Expansion of CD133-positive glioma cells in recurrent de novo glioblastomas after radiotherapy and chemotherapy

Laboratory investigation

Restricted access

Object

Recent evidence suggests that a glioma stem cell subpopulation may determine the biological behavior of tumors, including resistance to therapy. To investigate this hypothesis, the authors examined varying grades of gliomas for stem cell marker expressions and histopathological changes between primary and recurrent tumors.

Methods

Tumor samples were collected during surgery from 70 patients with varying grades of gliomas (Grade II in 12 patients, Grade III in 16, and Grade IV in 42) prior to any adjuvant treatment. The samples were subjected to immunohistochemistry for MIB-1, factor VIII, GFAP, and stem cell markers (CD133 and nestin). Histopathological changes were compared between primary and recurrent tumors in 31 patients after radiation treatment and chemotherapy, including high-dose irradiation with additional stereotactic radiosurgery.

Results

CD133 expression on glioma cells was confined to de novo glioblastomas but was not observed in lower-grade gliomas. In de novo glioblastomas, the mean percentage of CD133-positive glioma cells in sections obtained at recurrence was 12.2% ± 10.3%, which was significantly higher than that obtained at the primary surgery (1.08% ± 1.78%). CD133 and Ki 67 dual-positive glioma cells were significantly increased in recurrent de novo glioblastomas as compared with those in primary tumors (14.5% ± 6.67% vs 2.16% ± 2.60%, respectively). In contrast, secondary glioblastomas rarely expressed CD133 antigen even after malignant progression following radiotherapy and chemotherapy.

Conclusions

The authors' results indicate that CD133-positive glioma stem cells could survive, change to a proliferative cancer stem cell phenotype, and cause recurrence in cases with de novo glioblastomas after radiotherapy and chemotherapy.

Abbreviations used in this paper:EBRT = external-beam radiation therapy; GKS = Gamma Knife surgery.

Article Information

Address correspondence to: Kaoru Tamura, M.D., Ph.D., Department of Neurosurgery, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. email: kaoru-tmd@umin.ac.jp.

Please include this information when citing this paper: published online August 30, 2013; DOI: 10.3171/2013.7.JNS122417.

© AANS, except where prohibited by US copyright law.

Headings

Figures

  • View in gallery

    Immunohistochemical analysis for CD133 in gliomas of different grades at primary surgery. A: CD133 immunohistochemistry (brown, DAB) on paraffin section from Case 6 (de novo glioblastoma). Positive staining is seen in clusters. Counterstained with hematoxylin; bar = 100 μm. B: Nonspecific isotype control staining on the serial section of that shown in panel A. Bar = 100 μm. C: CD133-positive glioma cells around the tumor vasculature designated by the arrowhead. Bar = 20 μm. D: Double immunostaining for CD133 (brown, DAB) and Ki 67 (purple, Vector VIP substrate) on a section from Case 16 (de novo glioblastoma). CD133-postive staining surrounds areas of necrosis with few Ki 67–positively stained cells. Bar = 100 μm. E: Percentage of CD133-positive glioma cells per tumor area in 70 patients with different grades of gliomas. Closed squares and error bar represent the mean ± SD of each group.

  • View in gallery

    Increased frequency of CD133 in recurrent de novo glioblastomas after radiotherapy and chemotherapy. A: Immunohistochemistry for CD133 in sections from primary tumors after EBRT and GKS in Case 7. Arrowhead designates CD133-positive cells. Bar = 100 μm. B: Immunohistochemistry for CD133 in sections from recurrent tumors after EBRT and GKS in Case 7. Bar = 100 μm. C–H: Case 9. Contrast-enhanced axial MR images at the initial diagnosis (C), at recurrence after resection and irradiation (D), and in the terminal stage (E). There is no CD133 immunopositivity in the primary surgery specimen (F), but extensive immunopositivity is seen in pseudopalisade formations in the section obtained from the remote-site recurrence (red arrow in E) at autopsy (G and H). Bar = 100 μm (F–H).

  • View in gallery

    Expansion of CD133-positive glioma cells after radiotherapy and chemotherapy in de novo glioblastomas. Upper: Percentage of CD133-positive glioma cells in histological sections from primary and recurrent tumors of de novo glioblastomas. Lower: Percentage of CD133-positive glioma cells in histological sections from primary and recurrent tumors of secondary glioblastomas following malignant progression. Closed squares and error bars represent the mean ± SD of each group. *p < 0.00006; n.s. = not significant versus primary tumor by paired t-test.

  • View in gallery

    Effects of high-dose radiation on tumor blood vascular density. A and B: Immunohistochemistry for factor VIII in sections obtained during the primary surgery (A) and the second surgery 26.2 months after high-dose radiation (B) in a patient with glioblastoma (Case 5). Bar = 100 μm. C: Graphs showing tumor blood vessel density before and after high-dose radiation (GKS plus EBRT, Cases 1–8) and EBRT alone (Cases 11–20) in de novo glioblastomas with local failure. Tumor blood vessel density was significantly decreased in sections obtained after high-dose radiation, while specimens obtained after EBRT alone did not differ significantly from the primary surgery. Closed squares and error bars represent the mean ± SD of each group. *p < 0.005; n.s. = not significant versus primary tumor by paired t-test.

  • View in gallery

    Proliferative activity in CD133-positive glioma cells by double immunostaining for CD133 and Ki 67. A and B: Double immunostaining for CD133 (brown) and Ki 67 (purple) on sections from primary (A) and recurrent (B) tumors in a patient with glioblastoma (Case 8). Green arrows indicate Ki 67–positive and CD133-negative cells and red arrows indicate Ki 67 and CD133 dual-positive cells. Bar = 20 μm. C: Ki 67 indices of CD133-positive glioma cells in primary and recurrent tumors. Closed squares and error bars represent the mean ± SD of each group. *p < 0.00000005 versus primary tumor by unpaired t-test. D: Ki 67 indices of CD133-negative glioma cells. **p < 0.02 versus primary tumor by paired t-test.

References

  • 1

    Bao SWu QMcLendon REHao YShi QHjelmeland AB: Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444:7567602006

    • Search Google Scholar
    • Export Citation
  • 2

    Baumann MKrause MHill R: Exploring the role of cancer stem cells in radioresistance. Nat Rev Cancer 8:5455542008

  • 3

    Beier DHau PProescholdt MLohmeier AWischhusen JOefner PJ: CD133(+) and CD133(−) glioblastoma-derived cancer stem cells show differential growth characteristics and molecular profiles. Cancer Res 67:401040152007

    • Search Google Scholar
    • Export Citation
  • 4

    Bidlingmaier SZhu XLiu B: The utility and limitations of glycosylated human CD133 epitopes in defining cancer stem cells. J Mol Med (Berl) 86:102510322008

    • Search Google Scholar
    • Export Citation
  • 5

    Calabrese CPoppleton HKocak MHogg TLFuller CHamner B: A perivascular niche for brain tumor stem cells. Cancer Cell 11:69822007

    • Search Google Scholar
    • Export Citation
  • 6

    Chang CJHsu CCYung MCChen KYTzao CWu WF: Enhanced radiosensitivity and radiation-induced apoptosis in glioma CD133-positive cells by knockdown of SirT1 expression. Biochem Biophys Res Commun 380:2362422009

    • Search Google Scholar
    • Export Citation
  • 7

    Christensen KSchrşder HDKristensen BW: CD133 identifies perivascular niches in grade II-IV astrocytomas. J Neurooncol 90:1571702008

    • Search Google Scholar
    • Export Citation
  • 8

    Corbeil DRöper KHellwig ATavian MMiraglia SWatt SM: The human AC133 hematopoietic stem cell antigen is also expressed in epithelial cells and targeted to plasma membrane protrusions. J Biol Chem 275:551255202000

    • Search Google Scholar
    • Export Citation
  • 9

    Günther HSSchmidt NOPhillips HSKemming DKharbanda SSoriano R: Glioblastoma-derived stem cell-enriched cultures form distinct subgroups according to molecular and phenotypic criteria. Oncogene 27:289729092008

    • Search Google Scholar
    • Export Citation
  • 10

    Hambardzumyan DBecher OJRosenblum MKPandolfi PPManova-Todorova KHolland EC: PI3K pathway regulates survival of cancer stem cells residing in the perivascular niche following radiation in medulloblastoma in vivo. Genes Dev 22:4364482008

    • Search Google Scholar
    • Export Citation
  • 11

    Hermansen SKChristensen KGJensen SSKristensen BW: Inconsistent immunohistochemical expression patterns of four different CD133 antibody clones in glioblastoma. J Histochem Cytochem 59:3914072011

    • Search Google Scholar
    • Export Citation
  • 12

    Jordan CTGuzman MLNoble M: Cancer stem cells. N Engl J Med 355:125312612006

  • 13

    Kang MKHur BIKo MHKim CHCha SHKang SK: Potential identity of multi-potential cancer stem-like subpopulation after radiation of cultured brain glioma. BMC Neurosci 9:152008

    • Search Google Scholar
    • Export Citation
  • 14

    Keunen OJohansson MOudin ASanzey MRahim SAFack F: Anti-VEGF treatment reduces blood supply and increases tumor cell invasion in glioblastoma. Proc Natl Acad Sci U S A 108:374937542011

    • Search Google Scholar
    • Export Citation
  • 15

    Kleihues PBurger PCCollins VPNewcomb EWOhgaki HCavenee WKGlioblastoma. Kleihues PCavenee WK: Pathology and Genetics of Tumors of the Nervous System ed 2LyonIARC Press2000. 2939

    • Search Google Scholar
    • Export Citation
  • 16

    Kleihues POhgaki H: Primary and secondary glioblastomas: from concept to clinical diagnosis. Neuro Oncol 1:44511999

  • 17

    Ma YHMentlein RKnerlich FKruse MLMehdorn HMHeld-Feindt J: Expression of stem cell markers in human astrocytomas of different WHO grades. J Neurooncol 86:31452008

    • Search Google Scholar
    • Export Citation
  • 18

    Miraglia SGodfrey WYin AHAtkins KWarnke RHolden JT: A novel five-transmembrane hematopoietic stem cell antigen: isolation, characterization, and molecular cloning. Blood 90:501350211997

    • Search Google Scholar
    • Export Citation
  • 19

    Nandi SUlasov IVTyler MASugihara AQMolinero LHan Y: Low-dose radiation enhances survivin-mediated virotherapy against malignant glioma stem cells. Cancer Res 68:577857842008

    • Search Google Scholar
    • Export Citation
  • 20

    Ogden ATWaziri AELochhead RAFusco DLopez KEllis JA: Identification of A2B5+CD133-tumor-initiating cells in adult human gliomas. Neurosurgery 62:5055152008

    • Search Google Scholar
    • Export Citation
  • 21

    Pàez-Ribes MAllen EHudock JTakeda TOkuyama HViñals F: Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell 15:2202312009

    • Search Google Scholar
    • Export Citation
  • 22

    Pallini RRicci-Vitiani LBanna GLSignore MLombardi DTodaro M: Cancer stem cell analysis and clinical outcome in patients with glioblastoma multiforme. Clin Cancer Res 14:820582122008

    • Search Google Scholar
    • Export Citation
  • 23

    Phillips TMMcBride WHPajonk F: The response of CD24(-/low)/CD44+ breast cancer-initiating cells to radiation. J Natl Cancer Inst 98:177717852006

    • Search Google Scholar
    • Export Citation
  • 24

    Reya TMorrison SJClarke MFWeissman IL: Stem cells, cancer, and cancer stem cells. Nature 414:1051112001

  • 25

    Riemenschneider MJJeuken JWWesseling PReifenberger G: Molecular diagnostics of gliomas: state of the art. Acta Neuropathol 120:5675842010

    • Search Google Scholar
    • Export Citation
  • 26

    Sakariassen POPrestegarden LWang JSkaftnesmo KOMahesparan RMolthoff C: Angiogenesis-independent tumor growth mediated by stem-like cancer cells. Proc Natl Acad Sci U S A 103:16466164712006

    • Search Google Scholar
    • Export Citation
  • 27

    Singh SKClarke IDTerasaki MBonn VEHawkins CSquire J: Identification of a cancer stem cell in human brain tumors. Cancer Res 63:582158282003

    • Search Google Scholar
    • Export Citation
  • 28

    Singh SKHawkins CClarke IDSquire JABayani JHide T: Identification of human brain tumour initiating cells. Nature 432:3964012004

    • Search Google Scholar
    • Export Citation
  • 29

    Son MJWoolard KNam DHLee JFine HA: SSEA-1 is an enrichment marker for tumor-initiating cells in human glioblastoma. Cell Stem Cell 4:4404522009

    • Search Google Scholar
    • Export Citation
  • 30

    Stupp RMason WPvan den Bent MJWeller MFisher BTaphoorn MJ: Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med 352:9879962005

    • Search Google Scholar
    • Export Citation
  • 31

    Tamura KAoyagi MWakimoto HAndo NNariai TYamamoto M: Accumulation of CD133-positive glioma cells after high-dose irradiation by Gamma Knife surgery plus external beam radiation. Clinical article. J Neurosurg 113:3103182010

    • Search Google Scholar
    • Export Citation
  • 32

    Vlashi EKim KLagadec CDonna LDMcDonald JTEghbali M: In vivo imaging, tracking, and targeting of cancer stem cells. J Natl Cancer Inst 101:3503592009

    • Search Google Scholar
    • Export Citation
  • 33

    Wakimoto HAoyagi MNakayama TNagashima GYamamoto STamaki M: Prognostic significance of Ki-67 labeling indices obtained using MIB-1 monoclonal antibody in patients with supratentorial astrocytomas. Cancer 77:3733801996

    • Search Google Scholar
    • Export Citation
  • 34

    Wen PYKesari S: Malignant gliomas in adults. N Engl J Med 359:4925072008

  • 35

    Zeppernick FAhmadi RCampos BDictus CHelmke BMBecker N: Stem cell marker CD133 affects clinical outcome in glioma patients. Clin Cancer Res 14:1231292008

    • Search Google Scholar
    • Export Citation

TrendMD

Metrics

Metrics

All Time Past Year Past 30 Days
Abstract Views 379 379 22
Full Text Views 214 214 7
PDF Downloads 63 63 4
EPUB Downloads 0 0 0

PubMed

Google Scholar