression of GD3S in GBM tissues. Representative images of normalbrain tissues (A) and GBM (B) after immunohistochemical staining. (Scalebars, 20 μm.) (C) Statistical results of GD3S immunohistochemistry. Grade I(n = 9), grade II (n = 12), grade III (n = 7), grade IV (GBM, n = 46), and normalbrain tissues (n = 10) were counterstained with hematoxylin after immu-nohistochemistry. The staining intensity of the tissues was scored as 0 (neg-ative), 1+ (weak), 2+ (moderate), and 3+ (strong).

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address whether sphere formation regulated by GD3S was throughactivation of c-Met, we treated the GD3S O/E cells with SU11274,a c-Met inhibitor, and found that either 1 μM or 10 μM SU11274was capable of reducing the p-c-Met expression (Fig. 5B) and thesphere formation capacity of O/E control cells and GD3S O/Ecells (Fig. 5C), indicating that c-Met signaling functions down-stream of GD3S.

GD3 Antibody Mediates CDC Against GBM Cells and SuppressesTumor Growth in Vivo. To examine whether targeting GD3would trigger CDC in GBM cells, cells were treated with a GD3antibody (R24) and rabbit complement. It was found that in thepresence of complement, R24 reduced the number of viableGBM cells significantly (Fig. S5A). We observed an obvious R24-mediated CDC in GD3-expressed GBM cell lines and not in theGBM cells, which expressed no detectable GD3. Therefore, thelevel of R24-mediated CDC positively correlated with the ex-pression level of GD3 in GBM cells.To examine if R24 was able to suppress GBM tumor growth

in vivo, R24 was administered to nude mice inoculated s.c. withDBTRG cells when the tumor size reached 15–30 mm3 at day 28postinjection. The experiment showed that the administration ofR24 could suppress DBTRG tumor growth (Fig. S5B). In com-parison to mice receiving R24 treatment, DBTRG tumors grewaggressively in the control group treated with mouse IgG3. Theseresults indicated that R24 can suppress the growth of GBM tumors.

DiscussionGBM displays a complex genetic and remarkable heterogeneity,so it is unlikely that the expression of a single marker can ab-solutely enrich and define GSCs in every tumor; hence, a com-bination of markers to enrich GSCs specifically and sufficiently isnecessary. CD133 has been proven not to be a definitive markerfor GSCs because CD133 expression is not detectable in manyglioma cell lines and fresh GBM specimens (11, 12, 16), and bothCD133− and CD133+ cells separated from the same tumorspecimen can be cultured as neurospheres and are able to self-renew and initiate tumor formation (13). In addition, the tumorinitiation assay required the injection of 100 or more CD133+

cells in immunodeficient mice. In this study, we evaluated theexpression levels of glycan-related molecules in various GBMneurospheres and found the ganglioside GD3 is highly expressedin neurospheres. Moreover, the cells segregated by GD3, or incombination with CD133, showed a high self-renewal ability androbust tumor initiation potency.Of all the gangliosides examined, neurospheres displayed the

highest GD2 expression and medium GD3 expression in flowcytometry, but they showed similar expression levels of GD2 andGD3 in MALDI-MS assays, indicating that the antibody-bindingaffinity for GD2 and GD3 is different. The functional role of

GD3 in GSCs is not clear. Recent studies suggest that ganglio-sides are widely expressed on tumor cells (36) and disialyl gan-gliosides enhance tumor phenotype with multiple modalities(37). These reports demonstrate that GD3 promotes cell growthand invasion through phosphorylation of p130Cas, paxillin, andfocal adhesion kinase (FAK) in melanoma cells (38). Further-more, the Src family kinase Yes was found to link with p130Casor FAK as an activated form to promote malignant properties ofhuman melanoma cells expressing GD3 (37, 39). It was alsorevealed that gangliosides, including GD1a, GD1b, GD3, andGM3, may facilitate tumor cell escape from the immune systemwithin the tumor microenvironment (40). It has been shown thatGM3 and GD3 induce apoptosis in immune cells, includingnatural killer (NK) and T cells (41). The b-series gangliosides,including GD3 and GD2, but not other ganglioseries ganglio-sides, were shown to bind to sialic acid-binding Ig-like lectin 7(Siglec-7), an inhibitory receptor on human NK cells, and thatcells that engineered to overexpress GD3S inhibit NK cell-modulated cytotoxicity via a Siglec-7–dependent mechanism (42).Considering the fact that CSCs have to overcome a complex im-mune system to survive and form tumors in the harsh microenvi-ronment, the immunosuppressive function of GD3 may be criticalfor their tumorigenicity.We previously showed that the globoseries glycolipids SSEA3

and SSEA4 are highly expressed on GBM cells and that an anti-SSEA4 antibody was effective against GBM (21). As a tumor-associated antigen, GD3 has been an attractive target in immu-notherapy (43). The anti-GD3 mAb R24 has been described tomediate in vitro effector functions, including CDC and antibody-dependent cell-mediated cytotoxicity (ADCC), and to suppresstumor growth of melanoma in mice models, and it was used forpatients with melanoma (44, 45). We found that R24 also showeda strong CDC effect on the GBM cells expressing high GD3, butnot on the cells with low expression, suggesting that R24 could beused in GBM immunotherapy.We also noted that the key enzyme regulating the expression

levels of GD3 or GD2 is GD3S, but not GD2S, even thoughGD2S is the intermediate enzyme converting GD3 to GD2. Inaddition, the expression of GD3S mRNA is up-regulated invarious GBM neurospheres and in GD3hi cells from GBM xe-nograft tumors. Recently, clinical studies showed that high ex-pression of GD3S was found in estrogen receptor (ER)-negativebreast cancer and was associated with poor histological grade inER-negative tumors (29). GD3S can enhance proliferation ofMDA-MB-231 breast cancer cells through the constitutive acti-vation of the c-MET receptor and downstream mitogen-acti-vated protein kinase/extracellular signal-regulated kinase andphosphoinositide-3 kinase/Akt signaling pathways (35). With respectto breast CSCs, GD3S not only regulates epithelial-mesenchymaltransition and CSC properties but also metastasis in vivo (46). In

Fig. 5. GD3S regulates sphere formation through activation of c-Met signaling. (A) Western blot analysis of p-c-Met and c-Met expression in DBTRG parentalcells, neurospheres, and both of them transfected with KD control and GD3S KD plasmid. (B) Expression of p-c-Met and c-Met in DBTRG GD3S O/E cells treatedwith or without SU11274. (C) SU11274 treatment in DBTRG O/E control and DBTRG GD3S O/E cells was used for sphere formation assay. The mean ± SD foreach group (n = 10) is shown. The P value was obtained by one-way ANOVA. Ctrl, control; SU, SU11274. *P < 0.05; **P < 0.01.

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summary, these findings uncovered a significant role of GD3 andthe enzyme GD3S in GBM, and GD3 can be combined with CD133for the enrichment of GSCs, suggesting GD3 and GD3S as thera-peutic targets against GSCs and GBM.

Materials and MethodsNeurosphere Formation Assay. Cultured cells or tumor cells (xenograft tu-mors) were trypsinized, and single-cell suspensions with the indicated cellnumber (5, 20, 50, and 100 cells per well) were cultured in 96-well ultra-lowattachment plates (Corning, USA) containing Neurobasal medium (Invi-trogen) supplemented with B27 (Invitrogen), 20 ng/mL EGF, and 20 ng/mLbasic FGF (Peprotech). After 20 d, the number of wells with neurosphereswas quantified. For secondary neurosphere formation assay, the estab-lished neurospheres were dissociated into single cells and were cultured in96-well ultra-low attachment plates. The number of wells with neuro-spheres >100 μm in diameter was counted, and the frequency for sphereformation was generated and computed using the ELDA online algorithm(47) (bioinf.wehi.edu.au/software/elda/).

Tumor Formation in Vivo. Four-week-old nonobese diabetic-scid IL2rγnull micewere obtained and maintained under specific pathogen-free conditions atthe Genomics Research Center of Academia Sinica. Procedures involvinganimals and their care were conducted according to the protocols of theAcademia Sinica Institutional Animal Care and Utilization Committee incompliance with national and international laws and policies. For xenograft

tumor preparation, DBTRG cells with or without the luciferase gene (1 × 107

cells in 200 μL of PBS) were injected into the flank regions of mice (around4 wk old), and mice were killed when the tumor size reached 1 cm3. Singlecells were harvested from the tumors, mechanically minced, and enzymati-cally treated with RPMI medium supplemented with 10% (vol/vol) FBS, 200U/mL collagenase IV, and 0.6 U/mL dispase. For the limiting dilution assay oftumorigenicity, DBTRG tumor cells (six partitioned cell populations) carryingthe luciferase gene were injected into the flank regions of mice (around4 wk old) with the indicated cell number to generate the tumor, and tumorgrowth was monitored by bioluminescence every 4 d using the IVIS 200imaging system (PerkinElmer). For the function of GD3S in tumor growth,DBTRG cells carrying the KD control or GD3S KD were injected into the flankregions of mice; the tumor size was determined using a vernier caliper bymeasuring the length (L) and width (W), and the tumor volume was calcu-lated (in cubic millimeters) as 1/2 × LW2. For tumor growth inhibited by R24,BALB/c nude mice were purchased from the National Laboratory AnimalCenter (Taiwan) and maintained under specific pathogen-free conditions.DBTRG cells (107 cells per 200 μL of PBS) were injected s.c. into the flankregions of 4-wk-old mice to generate the xenograft model. On days 28, 32,36, and 40, each mouse was injected i.p. with 150 μg of R24 or with mouseIgG3 isotype antibody.

ACKNOWLEDGMENTS. We thank Dr. Hua-Chien Chen for providing variouscancer cell lines. We also thank Dr. Chia-Ning Shen, Dr. Patrick C. H. Hsieh,and Tsung-Ching Lai for assistance with experiments. This research wassupported by the Genomics Research Center, Academia Sinica, Taiwan.

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