Identification of tumor stem-like cells in admanatimomatous craniopharyngioma and determination of these cells’ pathological significance

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OBJECTIVE

Nuclear β-catenin, a hallmark of active canonical Wnt signaling, can be histologically detected in a subset of cells and cell clusters in up to 94% of adamantinomatous craniopharyngioma (ACP) samples. However, it is unclear whether nuclear β-catenin–containing cells within human ACPs possess the characteristics of tumor stem cells, and it is unknown what role these cells have in ACP.

METHODS

Primary ACP cells were cultured from 12 human ACP samples. Adamantinomatous CP stem cell–like cells (CSLCs) showing CD44 positivity were isolated from the cultured primary ACP cells by performing magnetic-activated cell sorting. The tumor sphere formation, cell cycle distribution, stemness marker expression, and multidifferentiation potential of the CD44− cells and the CSLCs were analyzed.

RESULTS

Compared with the CD44− cells, the cultured human CSLCs formed tumor spheres and expressed CD44 and CD133; moreover, these cells demonstrated nuclear translocation of β-catenin. In addition, the CSLCs demonstrated osteogenic and adipogenic differentiation capacities compared with the CD44− cells. The CSLCs also displayed the capacity for tumor initiation in human–mouse xenografts.

CONCLUSIONS

These results indicate that CSLCs play an important role in ACP development, calcification, and cystic degeneration.

ABBREVIATIONS ACP = adamantinomatous craniopharyngioma; bFGF = basic fibroblast growth factor; CP = craniopharyngioma; CSLC = CP stem cell–like cell; DMEM = Dulbecco’s modified Eagle’s medium; EDTA = ethylenediaminetetraacetic acid; EGF = epidermal growth factor; ExoSAP = exonuclease I and shrimp alkaline phosphatase; FBS = fetal bovine serum; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; MACS = magnetic-activated cell sorting; Pan-CK = pan-cytokeratin; PBS = phosphate-buffered saline; PVDF = polyvinylidene difluoride; qPCR = quantitative polymerase chain reaction; SDS-PAGE = sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

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Article Information

Correspondence Yi Liu: Nanfang Hospital, Southern Medical University, Guangzhou, China. liuyi86818@126.com.

INCLUDE WHEN CITING Published online August 30, 2019; DOI: 10.3171/2019.5.JNS19565.

C.H.W., S.T.Q., J.F., J.P., and J.X.P. contributed equally to this study.

Disclosures This work is supported by grants from the Science and Technology Program of Guangdong (2016A020213006, 2017A020215048, and 2017A020215191); the Natural Science Foundation of Guangdong (2016A030310377); the Science and Technology Program of Guangzhou (201707010149); and the President Foundation of Nanfang Hospital, Southern Medical University (2015C018, 2016L002, and 2017Z009).

© AANS, except where prohibited by US copyright law.

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Figures

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    Stem cell markers expressed in ACP tissues. Coexpression of nuclear β-catenin (green) and the tumor stem-cell markers CD44 (red) (A–C), CD133 (red) (D–F), and KLF4 (red) (G–I). Coexpression of the tumor stem-cell markers OCT4 (green) and Notch1 (red) (J–L). Figure is available in color online only.

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    Comparison of stem cell markers expressed in primary ACP cells and in CSLCs. A–C: Reflection microscopy images of primary ACP cells (A) treated with antibodies against pan-CK (green, B) and β-catenin (red, C). D: Representative image of a sphere of CSLCs. E and F: Reflection microscopy images of CSLCs treated with antibodies against pan-CK (green, E) and β-catenin (red, F). G–I: Dual immunofluorescence staining of CD133 (green, G) and CD44 (red, H) showing their colocalization within the same cells (merged, I). J–L: Quantitative PCR analysis of mRNA expression of the genes encoding β-catenin (J), CD44 (K), and CD133 (L) in CSLCs and CD44− ACP cells. M and N: Immunoblotting analysis (M) and quantification of the protein expression (N) of CD44, CD133, and β-catenin in CSLCs and CD44− ACP cells. * p < 0.05; *** p < 0.01. O: Quantitative PCR analysis of mRNA expression of the genes encoding KLF-4, OCT4, and Notch1 in the CSLCs and CD44− ACP cells. Figure is available in color online only.

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    Comparison of CD44− ACP cells and CSLCs by performing flow cytometry analysis. A: The protocol of MACS of CSLCs by using CD44. B–G: Images showing flow cytometry analyses of CD44+ and CD133+ CSLCs and CD44− ACP cells, which were isolated by performing MACS. H: Proliferation of CSLCs and CD44− ACP cells over a 6-day period. * p < 0.05; *** p < 0.01. I and J: Analysis of the cell cycle by performing flow cytometry indicated that the CSLCs and CD44− ACP cells were arrested in the S phase. Figure is available in color online only.

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    Differentiation capacity of CSLCs. A and B: Alizarin red S staining and quantification of CSLCs and CD44− ACP cells on days 3, 7, 14, and 21. C and D: Immunoblotting analysis (C) and quantification of protein expression (D) of Runx2, DMP-1, DSPP, BMP-2, and ALP in the CSLCs and in normal ACP cells (CD44− ACP cells). E: Quantitative PCR analysis of mRNA expression of the genes encoding Runx2, DSPP, DMP-1, BMP-2, and ALP in CSLCs and CD44− ACP cells. F and G: After CSLCs and CD44− ACP cells had been stained with Oil Red O, they were observed under a microscope. H: Immunoblotting analysis of protein expression of PPAR-γ in the CSLCs and CD44− ACP cells. I: Quantitative PCR analysis of the gene encoding PPAR-γ in the CSLCs and CD44− ACP cells. ** p < 0.05; *** p < 0.01. Figure is available in color online only.

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    Differentiation of CSLCs may induce adamantinomatous craniopharyngioma (ACP) calcification and cystic fluid formation. A–C: Nuclear β-catenin–containing cells (blue arrowheads) will gradually transform into ghost cells (green arrowheads) and eventually undergo calcification (black arrowheads) or osteogenic differentiation (black star); all stages can be observed in the same field of vision. D–F: Coexpression of nuclear β-catenin and Runx2 around the regions of calcification (white arrowheads). G–I: Coexpression of BMP-2 and DSPP around the calcification region (white stars). J–L: Nuclear β-catenin–containing cells (black arrow), numerous lipid droplets (yellow arrows), and several foam cells (green arrows) can be observed in the same field of vision. M–O: Coexpression of PPAR-γ and nuclear β-catenin around the calcification region. P–R: High expression of the scavenger receptor CD36 (green) in foam cells around the tumor. Figure is available in color online only.

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    Craniopharyngioma stem cell–like cells (CSLCs) can induce tumor initiation in human–mouse xenografts. A and B: MRIs of brain xenografts (red arrows) derived from CSLCs in the NOD/SCID mice. C and D: Brain xenografts derived from the CSLCs and H & E staining of the xenografts. E–H: All of the xenografts expressed pan-CK (E), and the whorl-like cells showed β-catenin nuclear translocation (F). The whorl-like cells also expressed the stem cell markers CD133 (G) and CD44 (H). Figure is available in color online only.

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