Lectins identify glycan biomarkers on glioblastoma-derived cancer stem cells

Abstract

Glioblastoma (GBM) is a highly aggressive primary brain tumor with a poor prognosis. Despite aggressive therapy with surgery, radiotherapy, and chemotherapy, nearly all patients succumb to disease within 2 years. Several studies have supported the presence of stem-like cells in brain tumor cultures that are CD133-positive, are capable of self-renewal, and give rise to all cell types found within the tumor, potentially perpetuating growth. CD133 is a widely accepted marker for glioma-derived cancer stem cells; however, its reliability has been questioned, creating a need for other identifiers of this biologically important subpopulation. We used a panel of 20 lectins to identify differences in glycan expression found in the glycocalyx of undifferentiated glioma-derived stem cells and differentiated cells that arise from them. Fluorescently labeled lectins that specifically recognize α-N-acetylgalactosamine (GalNAc) and α-N-acetylglucosamine (GlcNAc) differentially bound to the cell surface based on the state of cellular differentiation. GalNAc and GlcNAc were highly expressed on the surface of undifferentiated cells and showed markedly reduced expression over a 12-day duration of differentiation. Additionally, the GalNAc-recognizing lectin Dolichos biflorus agglutinin was capable of specifically selecting and sorting glioma-derived stem cell populations from an unsorted tumor stock and this subpopulation had proliferative properties similar to CD133(+) cells in vitro and also had tumor-forming capability in vivo. Our preliminary results on a single cerebellar GBM suggest that GalNAc and GlcNAc are novel biomarkers for identifying glioma-derived stem cells and can be used to isolate cancer stem cells from unsorted cell populations, thereby creating new cell lines for research or clinical testing.

FIG. 1.

Culturing, sorting, and differentiation of CD133-positive GBM-derived CSCs. (A) Schematic representation of CD133-positive cell sorting. (B) Sorted, dispersed, and expanded CD133-positive CSCs from patient with a high-grade glioma. Neurospheres were cultured in DMEM serum-free media with growth factors mentioned in the experimental procedures (scale bar=200 μm). (C) Differentiation of neurospheres in the absences of growth factors. Immunostaining shows that the protein GFAP [red (DAPI:blue)] is present after differentiation, indicating astrocytic differentiation. (D) Schematic drawing of CD133 protein, including the 8 putative glycosylation sites. Glycosylation is believed to shield epitopes on CD133 protein. CSCs, cancer stem cells; DBA, Dolichos biflorus agglutinin; DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle medium; GBM, glioblastoma; GFAP, glial fibrillary acidic protein.

FIG. 2.

Differentiation, α-N-acetylgalactosamine-recognizing lectin labeling, and flow cytometry of CD133-positive cells. (A) Sorted CD133-positive cells were grown as neurospheres and differentiated in the absence of growth factors on laminin-coated plates over a 12-day time course. At day 1, some cells with the rounded, undifferentiated stem cell phenotype were apparent. After 12 days of differentiation, in the absence of growth factors, cells with the rounded, undifferentiated phenotype were still present. (B) Primary GBM tumor cells were sorted for CD133 positivity, expanded as neurospheres (∼2 weeks), and differentiated on laminin-coated plates (12 days). Cells were gated base and analyzed base on SSC versus FSC. (C) Cells from (B) were analyzed for reactivity to fluorescently labeled DBA, PNA, SBA, GSL I, and VVA lectins by flow cytometry. Arrows indicate morphology commonly seen in cells with stemness. After 12 days of differentiation there are stem-like cells still present. PNA, Peanut agglutinin; SBA, Soybean agglutinin; GSL I, Griffonia simplificola lectin I; VVA, Vicia villosa agglutinin; SSC, side scatter; FSC, forward scatter; FITC, fluorescein isothiocyanate.

FIG. 3.

Differentiation, α-N-acetylglucosamine-recognizing lectin labeling, and flow cytometry of CD133-positive cells. (A) Primary GBM tumor cells were sorted for CD133 positivity, expanded as neurospheres (∼2 weeks), and differentiated on laminin-coated plates (12 days). Cells were gated base and analyzed base on SSC versus FSC. (B) Cells from (A) were analyzed for positive reactivity to fluorescently labeled GSL II, and ECL by flow cytometry. (C) Cells from (B) were analyzed for CD133 glycosylation, OCT4, and SOX2 expression. ECL, Erythrina cristagalli lectin; GSL II, Griffonia simplicifolia lectin II; PE, phycoerythrin.

FIG. 4.

Sorting, culturing, and measurement of neurosphere diameter. (A) Schematic representation of DBA-lectin-positive cell sorting. (B) Sorted, dispersed, and expanded DBA-lectin-positive and DBA-lectin-negative CSCs from a patient with a high-grade glioma. Neurospheres were cultured in DMEM serum-free media with growth factors mentioned in the experimental procedures. DBA-lectin-positive neurospheres (n=27) proliferate at a slower rate and therefore have smaller diameters when compared with the DBA-lectin-negative neurospheres (n=24). (C) Bar graph representation of data collected from (B); *P value ≤0.05.

FIG. 5.

GBM-CSCs at day 1 and day 12 of differentiation labeled for Ki67 and cleaved caspase-3. Bar graph representation of DBA-positive and DBA-negative sorted cells that were grown as neurospheres and differentiated on laminin-coated plates for 12 days. Cells were labeled at day 1 and day 12 of differentiation for (A) Ki67 (proliferation) and (B) cleaved caspase-3 (apoptosis) and analyzed using flow cytometry; *P value ≤0.05.

FIG. 6.

Quantitation of lectin binding on DBA-positive (A) and DBA-negative (B) GBM-CSC (day 1 and day 12) surfaces with 8 different lectins and 3 stem cells markers. The percent of cells with specific carbohydrate expression was determined by flow cytometry using 8 different lectins. The data are means±SD of 3 independent assays of CTB-1 GBM-CSCs. CTB-1 cell line was stained with CD133, OCT4, and SOX2 antibodies in addition to lectin staining. Statistical significance (P≤0.05) is indicated by asterisk. CON A, Concanavalin A; SD, standard deviation.

FIG. 7.

Photomicrograph of an H&E- and Ki67-stained high-grade glioma derived from CTB-1/DBA⁺ GBM neurospheres implanted in flank of NOD/SCID mice. (A) Solid growth pattern of high-grade glioma with high cell density and perivascular growth pattern. Ki67 labeling index was very high (greater than 40%). (B) High-grade malignancy showed features of a GBM including nuclear anaplasia, invasiveness, and proliferation of blood vessels. H&E, hematoxylin and eosin.

FIG. 8.

Sulforhodamine B assay for proliferation. (A) CTB/CD133⁺, CTB-1/DBA⁺, and CTB/CD133⁻ cell populations begin to form neurospheres within 24 h. (B) Quantitative representation of sulforhodamine B assay (n=9). *P value ≤0.05 versus CTB/CD133⁻ time equivalent. **P value ≤0.05 versus positive population (CTB/CD133⁺ or CTB-1/DBA⁺) time equivalent.