Co-Localization of CD133/EGFR and Antiangiogenic Activity Drive the Antitumor Effect of Nimotuzumab and Radiation in Human GBM U87MG Xenografted in Mice

Research Article

J Stem Cells Res, Rev & Rep. 2015;2(1): 1018.

Co-Localization of CD133/EGFR and Antiangiogenic Activity Drive the Antitumor Effect of Nimotuzumab and Radiation in Human GBM U87MG Xenografted in Mice

Diaz-Miqueli A¹*, Markelova MR², Lemm M² and Fichtner I²

¹Department of System Biology, Center of Molecular Immunology, Cuba

²Department of Experimental Pharmacology, Max Delbrück Center for Molecular Medicine, Germany

*Corresponding author: Diaz-Miqueli A, Department of System Biology, Center of Molecular Immunology, 216 St. Havana, Cuba, Tel: 537-271 5057; Fax: 537-273 3509; Email: [email protected]

Received: December 12, 2014; Accepted: February 23, 2015; Published: February 25, 2015


The targeting of CD133 radio and chemoresistant population have been suggested a crucial strategy to improve the local control disease in the treatment of brain tumors. In this study nimotuzumab, a monoclonal antibody specific to EGFR, showed no significant cytotoxic activity in vitro, neither alone nor in combination with radiotherapy against the human glioma cells U87MG assessed by MTT assays. In contrast, the co-administration of nimotuzumab and radiation significantly delayed subcutaneous tumor growth in NMRI nude mice, together with a significant antiangiogenic response. In vitro, U87MG cells formed tumor neurospheres when cultured in serum-free neural stem cell medium. Moreover, tumor neurospheres, but not adherent cells, showed an increased immunoreactivity to the neural CSC marker CD133. Immunofluorescence analysis performed to examine protein expression showed that CD133 was co-localized with the EGFR, suggesting a potential molecular mechanism by which nimotuzumab is able to target the CD133 radioresistant population in the U87MG cell line. In summary, the present study suggests that antitumor activity of nimotuzumab in combination with radiation against U87MG xenografts is mediated, at least in part, by a potent antiangiogenic response in addition to the ability of nimotuzumab to target the CD133 radioresistant population, suggesting a promising alternative to the failure of current available therapies in the treatment of glioblastoma.

Keywords: CD133; Epidermal growth factor receptor; Glioblastoma multiform; Nimotuzumab; Radiation; U87MG


CSC: Cancer Stem Cells; EGF: Epidermal Growth Factor; EGFR: EGF Receptor; GBM: Glioblastoma Multiform; MGMT: O6- Methylguanine-DNA Methyltransferase; VEGF: Vascular Endothelial Growth Factor; VEGFR: VEGF Receptor


Glioblastoma Multiform (GBM) is one of the most aggressive malignancies of the central nervous system and it is considered among the deadliest human cancers [1]. The incidence rate of GBM is 3.1 per 100000 person-years and accounts for the 18.5% of all brain tumors, despite has increased slightly over the last decades, being more frequent among Caucasians [2,3]. The standard therapy for patients with GBM consists of surgery, fractioned radiotherapy with concomitant temozolamide, followed by adjuvant temozolamide. However, despite current available aggressive treatments, the prognosis for patients with GBM remains daunting, with a median survival time of 14.6 months and only 3.4% of patients remains alive after 5 years with treatment [4,5]. This low median survival of patients with GBM has been ascribed to de novo or acquired resistance to ionizing radiation [6]. Moreover, the high toxicity profile inherent to these therapies becomes poorly tolerated and often limits their minimum benefits [7]. Therefore, novel therapeutic paradigms are urgently required to overcome the inherent limitations of conventional treatments currently available for patients with GBM [8].

Most of high-grade gliomas overexpress the Epidermal Growth Factor Receptor (EGFR), which distinguish from the very low levels found in normal brain [9]. Mutation and gene amplification of the EGFR are associated with a more aggressive phenotype and a worse clinical outcome [10]. This has led to develop new molecular-targeted therapies based on the inhibition of this molecule. Among different strategies exploited to target the EGFR, monoclonal antibodies that bind directly extracellular epitopes of the receptor and smallmolecule tyrosine kinase inhibitors that inactivate the tyrosine kinase domain of the EGFR have undergone the more successful approaches so far in the clinic [11].

Nimotuzumab is a humanized monoclonal antibody against the EGFR that has shown promising results in clinical trials [12]. Nimotuzumab has undergone an extensive evaluation in GBM, showing efficacy as a single agent therapy or combined with ionizing radiation or chemotherapy [13-18]. The low toxicity profile of the antibody, together with its proven efficacy becomes nimotuzumab a promising therapeutic option for patients with GBM, especially in pediatric population [19]. Moreover, its ability to improve the efficacy of conventional cytotoxic therapies and the possibility to use used under long-term schemes without a dose-limiting toxicity highlights the need to elucidate the basic mechanisms by which this antibody acts [16,19].

Previous studies conducted by our group have corroborated the antitumor efficacy of nimotuzumab against U87MG xenografts either alone or in combination with ionizing radiation [20,21]. These studies have suggested that the antitumor activity of nimotuzumab in U87MG xenografts might be in part due to its ability to target the CD133+ subpopulation, in addition to effectively block the EGFR [20,21]. However, molecular determinants accounting for the ability of nimotuzumab to target the CD133+ cells remains unclear. In the current study we investigated whether U87MG cells with ability to form neurospheres express the CD133+ CSC molecular marker and whether CD133 is co-localized with EGFR protein as analyzed by immunofluorescence. Moreover, the growth-inhibitory effects of nimotuzumab in combination with ionizing radiation in U87MG cells were evaluated either in cell culture or growing as xenografts in NMRI nude mice.

Materials and Methods

Cells and tumor spheres culture

U87MG (HTB-14, ATCC) is a human GBM cell line. Cells were grown in a 1+1 mixture of Eagle’s MEM and Basal medium (Sigma) containing 2mM L-glutamine and 10% Fetal Bovine Serum (FBS) under a humidified atmosphere of 5%CO2 at 37°C. For obtaining tumor spheres, the cells were cultured as described previously [22] with modifications. The half of the medium was replaced with an equal volume of serum-free neural stem cell medium containing MEM, recombinant human epidermal growth factor (10 ng/ml; Sigma), and endothelial cell growth supplement factor (1μg/ml). This procedure was repeated every 24 h until several primary tumor spheres were visible under microscopy. At this point, all cell culture medium was discarded and maintained in serum-free neural stem cell medium. For obtaining secondary spheres, primary tumor spheres were mechanically disaggregated and cultured for additional 72 h in conditioned serum-free media MEM supplemented with EGF 10 ng/ mL and endothelial cell growth supplement factor 1 ug/mL.


The humanized anti-EGFR monoclonal antibody nimotuzumab was generated at the Center of Molecular Immunology [12]. Primary and secondary antibodies were purchased from commercial sources as listed: rat monoclonal anti CD31/PECAM-1 antibody (BD Pharmingen), horseradish peroxidase (HRP)-conjugated anti-rat IgG1 (Southern Biotech), biotin-conjugated mouse monoclonal antibody to CD133/1 (AC133) (Miltenyi Biotec), and HRPconjugated anti-mouse IgG (DakoCytomation).

Drug cytotoxicity assay

U87MG cells were seeded and plated onto 96-well plates at a density of 5x103 cells/well in 0.1 mL culture medium containing 10% FBS, and incubated at 37°C and 5% CO2. Twenty four hours later cells were exposed to different concentrations of nimotuzumab for the required time period. On days 0, 1, 2, and 3, culture medium was removed and 0.2 mL 3-(4,5)-dimethylthiahiazo(-z-yl)-3,5-diphenytetrazoliumromide (MTT) solution (0.5 mg/mL; Sigma) was added. The cells were incubated for 4h and the medium was replaced by 0.15 mL DMSO. The plates were agitated for 15 min and the optical density was measured at 550 nm in a photometer (Bio-TEK).

Immunofluoresence staining

For examination of neural stem cell markers, CD133 staining was detected with immunofluorescence. Immunofluorescence staining was performed following a protocol described [23] with modifications. The cells were deposed in slides, fixed in 4% paraformaldehyde, and frozen at -20°C until analysis. For immunostaining the cells were pretreated with normal goat serum for 30 minutes and then incubated with biotin-conjugated antibody against CD133/1 (1:10) for 1h, followed by incubation with streptavidine. For double labeling of CD133/EGFR, primary and secondary antibodies were used 1h for CD133 and overnight for EGFR. The cells were counterstained with DAPI to reveal the nuclei. The slides were examined and photographed with a laser confocal scanning microscope (Leica).


All the specimens were embedded in Tissue-Tek OCT (optimal cutting temperature) compound, shock frozen and stored in nitrogen until analysis. Immunostaining was performed on 4% paraformaldehyde-fixed, cryostat 5-μm tissue sections placed on glass slides. To detect microvessels, sections were stained with an antibody against CD31/PECAM-1 (1:100) as described previously [20]. Negative controls consisted of duplicate sections of the same specimens in which the primary antibody had been excluded and replaced with PBS or negative control immunoglobulin. Sections were visualized with 3,3’-diaminobenzidine as a chromogen and counterstained with Mayer’s hematoxylin. Representative tumor sections were identified on a light microscope (Zeiss, Axioskop 40) with an ocular magnification of X40 evaluating 4 to 5 tumors from each group.

Animal experiments

Female nude mice (8-10 weeks old, nu/nu) were obtained from Charles River. The mice were housed and maintained under aseptic conditions in facilities approved by the German Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the German Animal Protection Law, and their use was approved by the local responsible authorities. Animals met the requirements of the UKCCCR guidelines [24]. To produce xenografts, tumor cells were harvested from subconfluent cultures by treatment with 0.25% trypsin and 0.05% EDTA. Only single-cell suspensions with >90% viability were used for injections. Animals were inoculated with 107 U87MG tumor cells s.c. into left flank. Tumor volume were determined from direct measurement with calipers and calculated according to the formula: 0.5 x (large diameter) x (small diameter)2. Treatments were initiated three days after tumor cell injection. The treatment group consisted of eight mice, which received the anti-EGFR monoclonal antibody nimotuzumab concomitant with radiation. The antibody was administered intraperitoneal three times per week with 1 mg per mouse (50mg/ kg), during three weeks. For radiation, animals were exposed to a total dose of 3.0 Gy of total body radiation fractioned in 1.0 Gy weekly. The control group consisted of ten mice which received saline solution instead of the antibody. The antibody was administered 6 h before radiation therapy. All animals were sacrificed by day 36 when tumor weight from the control group exceeded the ten percent of total animal weight. Subcutaneous tumors were snapping frozen and stored for additional analyses.

Statistical Analysis

Statistical analysis was performed using the GraphPAD In-Stat software for Windows, version 4.0 (GraphPAD). Statistical testing was determined by Student´s t-test and P<0.05 was considered as significant.


In vitro cytotoxicity of nimotuzumab in U87MG cells either alone or in combination with radiation

We first explored the cytotoxic activity of the anti-EGFR monoclonal antibody nimotuzumab alone or in combination with radiation on the growth of U87MG cell line. For that purposes U87MG cells were incubated with the antibody at different concentrations by 1, 2, or 3 days and its cytotoxic activity was assessed by MTT assay. Figure 1A shows the growth inhibition profiles upon antibody incubation at different time points. The maximal cytotoxic activity of nimotuzumab was reached at concentrations higher than 1 μg/mL, when cells were incubated with the antibody by 3 days. However, even under extreme conditions assessed (high antibody concentrations and large incubations) the cytotoxicity found in treated cells after antibody exposure was weak and non-significant compared to the control group.