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Expression of the stem cell marker CD133 in recurrent glioblastoma and its value for prognosis
Article first published online: 30 AUG 2010
Copyright © 2010 American Cancer Society
Volume 117, Issue 1, pages 162–174, 1 January 2011
How to Cite
Pallini, R., Ricci-Vitiani, L., Montano, N., Mollinari, C., Biffoni, M., Cenci, T., Pierconti, F., Martini, M., De Maria, R. and Larocca, L. M. (2011), Expression of the stem cell marker CD133 in recurrent glioblastoma and its value for prognosis. Cancer, 117: 162–174. doi: 10.1002/cncr.25581
- Issue published online: 16 DEC 2010
- Article first published online: 30 AUG 2010
- Manuscript Accepted: 14 JUL 2010
- Manuscript Revised: 22 JUN 2010
- Manuscript Received: 22 APR 2010
- cancer stem cells;
- tumor recurrence
Experimental data suggest that glioblastoma cells expressing the stem cell marker CD133 play a major role in radiochemoresistance and tumor aggressiveness. To date, however, there is no clinical evidence that the fraction of CD133-positive cells in glioblastoma that recurs after radiochemotherapy may be relevant for prognosis.
The authors used immunohistochemistry to assess CD133 expression in 37 paired glioblastoma samples, including 1 primary tumor sample and 1 recurrent tumor sample, after patients received adjuvant radiochemotherapy. To assess the actual composition of the CD133-positive glioblastoma cell population, fluorescence-associated cell sorting (FACS) analysis was used to sort CD133-positive/CD45-negative cells that were assayed for tumor-specific chromosomal aberrations using interphase fluorescence in situ hybridization. To rule out endothelial precursor cells, CD133-positive fractions also were assayed with anti-CD34 by FACS.
In recurrent glioblastomas, the percentage of CD133-positive cells was increased by 4.6-fold compared with the percentage in primary glioblastomas, although, in some tumors, it increased up to 10-fold and 20-fold. Unexpectedly, the increase in CD133 expression was associated significantly with longer survival after tumor recurrence. An analysis of tumor-specific chromosomal aberrations and in vivo studies revealed that the CD133-positive cell compartment of recurrent glioblastoma was composed of both cancer stem cells and nontumor neural stem cells. The latter cells represented from 20% to 60% of the CD133-positive cell population, and their relative percentage favorably affected the survival of patients with recurrent glioblastoma. Endothelial CD133-positive/CD34-positive precursors did not contribute to the CD133-positive cell population.
The authors hypothesized that, similar to the phenomenon described in glioblastoma models, neural stem/progenitor cells that are recruited by the tumor from surrounding brain may exert an antitumorigenic effect. Cancer 2011. © 2010 American Cancer Society.
Despite aggressive multimodal therapy, the prognosis for patients with glioblastoma remains very poor.1 Recently, tumor cells with stem-like features have been identified in glioblastoma.2, 3 These cells, which are referred to commonly as cancer stem cells (CSCs), express the transmembrane glycoprotein CD133 and have the ability to initiate tumor formation in vivo.2-4 It has been hypothesized that glioblastoma CSCs may arise from the malignant transformation of neural stem cells (SCs) that are resident in the subventricular zone and that the migration of these cells may lead to the development of glioma in different areas of the brain.5 Although tumorigenicity may be not confined entirely to the CD133-positive cell fraction of glioblastoma,6-9 growing data demonstrate that CD133 expression is related to an adverse prognosis10-14 and that the CD133-expressing glioblastoma cells contribute to radiochemoresistance and tumor aggressiveness.15-19 For example, Liu and colleagues demonstrated a positive selection for CD133-positive tumor cells in patients with glioblastoma after chemotherapy.15 The resistance of CD133-positive glioblastoma cells to cytotoxic drugs was related to high levels of drug transporter and inhibitor of apoptosis proteins and to enhanced methyl guanine-DNA methyl transferase (MGMT)-mediated DNA repair pathways.15 Bao and colleagues demonstrated that radiation treatment enriched glioblastoma xenografts with CD133-expressing cells and that radioresistant glioblastoma cells from irradiated xenografts formed secondary tumors with decreased latencies relative to untreated xenografts.19 In addition, CD133-positive cells that were isolated both from human glioma xenografts and from primary glioblastoma specimens activated the DNA damage checkpoint in response to radiation and repair radiation-induced DNA damage more effectively than CD133-negative tumor cells.19 Despite these results, there is no evidence that higher fractions of CD133-positive cells in glioblastoma that recurs after radiochemotherapy are correlated with a worse prognosis. It also remains to be determined whether and the extent to which the neural SCs that exhibit positive tropism both toward brain tumors and toward areas of brain inflammation20-22 may affect the composition of the CD133-positive cell fraction of glioblastoma.
MATERIALS AND METHODS
This study included 37 consecutive adult patients who underwent craniotomy for resection of histologically confirmed glioblastoma (World Health Organization grade IV)23 in the supratentorial compartment and who underwent reoperation for tumor recurrence after radiochemotherapy (Table 1). Both surgical procedures were performed at the Institute of Neurosurgery, Catholic University School of Medicine, Rome. All patients provided written informed consent according to the research proposals approved by the Ethical Committee of the Catholic University. Patients of pediatric age were not included. The patients were aged 28 to 74 years at the time of primary surgery (median age, 54 years; mean age [±standard deviation], 54.2 ± 10.3 years) and included 17 men and 20 women. In all patients, macroscopic total tumor resection was achieved both at primary surgery and at reoperation. After primary surgery, all patients received radiotherapy to limited fields (2 grays [Gy] per fraction, once daily 5 days a week; 60 Gy total dose) and adjuvant temozolomide.12 Overall survival was calculated from the date of primary surgery to the date of death; and survival after recurrence was calculated from the date of reoperation to the date of death.
|Patient No.||Age,ya||Sex||KPSa||Ki-67, %a||MGMTa,b||OS, mo||Survival After Disease Recurrence, mo||CD133, %||CD133 Recurrence/ Primary Ratio|
Immunohistochemistry and Immunofluorescence
Immunohistochemistry was performed on deparaffinized sections using the avidin-biotin-peroxidase complex method (ABC-Elite Kit; Vector Laboratories, Burlingame, Calif).24 Endogenous biotin was saturated with a biotin-blocking kit (Vector Laboratories). For antigen retrieval, paraffin sections were microwave-treated in 0.01 M citric acid buffer, pH 6.0 (2 cycles for 5 minutes each at 750 W), followed by inhibition of endogenous peroxidase with 3% H2O2 for 5 minutes. Then, the sections were incubated with antibody directed against CD133 (dilution 1:50; Biocare Medical, Concord, Calif). After a 1-hour incubation at room temperature, immunodetection was performed using goat antirabbit secondary antibody (Vector Laboratories) and freshly made diaminobenzidine as a chromogen. For human telomerase reverse transcriptase (hTERT) antigen retrieval, paraffin sections were microwave-treated in ethylene diamine tetracetic acid buffer, pH 8.0, for 10 minutes. The percentage of CD133-positive cells was evaluated independently by 2 pathologists (F.P. and L.M.L.) who were unaware of the clinical data. For each slide, a minimum of 10 nonsuperimposing fields was examined at high-power magnification (×400), and at least 2000 tumor cells were counted in areas that were devoid of necrosis, hemorrhage, and abundant new vessel formation. Interobserver agreement was reached in the first analysis in 90% of slides; for the remaining slides, a consensus was reached by a joint review of the slides. For immunophenotyping of sorted cells, the cells were fixed with 4% paraformaldehyde and stained with antibody directed against nestin (Chemicon, Temecula, Calif), sex-determining Y-box 2 (SOX2) (R&D Systems, Minneapolis, Minn), musashi homolog 1 (Musashi1) (R&D Systems), glial fibrillary acidic protein (GFAP) (Dako, Glostrup, Denmark), and hTERT (Novocastra Laboratories, Newcastle upon Tyne, United Kingdom). Appropriate secondary antibodies (affinity-purified goat antimouse tetra methyl Rhodamin isothiocyanate-conjugated immunoglobulin G [IgG] and goat antirabbit fluorescein isothiocyanate [FITC]-conjugated IgG; both from Chemicon) were used.
Flow Cytometry and Fluorescence-Activated Cell Sorting for CD133, CD45, and CD34
Glioblastoma cells obtained from freshly dissociated surgical specimens were washed and resuspended in phosphate-buffered saline (PBS) plus 0.5% bovine serum albumin (BSA) (PBS-BSA) then incubated with anti-CD133 phycoerythrin (PE)-conjugated antibody (clone AC133-PE, mouse IgG1; Miltenyi Biotec, Bergisch Gladbach, Germany) and with either anti-CD45 FITC-conjugated antibody (clone T29/33-FITC, mouse IgG1; DakoCytomation, Carpenteria, Calif) or anti-CD34 FITC-conjugated antibody (clone BIRMA-K3 mouse IgG1; DakoCytomation) antibody. PE-conjugated and an FITC-conjugated mouse IgG1 isotype antibodies were used as controls according to the manufacturer's instructions for 60 minutes at 4°C. Then, the cells were washed with 2 mL of PBS-BSA, centrifuged at 800 revolutions per minute (rpm) for 5 minutes, resuspended in 0.5 mL PBS-BSA, and analyzed by flow cytometry. Viable cells were identified using 7-amino actinomycin D. Cell sorting (BD fluorescence-associated cell sorting [FACS] Aria; Becton-Dickinson, Franklin Lakes, NJ) was performed using the same staining method described above for CD133-PE. Sorted cells were analyzed for purity by flow cytometry with a FACSCanto flow cytometer (Becton-Dickinson) and FACS Diva software (Becton-Dickinson). Sorted cells were fixed in a solution of methanol and acetic acid (3:1) for 10 minutes and then processed for fluorescence in situ hybridization (FISH) as described below.
Fluorescence In Situ Hybridization
Single-probe and dual-probe FISH was performed either on cell nuclei extracted from paraffin-embedded sections of the tumor or on the CD133-positive/CD45-negative and CD133-positive/CD45-positive sorted cells using locus-specific probes for the centromere of chromosome enumeration probe 10 (CEP 10) for telomere of chromosome 19 (tel 19q) and for a locus-specific probe on chromosome 22 (LSI22) (breakpoint cluster region locus q11.2; Vysis Inc. Abbot Laboratories SA, Downers Grove, Ill), as described elsewhere.25 These probes were chosen because loss of euploidy for chromosomes 10, 19, and 22 has been observed frequently in glioblastoma.12, 26, 27 Briefly, for nuclei extraction, 40-μm-thick sections were dewaxed with xylene, manually disaggregated, and digested with freshly prepared 0.005% proteinase K in TRIS 0.05 M, pH 7, for 30 minutes at 37°C. To aid with enzymatic digestion, the samples were vortexed for 5 seconds at 5-minute intervals during this incubation. Nuclei were pelleted using a microcentrifuge (6000 rpm) for 10 minutes. Proteinase K was carefully removed, and the nuclei were washed by resuspension with vortexing in 100 μL PBS. The PBS solution was removed, and the nuclear suspension was fixed in a solution of methanol and acetic acid (3:1). Eight microliters of nuclear suspension were placed on a slide that was positively charged within a 13-mm circle. The slides were then dried in a 65 °C oven for 15 minutes and treated in a microwave oven for 10 minutes in citrate buffer, pH 6.0, followed by enzymatic digestion with 4 mg/mL of pepsin in NaCl 0.9%, pH 1.5, for 20 minutes at 37 °C.28 Samples were then dehydrated in a graded ethanol series and subjected to FISH analysis. Sorted cells had been fixed previously in a solution of methanol and acetic acid (3:1) for 10 minutes. After specimen/probe denaturation at 73°C for 5 minutes, the probe (10 μL to the slide) was applied to the slides, and the slides were incubated at 42°C overnight for CEP10 and at 37 °C for 10 to 16 hours for LSI22/tel 19q. The posthybridization procedure included subsequent washing in 50% formamide/2 × standard saline citrate (SSC) (for 30 minutes at 46°C) and in 2 × SSC 0.1% NP40 (for 5 minutes at room temperature). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Vectashield mounting medium with DAPI; catalog number H-1200; Vector Laboratories). The slides were evaluated with an Axioplan fluorescent microscope (Karl Zeiss, Gottingen, Germany) equipped with appropriate filters (Vysis Inc.). In each hybridization, at least 500 cells in interphase were analyzed.
Intracranial Grafting of Glioblastoma Cancer Stem Cells in Immunodeficient Mice
Severe combined immunodeficient (SCID) mice (both sexes, aged 4 weeks; Charles River, Lecco, Italy) were used. Mouse research was approved by the Ethical Committee of the Catholic University School of Medicine in Rome. For intracranial grafting, 3 × 104 FACS-sorted CD133-positive/CD45-negative glioblastoma cells were resuspended in 4 μL Dulbecco modified Eagle medium. The mice were anesthetized (diazepam 2 mg/100 g intraperitoneally followed by ketamine 4 mg/100 g intramuscularly), the skull was immobilized in a stereotactic head frame, and a burr hole was made 2 mm to the right of the midline and 1 mm behind the coronal suture. The tip of a 10-μL Hamilton microsyringe was placed at a depth of 3.5 mm from the dura, and the cells were slowly injected. After grafting, mice were monitored and killed 16 weeks after grafting or when they presented with signs of tumor. The brain was removed, fixed in 4% paraformaldehyde, embedded in paraffin, and cut on the coronal plane either in 5-μm-thick sections for histology or in 40-μm-thick sections for cell nuclei extraction. The 5-μm-thick sections were stained with hematoxylin and eosin. Nuclei extraction was performed as described above.
The percentages of CD133-positive cells in glioblastoma at first diagnosis and at recurrence after radiochemotherapy were compared using the Student t test. Correlations between the expression of CD133 and either overall survival or survival after recurrence were studied using regression analysis and the Pearson correlation coefficient. Curves for overall survival and for survival after tumor recurrence were estimated by using the Kaplan-Meier method and were compared by using 2-sided log-rank tests. A Cox proportional-hazards model was fitted to assess the prognostic value of CD133 expression, Ki67 expression, MGMT methylation, and other potential prognostic factors, like patient age and preoperative performance status,29, 30 by generating different models that considered overall survival. Correlation analyses between variables were performed before including each variable in each model using MedCalc software, version 9.5.0 (MedCalc Software, Mariakerke, Belgium). The results are reported as 2-sided P values with 95% confidence intervals (CIs).
Increased CD133 Expression in Recurrent Glioblastoma and Its Relation to Survival
By using immunohistochemical staining, we assessed the percentage of CD133-positive cells in 37 paired glioblastoma tumors both at first diagnosis before any therapy and at tumor recurrence after surgery and adjuvant radiochemotherapy (Fig. 1, Table 1). In each patient, any change in CD133 expression was calculated as the CD133 ratio (ie, the ratio between the percentage of CD133-positive cells in recurrent glioblastoma and the percentage of CD133-positive cells in glioblastoma at first diagnosis). Compared with glioblastoma at first diagnosis, in recurrent tumors, the CD133-positive cell population was increased in 19 patients (51.3%; CD133 ratio, >1), unchanged in 12 patients (32.4%; CD133 ratio, 1), and reduced in 6 patients (16.2%; CD133 ratio, <1) (Fig. 1C). The mean CD133 ratio was 4.6; however, in some instances, it reached values of 10 to 20 (Table 1). Overall, the mean percentage (±standard error of the mean [SEM]) of CD133-positive cells was 2.34% ± 0.42% (range, 0.5%-10%) and 4.55 ± 0.65 (range, 0.5%-15%) in glioblastoma at first diagnosis and at recurrence after radiochemotherapy, respectively (P < .001; Student t test) (Fig. 1D).
The increased CD133 expression in glioblastoma that recurred after radiochemotherapy was not an unfavorable factor for prognosis; in fact, it was associated significantly with longer survival after tumor recurrence (Fig. 2). It is interesting to compare linear regressions that analyzed overall survival and percentages of CD133-positive cells in primary tumors and recurrent tumors (Fig. 2A,B). Although the phenomenon did not reach statistical significance, in primary glioblastomas, the percentage of CD133-positive cells was associated with shorter survival (Fig. 2A). In recurrent glioblastomas, however, the CD133-positive cell fraction appeared to acquire an opposite association (Fig. 2B,C). When survival after recurrence was considered in a regression analysis, then both the percentage of CD133-positive cells in recurrent tumor and the CD133 ratio were associated significantly with a better prognosis (Fig. 2C,E). The favorable prognostic value of increased CD133 expression in recurrent glioblastoma is demonstrated clearly by the survival curves for patients who had tumors with a CD133 ratio >1 (n = 18) relative to patients who had tumors with a CD133 ratio ≤1 (n = 19; P ≤ .0001 for both overall survival and survival after recurrence; log-rank test) (Fig. 3A,B). On multivariate analysis, a CD133 ratio >1 (P = .00013; 95% CI, 0.0394-0.3489), Ki-67 expression (P = .03365; 95% CI, 1.0024-1.0571), and Karnofsky performance status (P = .04635; 95% CI, 0.8981-0.9989) emerged as independent predictors of overall survival. Age was not related significantly to patient survival; however, this result does not necessarily suggest that our series is not a representative study sample, because it may depend on the exclusion of older patients for reoperation.
Heterogeneous Nature of the CD133-Positive Cell Population in Recurrent Glioblastoma
To explain why the increased CD133 expression in recurrent glioblastoma (after radiochemotherapy) was associated with longer survival, we hypothesized that, in recurrent glioblastoma, the CD133-positive cell population may not be constituted entirely by tumor cells and that it also may contain nontumor, stem-like cells. Experiments using homotopic human glioma xenografts have demonstrated a remarkable tropism of endogenous neural SCs toward the area where the tumor had been implanted.20, 21 In addition, postoperative glial scarring and radiotherapy may enhance the migration of endogenous neural SCs cells in the recurrent tumor.22 Therefore, in a subset of recurrent glioblastomas (n = 17), we used FACS analysis to sort the CD133-positive cells from freshly dissociated specimens. To rule out CD133-positive cells of hematopoietic origin,31 the cells also were labeled with anti-CD45 (Fig. 4A,C). Sorted CD133-positive/CD45-negative and CD133-positive/CD45-positive cells were then assessed with an interphase FISH assay using probes for chromosomes 10, 19, and 2225 (Tables 2 and 3). We observed that each glioblastoma that harbored aneuploidy for 1 or more of these chromosomes had a fraction of euploid CD133-positive/CD45-negative cells that which ranged from 20% to 60% (mean ± SEM, 38.1% ± 5.7%) (Fig. 4B,C; Table 3). In any given tumor, all CD133-positive/CD45-positive cells were euploid (Fig. 4B). To rule out the possibility that endothelial precursor cells were involved in tumor angiogenesis,32, 33 CD133-positive fractions also were assayed for CD34 by FACS analysis. In each tumor, however, the CD133-positive/CD34-positive cells were so rare that they could not substantially affect the composition of the CD133-positive cell fraction (Fig. 5).
|CD133+/ CD45− Cells, %||Chromosomal Aberration||Euploid CD133+/ CD45− Cells, %|
|2||1.2||Cep10 monosomy, LSI22 monosomy||20|
|16||1.4||Cep10 polysomy, Tel19 polysomy, LSI22 polysomy||30|
Because, in our series of recurrent glioblastomas, the percentage of CD133-positive cells that lacked chromosomal changes (38.1%) was higher than that reported by Bao and colleagues in 1 glioblastoma at first diagnosis (approximately 15%),19 we investigated whether this discrepancy had a technical explanation or reflected a specific feature of the recurrent tumors. Therefore, we used FACS analysis to sort out CD133-positive/CD45-negative cells from 3 primary glioblastomas (from Patients 27, 29, and 35); then, we assessed these cells for tumor-specific chromosomal changes using an interphase FISH assay. In primary glioblastomas, the percentage of CD133-positive/CD45-negative cells that did not harbor tumor-specific chromosomal aberrations ranged from 5% to 15% (mean, 9%). This result suggests that the euploid CD133-positive/CD45-negative cells increased remarkably in recurrent tumors.
We also assessed the phenotype of sorted CD133-positive/CD45-negative cells with immunocytochemistry, and we observed that the majority of these cells expressed the SC marker nestin (mean ± SEM, 61.2% ± 4%; range, 49%-78%) (Fig. 6A,B). A proportion of the CD133-positive/CD45-negative cells were not immunostained with either antinestin or anti-GFAP antibodies (mean ± SEM, 22.5% ± 2.6%; range, 13%-33%), whereas a minor fraction of these cells coexpressed both nestin and GFAP (mean ± SEM, 15.1% ± 2.7%; range, 6%-29%). The stem cell markers SOX2 and Musashi1 were expressed by 73.2% and 62.5% of the sorted CD133-positive/CD45-negative cells, respectively (Fig. 6C). Expression of hTERT, a feature that distinguishes tumorigenic human adult neural SCs cells from their normal counterparts,34 was absent in approximately 33% of CD133-positive/CD45-negative cells (mean ± SEM; 32.2% ± 6.4%; range 18%-47%) that sorted from hTERT-positive recurrent glioblastoma (Fig. 6D). This result provides additional evidence that a fraction of CD133-positive/CD45-negative cells in recurrent glioblastoma lacks the neoplastic features of the parent tumor.
Tumor and Nontumor CD133-Expressing Cells in Recurrent Glioblastoma
Glioblastomas are genetically heterogeneous neoplasms that are likely to acquire several mutations during the course of tumor progression. Therefore, the CD133-positive/CD45-negative cells sorted from recurrent glioblastomas that lacked aneuploidy for chromosome 10, 19, or 22 may have been tumor cells harboring mutations that were not detectable with the FISH probes used in our study. Alternatively, such euploid CD133-positive/CD45-negative cells may have been nontumor cells with stem-like features. To address this issue, in immunocompromised mice, we grafted CD133-positive/CD45-negative cells that were sorted from 3 recurrent glioblastomas in which FISH analysis had demonstrated aneuploidy for chromosome 10, 19, or 22 (Table 2, Fig. 7). In this experiment, the grafted cells were a mixed population that contained both euploid and aneuploid CD133-positive/CD45-negative cells. It is known that the ability to generate tumors in vivo is a landmark for glioblastoma CSCs that distinguishes these cells from normal neural SCs. If all of the CD133-positive/CD45-negative cells were neoplastic cells, the we expected that the tumor xenografts would contain both aneuploid and euploid cells. Five of 9 mice that were grafted with CD133-positive/CD45-negative cells developed highly infiltrating brain tumors that involved the entire striatum and piriform cortex with swelling of the hemisphere and compression of the lateral ventricle (Fig. 7). When the cell nuclei of the tumor xenografts were assessed by FISH using probes for human chromosome 10, 19, or 22, we observed that virtually all cell nuclei with positive FISH signals were aneuploid (Fig. 7), indicating that the euploid fraction of CD133-positive/CD45-negative cells sorted from recurrent glioblastoma was not tumorigenic. When we revisited the correlation between CD133 expression and survival in the 8 patients who had recurrent glioblastoma in which aneuploid CD133-positive/CD45-negative tumor cells could be dissected from euploid CD133-positive/CD45-negative stem-like cells, we observed that the relative presence of the latter cells was related significantly to a better outcome in terms of both overall survival and survival after recurrence (Fig. 8).
In the current study, we assessed CD133 expression in a group of 37 glioblastomas that recurred after patients had undergone surgery and received adjuvant radiochemotherapy, and we observed that the percentage of CD133-positive cells was increased up to 20-fold compared with the percentage in the paired primary tumors. Surprisingly, the increased CD133 expression in recurrent glioblastoma was not an adverse factor for prognosis, because it often was associated with longer survival. Analyses of tumor-specific chromosomal aberrations and of hTERT expression in CD133-positive/CD45-negative cells that were sorted from recurrent glioblastomas as well as intracerebral xenografts of these cells in immunocompromised mice suggested that diverse subsets of CD133-positive cells contribute toward populating recurrent glioblastoma.
The heterogeneous composition of the CD133-positive cell population may help to explain some recent data that appear to diverge from the cancer stem hypothesis for glioblastoma. For example, Joo et al9 reported less dramatic clinical outcomes for patients who had glioblastomas with high CD133 expression (≥3%) compared with patients who had tumors that expressed low levels of CD133 (<3%), as determined by FACS analysis. Their result may be explained by the presence of nontumor CD133-positive cells in the surgical specimens. In a previous study by our group, we were unable to demonstrate a direct relation between the percentage of immunostained CD133-positive cells in glioblastoma and patient prognosis, whereas such relation emerged for proliferating CD133-positive/Ki67-positive cells.12 It is likely that the CD133-positive/Ki67-positive cells may represent the actual tumorigenic population of glioblastoma more closely than the CD133-positive cells as a whole, assuming that the proliferation of neural SCs is a very rare event.
Tumor and nontumor CD133-positive cells may play completely different roles in glioblastoma biology. In a mouse model, the glioblastoma-induced attraction of endogenous neural precursor cells was associated with improved survival because of antiproliferative and proapoptotic actions of the neural precursors in glioblastoma cells.35 Defining the actual role of the neural SCs in human glioblastoma and their interactions with CSCs was beyond the objectives of the current study. However, for those patients in whom the aneuploid CD133-positive CSCs could be distinguished from the euploid CD133-positive neural SCs, the relative presence of the latter cells was linked significantly to a better outcome.
In conclusion, CD133-positive cells in recurrent glioblastomas are a heterogeneous cell population composed of both CSCs that are likely to represent the actual tumorigenic fraction responsible for tumor progression and nontumor cells—putatively, neural SCs. The latter cells are supposed to migrate from surrounding brain toward the tumor in which they may exert an antiglioma effect. The CD133-positive endothelial precursor cells do not contribute substantially toward populating recurrent glioblastoma. These observations warrant further studies to distinguish the CD133-positive CSCs from nontumor, CD133-positive cells that are recruited by glioblastoma and to establish the actual role of the latter cells in tumor biology.
CONFLICT OF INTEREST DISCLOSURES
Supported by grants from Fondi d'Ateneo (D1) and Mrs. Paola Krajnik (to R.P.), and from Associazione Italiana per la Ricerca sul Cancro and the Italian Health Ministry (to R.D.M.).
- 1European Organization for Research and Treatment of Cancer Brain Tumor and Radiotherapy Groups; National Cancer Institute of Canada Clinical Trials Group. Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma. N Engl J Med. 2005; 352: 987-996., , , et al;
- 23Diffusely infiltrating astrocytomas. In: KleihuesP, CaveneeCW, eds. Pathology and Genetics of Tumours of the Nervous System. World Health Organization Classification of Tumours. 2nd ed. Lyon, France: IARC Press; 2000: 10-21., , , , ,
- 29European Organisation for Research and Treatment of Cancer (EORTC) Brain and Radiotherapy Groups, National Cancer Institute of Canada (NCIC) Clinical Trials Group: clinical prognostic factors affecting survival in patients with newly diagnosed glioblastoma multiforme (GBM) [abstract]. J Clin Oncol. 2004; 22: 859s.Abstract 9599., , , et al.