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Keywords:

  • Oligodendroglioma;
  • Astrocytoma;
  • N-Glycolylneuraminic acid;
  • Brain tumor;
  • Asialo-GM1

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSLs
  5. High-performance TLC (HPTLC)
  6. TLC immunostaining
  7. RESULTS
  8. DISCUSSION
  9. Acknowledgements
  10. References

Abstract: Ganglioside sialic acid content was examined in the U87-MG human glioma grown as cultured cells and as a xenograft in severe combined imunodeficiency (SCID) mice. The cultured cells and the xenograft possessed N-glycolylneuraminic acid (NeuGc)-containing gangliosides, despite the inability of human cells to synthesize NeuGc. Human cells express only N-acetylneuraminic acid (NeuAc)-containing gangliosides, whereas mouse cells express both NeuAc- and NeuGc-containing gangliosides. Small amounts of NeuGc ganglioside sialic acid (2-3% of total ganglioside sialic acid) were detected in the cultured cells, whereas large amounts (66% of total ganglioside sialic acid) were detected in the xenograft. The NeuGc in gangliosides of the cultured cells was derived from gangliosides in the fetal bovine serum of the culture medium, whereas that in the U87-MG xenograft was derived from gangliosides of the SCID host. The chromatographic distribution of U87-MG gangliosides differed markedly between the in vitro and in vivo growth environments. The neutral glycosphingolipids in the U87-MG cells consisted largely of glucosylceramide, galactosylceramide, and lactosylceramide, and their distribution also differed in the two growth environments. Asialo-GM1 (Gg4Cer) was not present in the cultured tumor cells but was expressed in the xenograft, suggesting an origin from infiltrating cells (macrophages) from the SCID host. The infiltration of mouse host cells and the expression of mouse sialic acid on human tumor cell glycoconjugates may alter the biochemical and immunogenic properties of xenografts.

Glycosphingolipids (GSLs) are glycoconjugates present on the outer leaflet of the cell membrane and consist of a lipophilic ceramide conjugated to a hydrophilic oligosaccharide chain. GSLs are classified into two major groups: gangliosides and neutral glycosphingolipids (NGSLs). The presence of sialic acid on gangliosides distinguishes them from NGSLs. Sialic acids are negatively charged derivatives of neuraminic acid, which function in receptor mechanisms, cell—cell interactions, and other cellular and molecular processes (Jeanloz and Codington, 1976; Schauer, 1985, 1988; Hakomori, 1996; Varki, 1997). Human gangliosides possess only the N-acetylneuraminic acid (NeuAc) form of sialic acid, whereas mouse gangliosides possess two forms of sialic acid, NeuAc and N-glycolylneuraminic acid (NeuGc) (Yu and Ledeen, 1970; Seyfried et al., 1978a, 1987; Chou et al., 1998). NeuAc differs from NeuGc in having an acetyl group instead of a glycolyl group attached to the nitrogen on carbon number 5 (Schauer, 1985). The absence of NeuGc-containing gangliosides in human tissues arises from a partial deletion in the gene that encodes cytidine monophospho-N-acetylneuraminic acid hydroxylase, which is necessary for synthesizing NeuGc from the precursor NeuAc (Chou et al., 1998; Irie and Suzuki, 1998).

The GSL composition of brain tumors differs markedly from that of normal brain. This arises from GSL abnormalities in proliferating tumor cells and from tumor-infiltrating host cells (Seyfried et al., 1996, 1998). The enrichment of GSLs on the cell surface and their considerable structural diversity make them potential targets for tumor immunotherapy and diagnosis (Irie and Morton, 1986; Murray et al., 1994, 1996; Sung et al., 1994; Wikstrand et al., 1994a, b; Suetake et al., 1995; Uttenreuther Fischer et al., 1995; Nishinaka et al., 1996). The utility of GSLs for tumor diagnosis and therapy will depend to a large extent on their origin and cellular location. In this regard, it is essential to understand the influence of growth environment on tumor GSLs.

Fredman and co-workers (Mansson et al., 1986; Fredman, 1988) originally showed that NeuGc is the predominant sialic acid present on gangliosides isolated from a human glioma grown as a xenograft in nude mice. Kawashima and co-workers (1993) suggested that some NeuGc-containing gangliosides found in human melanomas grown as nude mouse xenografts were derived from gangliosides in mouse plasma. In this report, we show that GSLs from the mouse host can significantly alter the content and distribution of gangliosides and NGSLs of a human brain tumor grown as a xenograft in severe combined immunodeficiency (SCID) mice. The implications of these findings for the diagnosis and therapy of brain tumors are discussed.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSLs
  5. High-performance TLC (HPTLC)
  6. TLC immunostaining
  7. RESULTS
  8. DISCUSSION
  9. Acknowledgements
  10. References

Mice

The C57BL/6J-scid/SzJ (B6-SCID) mice were obtained from the Jackson Laboratory (Bar Harbor, ME, U.S.A.) and were propagated in the Boston College Animal Care Facility. Animal husbandry conditions were as previously described (Flavin et al., 1991). The mice were housed in plastic microisolator cages placed in a horizontal positive airflow system (Stay-Clean System B 30460; Lab Products). B6-SCID mice, ∼2-3 months of age, were used as tumor recipients. All animal procedures were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the institutional animal care committee.

Tumors

The human cell line U87-MG was originally derived from a malignant glioma (glioblastoma) (Ponten and Macintyre, 1968; Beckman et al., 1971). The cultured cells were grown in T-25 or T-75 culture flasks (Corning Costar Corp.) with Dulbecco’s modified Eagle’s medium (Life Technologies) containing 10% fetal bovine serum (Hyclone Laboratories) and 0.1% penicillin/streptomycin (5,000 U/ml each; Life Technologies). The culture medium was replaced once every 2 days. Cells were maintained at 37°C with 5% CO2 and 100% humidity in a Cellstar Laboratory CO2 incubator (Queue Systems). Confluent cells were removed from plates with trypsin/EDTA (0.05% trypsin with 0.53 mM EDTA; Life Technologies). In addition to cell culture, the tumors were maintained in B6-SCID mice by serial subcutaneous injections in the flank as previously described (Cotterchio and Seyfried, 1993). At the time of collection, the tumors reached a volume of ∼2 cm3.

GSLs

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSLs
  5. High-performance TLC (HPTLC)
  6. TLC immunostaining
  7. RESULTS
  8. DISCUSSION
  9. Acknowledgements
  10. References

The solid tumors (in vivo) or the cultured tumor cells (in vitro) were frozen at —80°C and lyophilized. After determining the dry weight, total lipids were extracted by placing the samples in 5 ml of chloroform/methanol (1:1 vol/vol) with 0.5 ml of H2O and stirring for 12 h at 22-25°C. The total lipid extract was separated into upper and lower phases by Folch partitioning (Folch et al., 1957). The upper phase (gangliosides) was applied to A-25 Sephadex ion-exchange columns (Pharmacia), as described previously (Seyfried et al., 1978b, 1987). The gangliosides were treated with 1 ml of 0.5 M aqueous NaOH for 1.5 h at 37°C. The ganglioside samples were desalted using C-18 reverse-phase Bond Elut columns (Varian) and were further purified by placing them over Sephadex LH-20 columns (Pharmacia), as described previously (Brigande et al., 1998). The lower phase (neutral lipids) was applied to A-25 Sephadex ion-exchange columns, followed by treatment in 1 ml of 0.5 M methanolic NaOH for 1.5 h at 37°C. The samples were applied to Sephadex LH-20 columns to remove base. The NGSLs were further purified by solubilizing them in 1 ml of chloroform and applying them to Iatrobeads silicic acid columns (Wako Pure Chemicals), as described previously (Seyfried and Ariga, 1992; Brigande et al., 1998).

High-performance TLC (HPTLC)

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSLs
  5. High-performance TLC (HPTLC)
  6. TLC immunostaining
  7. RESULTS
  8. DISCUSSION
  9. Acknowledgements
  10. References

Gangliosides and NGSLs were spotted on 10 × 10-cm E. Merck silica gel 60 HPTLC plates using a Camag Linomat III TLC spotter (Camag Scientific), as we described (Seyfried et al., 1994). The HPTLC solvent systems used are described in the figure legends. NGSLs were also spotted on plates pretreated for 30 s with 2.5% Na2B5O7 in methanol to separate glucosylceramide (GlcCer) from galactosylceramide (GalCer) (Kean, 1966; Brigande et al., 1998). Ganglioside and NGSL standards were received as gifts from Dr. Robert Yu (Medical College of Virginia, Richmond, VA, U.S.A.) or obtained from Matreya Inc. GSLs were visualized by spraying plates with resorcinol reagent (gangliosides) or orcinol reagent (NGSLs) and heating to 100°C for 30 min (Seyfried et al., 1978b; Sekine et al., 1984; Seyfried and Ariga, 1992).

TLC immunostaining

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSLs
  5. High-performance TLC (HPTLC)
  6. TLC immunostaining
  7. RESULTS
  8. DISCUSSION
  9. Acknowledgements
  10. References

Immunostaining for Gg4Cer (asialo-GM1) was performed on Polygram Sil G plastic-backed plates (Macherey-Nagel), as previously described (Hirabayashi et al., 1990; Seyfried et al., 1994). Gg4Cer antiserum was obtained from Dr. Robert Yu. Peroxidase-conjugated anti-rabbit IgG secondary antibodies (Sigma) were used to visualize Gg4Cer.

Quantitation of ganglioside sialic acid

Gangliosides NeuAc and NeuGc were quantitated using gas chromatography according to previously described methods (Yu and Ledeen, 1970). Sialic acid concentration was expressed as micrograms per 100 milligrams of dry weight.

Neuraminidase treatment

Purified tumor gangliosides were treated with neuraminidase, as previously described (Seyfried et al., 1994). In brief, gangliosides were dissolved in 0.1 M sodium acetate buffer (pH 5.0) and mixed with Clostridium perfringens neuraminidase (1 U/ml; Sigma grade V) with no added detergent. The mixture was incubated overnight at 37°C. The reaction products were purified on C-18 reverse-phase Bond Elute columns and were analyzed by HPTLC with chloroform/methanol/water (65:35:8 by vol) containing 0.02% CaCl2.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSLs
  5. High-performance TLC (HPTLC)
  6. TLC immunostaining
  7. RESULTS
  8. DISCUSSION
  9. Acknowledgements
  10. References

The influence of the growth environment on the ganglioside content of the U87-MG glioma is shown in Table 1. In striking contrast to the cultured cells where NeuGc constituted only ∼2.5% of the ganglioside sialic acid, NeuGc constituted almost 66% of the ganglioside sialic acid content of the xenograft. The ratio of NeuAc to NeuGc was only 36.4 in the cultured cells, but it was 0.53 in the xenograft. These findings indicate that growth environment significantly influenced the ganglioside sialic acid composition of the U87-MG glioma.

Table 1. Influence of growth environment on ganglioside content in U87-MG glioma
  Ganglioside neuraminic acid content (μg/100 mg of dry wt)
Growth environmentaTotalNeuAcNeuGc% NeuGc
  1. Values are expressed as means ± SEM from three independent samples.

  2. aThe tumors were grown subcutaneously in the flanks of B6-SCID mice or as cultured cells.

B6-SCID47.4 ± 6.916.4 ± 3.031.0 ± 4.665.6 ± 3.9
Cell culture59.9 ± 4.858.3 ± 4.11.6 ± 0.72.5 ± 1.1

The U87-MG cultured cells expressed GM3-NeuAc and GM2-NeuAc as major gangliosides with lower amounts of GD3 and GD1b (Fig. 1). Most of the gangliosides migrated as double bands due to the structural heterogeneity of the ceramide. In contrast to the ganglioside pattern of the cultured cells, the pattern of the U87-MG xenograft contained greater proportions of the more structurally complex gangliosides and smaller proportions of GM3 and GM2. Furthermore, the xenograft GM3 and GM2 gangliosides migrated with NeuGc-containing standards rather than with NeuAc-containing standards (Fig. 1).

image

Figure 1. HPTLC of gangliosides in the U87-MG human glioma grown as cultured cells (Vitro) and as a solid tumor subcutaneously in the flank of a B6-SCID mouse (Vivo). Std1, NeuAc-containing ganglioside standards; Std2, NeuGc-containing GM3 and GM2. The arrowheads and arrows indicate the locations of GM3 and GM2, respectively. Approximately 2 μg of ganglioside sialic acid was spotted for each lane. The plate was developed by one ascending run with chloroform/methanol/water (55:45:10 by vol) containing 0.02% CaCl2.

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Most of the complex xenograft gangliosides were neuraminidase sensitive and produced GSL products migrating with lactosylceramide (LacCer), GM2, and GM1 in the neutral solvent system (data not shown). In addition, a significant NGSL band was produced that migrated on the HPTLC slightly faster than GM3 in the region of asialo-GM1/GM2. These results suggest that the gangliosides in the xenograft comprised in part GM3, GD3, GD2, and gangliosides with a gangliotetraose backbone. In addition, the neuraminidase digestion of GM1b and GD1α, derived from tumor host-infiltrating macrophages, could contribute to the asialo-GM1 band (Ecsedy et al., 1998). Besides these host cell gangliosides, we do not exclude the possibility that some of the NGSL in the asialo-GM1/GM2 region is derived from digestion of tumor cell lactotetraose series gangliosides, as previously described (Mansson et al., 1986; Fredman et al., 1990).

The NGSLs of the U87-MG cultured cells comprised GlcCer, GalCer, LacCer, and several more complex structures (Fig. 2A and B). As seen for the gangliosides, many of the NGSLs appeared as double bands due to structural heterogeneity of the ceramide. Unlike the gangliosides, no migration variations were observed for the NGSLs between the cultured cells and the xenograft NGSLs. However, there were differences in relative distribution. For example, the xenograft contained lower amounts of GlcCer and Gb3Cer and greater amounts of GalCer relative to the cultured cells (Fig. 2A and B). In addition, Gg4Cer (asialo-GM1) was a prominent species in the xenograft but was undetectable in the cultured cells (Figs. 2A and 3).

image

Figure 2. HPTLC of NGSLs in the U87-MG human glioma grown as cultured cells (Vitro) and as a solid tumor subcutaneously in the flank of a B6-SCID mouse (Vivo). Std1, GalCer, LacCer, Gb3Cer, and Gb4Cer; Std2, GlcCer, Gg3Cer (asialo-GM2), and Gg4Cer (asialo-GM1). Equal amounts of NGSL were spotted for each lane. The plate was developed by one ascending run with chloroform/methanol/water (65:35:8 by vol) containing 0.02% CaCl2 (A), or the plate was pretreated with 2.5% Na2B4O7 in methanol and was developed by one ascending run with chloroform/methanol/25% ammonium hydroxide/water (65:35:4:4 by vol) (B).

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image

Figure 3. TLC immunostaining for Gg4Cer in the U87-MG human glioma grown as cultured cells (Vitro) and as a solid tumor subcutaneously in the flank of a B6-SCID mouse (Vivo). Aliquots of NGSL equivalent to 3 × 106 cells were spotted for the U87-MG lanes. Gg4Cer and Gg3Cer were used as positive and negative controls, respectively. The plate was developed by one ascending run with chloroform/methanol/water (65:35:8 by vol) containing 0.02% CaCl2.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSLs
  5. High-performance TLC (HPTLC)
  6. TLC immunostaining
  7. RESULTS
  8. DISCUSSION
  9. Acknowledgements
  10. References

Despite the inability of human cells to synthesize NeuGc, our results showed that NeuGc is the major sialic acid expressed on gangliosides of the human U87-MG glioma when grown as a xenograft in SCID mice. These results support the previous findings of Fredman and co-workers (Mansson et al., 1986; Fredman, 1988) with the human D54MG glioma grown as a xenograft in nude mice. In marked contrast to the U87-MG xenograft, only trace levels of NeuGc-containing gangliosides were found in the U87-MG cultured cells. Previous studies showed that low levels of NeuGc-containing gangliosides in cultured mouse and human tumor cells were derived exogenously from NeuGc-containing gangliosides in bovine serum (Hof and Faillard, 1973; Furukawa et al., 1988; El-Abbadi and Seyfried, 1994; Seyfried et al., 1998). Kawashima and co-workers (1993) also found that NeuGc-containing gangliosides in human melanoma xenografts grown in nude mice were derived from the uptake and metabolism of NeuGc-containing gangliosides present in the nude mouse plasma. As NeuGc accounts for >90% of the ganglioside sialic acid content in mouse plasma (Cotterchio and Seyfried, 1994), much of the NeuGc present in the gangliosides in the U87-MG xenograft is likely derived from the uptake and metabolism of gangliosides present in mouse serum.

The addition of CMP is necessary for the activation of sialic acid as a donor substrate for sialyltransferases. This reaction is catalyzed by CMP-sialic acid synthase, which recognizes both NeuAc and NeuGc as precursors for the formation of CMP-sialic acid (Kean, 1991). Furthermore, mammalian sialyltransferases can utilize equally well CMP-NeuAc and CMP-NeuGc as donor substrates for the synthesis of sialylated glycoconjugates (Higa and Paulson, 1985; Muchmore et al., 1989; Hamamoto et al., 1995). Muchmore and co-workers (1989) previously described the pathways by which exogenous NeuGc can be utilized in the synthesis of glycoconjugates. The U87-MG cells can therefore activate NeuGc acquired from mouse plasma and can add it to partially synthesized glycoconjugates.

Trace amounts of NeuGc-containing gangliosides have been detected in some human tissues and in several types of naturally occurring human tumors (Ohashi et al., 1983; Higashi et al., 1985; Hirabayashi et al., 1987a; b; Miyake et al., 1990; Kawai et al., 1991; Marquina et al., 1996; Chou et al., 1998). As NeuGc is not actively synthesized by human cells, Schauer (1985) suggested that the expression of NeuGc on human glycoconjugates may be derived from dietary sources. Indeed, bovine and porcine tissues, which are consumed by humans, contain significant amounts of NeuGc-containing gangliosides (Yu and Ledeen, 1970; Ariga et al., 1983; Suzuki et al., 1985). It is therefore possible that the low levels of NeuGc found on human tumor glycoconjugates are derived exogenously from the diet. Based on our findings, we predict that NeuGc-containing gangliosides will be incorporated more into neoplastic tumor cells than into normal cells. Hence, treating patients with NeuGc may result in the formation of tumor-specific gangliosides that could be targeted for tumor therapy.

In addition to acquisition from mouse plasma, some of the NeuGc-containing gangliosides in the U87-MG xenograft are also derived from tumor-infiltrating cells of the SCID mouse host. Although SCID mice lack T cells and B cells, they contain a normal complement of macrophages (Seyfried et al., 1998). Mouse macrophage gangliosides contain both NeuGc and NeuAc and contribute to the total GSL composition of brain tumors (Seyfried et al., 1996, 1998; Ecsedy et al., 1998). The enrichment of asialo-GM1 (Gg4Cer) in the xenograft but its absence in the cultured cells also reflect the presence and absence, respectively, of tumor-infiltrating macrophages from the mouse host. We recently showed that asialo-GM1 is a major NGSL of activated mouse macrophages and that its expression in mouse brain tumors is correlated with the presence of tumor-infiltrating macrophages (Seyfried et al., 1996; Ecsedy et al., 1998). Our findings therefore suggest that activated mouse macrophages contribute to the GSL composition of the U87-MG xenograft.

Although tumor-infiltrating host cells contribute to the ganglioside composition of the xenograft, it is unlikely that these cells account for the major differences in ganglioside composition between the U87-MG grown in vivo and in vitro. NeuAc-containing GM1a, GM1b, and GD1α, which are prominent gangliosides in mouse immune cells (Ecsedy et al., 1998), were not major species in the xenograft. As the U87-MG is not highly vascularized, it is also unlikely that endothelial cells contribute significantly to the total tumor ganglioside composition. We suggest that the NeuGc expressed on the gangliosides in the U87-MG xenograft is derived more from the mouse plasma than from the tumor-infiltrating host cells.

The substitution of NeuAc for NeuGc caused significant shifts in the chromatographic mobility of gangliosides in the xenograft. These shifts arise from the slower migration of NeuGc- than NeuAc-containing gangliosides on HPTLC (Seyfried et al., 1987, 1992). However, sialic acid-switching and tumor-infiltrating host cells cannot account for all of the changes in ganglioside distribution observed between the U87-MG cultured cells and the xenograft. For example, GM3-NeuAc and GM2-NeuAc were major ganglioside species in the cultured U87-MG cells, but their NeuGc counterparts were minor species in the xenograft. Fredman and co-workers (Mansson et al., 1986; Fredman, 1988; Fredman et al., 1990) previously reported that xenograft-grown human gliomas expressed lactotetraose series gangliosides that were not present in the cultured glioma cells. Furthermore, they later showed that the in vivo growth environment of the nude mouse host could alter ganglioside biosynthetic gene expression in the D54MG human glioma xenograft (Fredman et al., 1996). These findings in human gliomas, together with those in human melanomas, indicate that the growth environment of immunodeficient nude or SCID murine hosts causes striking changes in the content and composition of xenograft gangliosides.

Most of the major NGSLs were similar in the U87-MG cultured cells and in the xenograft, but the relative distribution of these differed markedly in the two growth environments. Our findings of GalCer expression in the U87-MG glioma support previous studies in this tumor (Joshi and Mishra, 1992). GalCer expression is uncommon in human gliomas but is a reliable marker for oligodendrocytes (Kennedy et al., 1987; Westphal et al., 1988; Louis et al., 1992). It is therefore possible that U87-MG may be an oligodendroglioma or may share lineage with oligodendrocytes.

The substitution of human sialic acid for mouse sialic acid on human tumor cells and the interaction of these tumor cells with mouse macrophages make the xenograft a bizarre biological system. As changes in cell surface sialic acid are associated with changes in cell adhesion, differentiation, and antigenicity, the expression of mouse sialic acid on human tumor cells may significantly change the biochemical and immunogenic properties of the tumor cell glycocalyx. It has been well documented that the response of xenografts to various tumor therapies can differ markedly from that of cultured tumor cells or from that of naturally occurring human tumors (Gura, 1997). Our findings may be relevant to the capricious response of xenografts to certain therapies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. GSLs
  5. High-performance TLC (HPTLC)
  6. TLC immunostaining
  7. RESULTS
  8. DISCUSSION
  9. Acknowledgements
  10. References

We thank Dr. Robert Yu for the generous gifts of GSL standards and antibodies. This work was supported by an NIH RO1 grant (NSCA33640), the Boston College Research Expense Fund, and a Veterans Administration Merit Review (to H.C.Y.).

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  2. Abstract
  3. MATERIALS AND METHODS
  4. GSLs
  5. High-performance TLC (HPTLC)
  6. TLC immunostaining
  7. RESULTS
  8. DISCUSSION
  9. Acknowledgements
  10. References
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