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Targeted therapy of glioblastoma stem-like cells and tumor non-stem cells using cetuximab-conjugated iron-oxide nanoparticles.

Kaluzova M, Bouras A, Machaidze R, Hadjipanayis CG - Oncotarget (2015)

Bottom Line: Transmission electron microscopy revealed cetuximab-IONP GBM cell binding and internalization.Treatment with cetuximab-IONPs resulted in a significant antitumor effect that was greater than with cetuximab alone due to more efficient, CD133-independent cellular targeting and uptake, EGFR signaling alterations, EGFR internalization, and apoptosis induction in EGFR-expressing GSCs and neurospheres.A significant increase in survival was found after cetuximab-IONP convection-enhanced delivery treatment of 3 intracranial rodent GBM models employing human EGFR-expressing GBM xenografts.

View Article: PubMed Central - PubMed

Affiliation: Brain Tumor Nanotechnology Laboratory, Department of Neurosurgery, Emory University School of Medicine, Winship Cancer Institute of Emory University, Atlanta, GA, USA.

ABSTRACT
Malignant gliomas remain aggressive and lethal primary brain tumors in adults. The epidermal growth factor receptor (EGFR) is frequently overexpressed in the most common malignant glioma, glioblastoma (GBM), and represents an important therapeutic target. GBM stem-like cells (GSCs) present in tumors are felt to be highly tumorigenic and responsible for tumor recurrence. Multifunctional magnetic iron-oxide nanoparticles (IONPs) can be directly imaged by magnetic resonance imaging (MRI) and designed to therapeutically target cancer cells. The targeting effects of IONPs conjugated to the EGFR inhibitor, cetuximab (cetuximab-IONPs), were determined with EGFR- and EGFRvIII-expressing human GBM neurospheres and GSCs. Transmission electron microscopy revealed cetuximab-IONP GBM cell binding and internalization. Fluorescence microscopy and Prussian blue staining showed increased uptake of cetuximab-IONPs by EGFR- as well as EGFRvIII-expressing GSCs and neurospheres in comparison to cetuximab or free IONPs. Treatment with cetuximab-IONPs resulted in a significant antitumor effect that was greater than with cetuximab alone due to more efficient, CD133-independent cellular targeting and uptake, EGFR signaling alterations, EGFR internalization, and apoptosis induction in EGFR-expressing GSCs and neurospheres. A significant increase in survival was found after cetuximab-IONP convection-enhanced delivery treatment of 3 intracranial rodent GBM models employing human EGFR-expressing GBM xenografts.

No MeSH data available.


Related in: MedlinePlus

Apoptosis in human GBM neurospheres containing GSCs treated with cetuximab-IONPsTransport of EGFR to the cytoskeletal structures. Neurospheres were treated with free IONPs (0.2 mg/ml), cetuximab-IONPs (0.2 mg/ml), control vehicle, or cetuximab alone (50 μg/ml) and expression of apoptotic proteins was evaluated by Western blotting. Elevated levels of cleaved caspase 3 and cleaved PARP were found in neurospheres N08-74 and N08-30 after treatment with cetuximab-IONPs for 3 (A, left) and in neurospheres N08-74 for 14 hs (A, right). Treatment with cetuximab-IONPs was most effective in inducing cleavage of caspase 3 and PARP although some caspase 3 cleavage was also induced by free IONPs in N08-30. In neurospheres N08-1002, induction of caspase 3 and PARP cleavage, and decreased phosphorylation of ERK 44/42 was found after 3 h treatment with cetuximab-IONPs and cetuximab alone, both in the presence and absence of EGF and FGF, caspase 3 was used as a control (B, top). Treatment with cetuximab-IONPs (but not the control conjugated antibody) increased cleavage of PARP in neurospheres N08-1002 whereas no cleavage was observed in NHPC (B, bottom). (C) N08-30 neurospheres were treated as above for 5 hs, lysates were subcellularly fractionated, and analyzed by Western blotting. Elevated levels of wtEGFR were found in the cytoskeletal fraction after cells were treated with cetuximab-IONPs. (D) U87MG and U87MGwtEGFR human GBM cell lines were treated with free IONPs, cetuximab-IONPs, or cetuximab alone. Apoptosis, as indicated by activation of caspase 3 cleavage, was seen only in the U87MGwtEGFR cell line treated with cetuximab-IONPs.
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Figure 3: Apoptosis in human GBM neurospheres containing GSCs treated with cetuximab-IONPsTransport of EGFR to the cytoskeletal structures. Neurospheres were treated with free IONPs (0.2 mg/ml), cetuximab-IONPs (0.2 mg/ml), control vehicle, or cetuximab alone (50 μg/ml) and expression of apoptotic proteins was evaluated by Western blotting. Elevated levels of cleaved caspase 3 and cleaved PARP were found in neurospheres N08-74 and N08-30 after treatment with cetuximab-IONPs for 3 (A, left) and in neurospheres N08-74 for 14 hs (A, right). Treatment with cetuximab-IONPs was most effective in inducing cleavage of caspase 3 and PARP although some caspase 3 cleavage was also induced by free IONPs in N08-30. In neurospheres N08-1002, induction of caspase 3 and PARP cleavage, and decreased phosphorylation of ERK 44/42 was found after 3 h treatment with cetuximab-IONPs and cetuximab alone, both in the presence and absence of EGF and FGF, caspase 3 was used as a control (B, top). Treatment with cetuximab-IONPs (but not the control conjugated antibody) increased cleavage of PARP in neurospheres N08-1002 whereas no cleavage was observed in NHPC (B, bottom). (C) N08-30 neurospheres were treated as above for 5 hs, lysates were subcellularly fractionated, and analyzed by Western blotting. Elevated levels of wtEGFR were found in the cytoskeletal fraction after cells were treated with cetuximab-IONPs. (D) U87MG and U87MGwtEGFR human GBM cell lines were treated with free IONPs, cetuximab-IONPs, or cetuximab alone. Apoptosis, as indicated by activation of caspase 3 cleavage, was seen only in the U87MGwtEGFR cell line treated with cetuximab-IONPs.

Mentions: Next, we examined the mechanism of cytotoxicity of the cetuximab-IONPs to GBM neurospheres, focusing on apoptosis and autophagy as putative mechanisms of cell death. In human GBM neurospheres N08-74, N08-30, and N08-1002, none of the treatments induced conversion of LC3B-I to LCB3-II, the hallmark of autophagy (data not shown), suggesting that autophagy is not a likely mode of cell death. In contrast, treatment with cetuximab-IONPs resulted in elevated levels of cleaved caspase 3 and cleaved PARP in GBM neurospheres N08-74, without any cleavage observed in cells treated with free IONPs and cetuximab for 3 hs (Figure 3A, left). After a 14 h incubation, induction of caspase 3 cleavage was even more pronounced (Figure 3A, right). In neurospheres N08-30, treatment with cetuximab-IONPs also resulted in elevated levels of cleaved PARP and cleaved caspase 3. Free IONPs also increased caspase 3 cleavage (Figure 3A, left), most likely due to nonspecific uptake as evidenced in Figure 1C, bottom. On the other hand, in neurospheres N08-1002, both cetuximab-IONPs and cetuximab alone caused apoptosis through cleavage of PARP and caspase 3. Cetuximab-IONPs and cetuximab alone inhibited phosphorylation of Y1068 in EGFR in N08-74 (data not shown). A concomitant decrease in phospho-ERK44/42 levels was also observed in the presence/absence of EGF and FGF (Figure 3B, top) (cetuximab has up to 10-fold higher affinity for EGFR than the EGF and can thus competitively inhibit EGF binding to the receptor [41]). When comparing GBM neurospheres N08-1002 and NHPC, elevated PARP cleavage was observed only in neurospheres treated with cetuximab-IONPs (Figure 3B, bottom). Consistently, U0126 (inhibitor of the ERK pathway) also increased PARP cleavage in cetuximab-IONP-treated cells (data not shown). By subcellular protein fractionation we found that treatment with cetuximab-IONPs for 2 (data not shown) and 5 hs promoted translocation of wtEGFR to the cytoplasmic (data not shown) and predominantly to the cytoskeletal fraction (Figure 3C, 60-fold increase over control as indicated by densitometric analysis). Compared with control, cetuximab-IONP treatment induced increased translocation of wtEGFR to the lysosomes (data not shown), suggesting increased lysosomal degradation of EGFR in the presence cetuximab-IONPs. In U87MG cells, cetuximab-IONP treatment resulted in elevated levels of cleaved caspase 3 only in U87MGwtEGFR cells but not in the parental U87MG cell line with basal level of wtEGFR (Figure 3D). These data highlight the necessity of EGFR for biological activity of the cetuximab-IONPs.


Targeted therapy of glioblastoma stem-like cells and tumor non-stem cells using cetuximab-conjugated iron-oxide nanoparticles.

Kaluzova M, Bouras A, Machaidze R, Hadjipanayis CG - Oncotarget (2015)

Apoptosis in human GBM neurospheres containing GSCs treated with cetuximab-IONPsTransport of EGFR to the cytoskeletal structures. Neurospheres were treated with free IONPs (0.2 mg/ml), cetuximab-IONPs (0.2 mg/ml), control vehicle, or cetuximab alone (50 μg/ml) and expression of apoptotic proteins was evaluated by Western blotting. Elevated levels of cleaved caspase 3 and cleaved PARP were found in neurospheres N08-74 and N08-30 after treatment with cetuximab-IONPs for 3 (A, left) and in neurospheres N08-74 for 14 hs (A, right). Treatment with cetuximab-IONPs was most effective in inducing cleavage of caspase 3 and PARP although some caspase 3 cleavage was also induced by free IONPs in N08-30. In neurospheres N08-1002, induction of caspase 3 and PARP cleavage, and decreased phosphorylation of ERK 44/42 was found after 3 h treatment with cetuximab-IONPs and cetuximab alone, both in the presence and absence of EGF and FGF, caspase 3 was used as a control (B, top). Treatment with cetuximab-IONPs (but not the control conjugated antibody) increased cleavage of PARP in neurospheres N08-1002 whereas no cleavage was observed in NHPC (B, bottom). (C) N08-30 neurospheres were treated as above for 5 hs, lysates were subcellularly fractionated, and analyzed by Western blotting. Elevated levels of wtEGFR were found in the cytoskeletal fraction after cells were treated with cetuximab-IONPs. (D) U87MG and U87MGwtEGFR human GBM cell lines were treated with free IONPs, cetuximab-IONPs, or cetuximab alone. Apoptosis, as indicated by activation of caspase 3 cleavage, was seen only in the U87MGwtEGFR cell line treated with cetuximab-IONPs.
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Figure 3: Apoptosis in human GBM neurospheres containing GSCs treated with cetuximab-IONPsTransport of EGFR to the cytoskeletal structures. Neurospheres were treated with free IONPs (0.2 mg/ml), cetuximab-IONPs (0.2 mg/ml), control vehicle, or cetuximab alone (50 μg/ml) and expression of apoptotic proteins was evaluated by Western blotting. Elevated levels of cleaved caspase 3 and cleaved PARP were found in neurospheres N08-74 and N08-30 after treatment with cetuximab-IONPs for 3 (A, left) and in neurospheres N08-74 for 14 hs (A, right). Treatment with cetuximab-IONPs was most effective in inducing cleavage of caspase 3 and PARP although some caspase 3 cleavage was also induced by free IONPs in N08-30. In neurospheres N08-1002, induction of caspase 3 and PARP cleavage, and decreased phosphorylation of ERK 44/42 was found after 3 h treatment with cetuximab-IONPs and cetuximab alone, both in the presence and absence of EGF and FGF, caspase 3 was used as a control (B, top). Treatment with cetuximab-IONPs (but not the control conjugated antibody) increased cleavage of PARP in neurospheres N08-1002 whereas no cleavage was observed in NHPC (B, bottom). (C) N08-30 neurospheres were treated as above for 5 hs, lysates were subcellularly fractionated, and analyzed by Western blotting. Elevated levels of wtEGFR were found in the cytoskeletal fraction after cells were treated with cetuximab-IONPs. (D) U87MG and U87MGwtEGFR human GBM cell lines were treated with free IONPs, cetuximab-IONPs, or cetuximab alone. Apoptosis, as indicated by activation of caspase 3 cleavage, was seen only in the U87MGwtEGFR cell line treated with cetuximab-IONPs.
Mentions: Next, we examined the mechanism of cytotoxicity of the cetuximab-IONPs to GBM neurospheres, focusing on apoptosis and autophagy as putative mechanisms of cell death. In human GBM neurospheres N08-74, N08-30, and N08-1002, none of the treatments induced conversion of LC3B-I to LCB3-II, the hallmark of autophagy (data not shown), suggesting that autophagy is not a likely mode of cell death. In contrast, treatment with cetuximab-IONPs resulted in elevated levels of cleaved caspase 3 and cleaved PARP in GBM neurospheres N08-74, without any cleavage observed in cells treated with free IONPs and cetuximab for 3 hs (Figure 3A, left). After a 14 h incubation, induction of caspase 3 cleavage was even more pronounced (Figure 3A, right). In neurospheres N08-30, treatment with cetuximab-IONPs also resulted in elevated levels of cleaved PARP and cleaved caspase 3. Free IONPs also increased caspase 3 cleavage (Figure 3A, left), most likely due to nonspecific uptake as evidenced in Figure 1C, bottom. On the other hand, in neurospheres N08-1002, both cetuximab-IONPs and cetuximab alone caused apoptosis through cleavage of PARP and caspase 3. Cetuximab-IONPs and cetuximab alone inhibited phosphorylation of Y1068 in EGFR in N08-74 (data not shown). A concomitant decrease in phospho-ERK44/42 levels was also observed in the presence/absence of EGF and FGF (Figure 3B, top) (cetuximab has up to 10-fold higher affinity for EGFR than the EGF and can thus competitively inhibit EGF binding to the receptor [41]). When comparing GBM neurospheres N08-1002 and NHPC, elevated PARP cleavage was observed only in neurospheres treated with cetuximab-IONPs (Figure 3B, bottom). Consistently, U0126 (inhibitor of the ERK pathway) also increased PARP cleavage in cetuximab-IONP-treated cells (data not shown). By subcellular protein fractionation we found that treatment with cetuximab-IONPs for 2 (data not shown) and 5 hs promoted translocation of wtEGFR to the cytoplasmic (data not shown) and predominantly to the cytoskeletal fraction (Figure 3C, 60-fold increase over control as indicated by densitometric analysis). Compared with control, cetuximab-IONP treatment induced increased translocation of wtEGFR to the lysosomes (data not shown), suggesting increased lysosomal degradation of EGFR in the presence cetuximab-IONPs. In U87MG cells, cetuximab-IONP treatment resulted in elevated levels of cleaved caspase 3 only in U87MGwtEGFR cells but not in the parental U87MG cell line with basal level of wtEGFR (Figure 3D). These data highlight the necessity of EGFR for biological activity of the cetuximab-IONPs.

Bottom Line: Transmission electron microscopy revealed cetuximab-IONP GBM cell binding and internalization.Treatment with cetuximab-IONPs resulted in a significant antitumor effect that was greater than with cetuximab alone due to more efficient, CD133-independent cellular targeting and uptake, EGFR signaling alterations, EGFR internalization, and apoptosis induction in EGFR-expressing GSCs and neurospheres.A significant increase in survival was found after cetuximab-IONP convection-enhanced delivery treatment of 3 intracranial rodent GBM models employing human EGFR-expressing GBM xenografts.

View Article: PubMed Central - PubMed

Affiliation: Brain Tumor Nanotechnology Laboratory, Department of Neurosurgery, Emory University School of Medicine, Winship Cancer Institute of Emory University, Atlanta, GA, USA.

ABSTRACT
Malignant gliomas remain aggressive and lethal primary brain tumors in adults. The epidermal growth factor receptor (EGFR) is frequently overexpressed in the most common malignant glioma, glioblastoma (GBM), and represents an important therapeutic target. GBM stem-like cells (GSCs) present in tumors are felt to be highly tumorigenic and responsible for tumor recurrence. Multifunctional magnetic iron-oxide nanoparticles (IONPs) can be directly imaged by magnetic resonance imaging (MRI) and designed to therapeutically target cancer cells. The targeting effects of IONPs conjugated to the EGFR inhibitor, cetuximab (cetuximab-IONPs), were determined with EGFR- and EGFRvIII-expressing human GBM neurospheres and GSCs. Transmission electron microscopy revealed cetuximab-IONP GBM cell binding and internalization. Fluorescence microscopy and Prussian blue staining showed increased uptake of cetuximab-IONPs by EGFR- as well as EGFRvIII-expressing GSCs and neurospheres in comparison to cetuximab or free IONPs. Treatment with cetuximab-IONPs resulted in a significant antitumor effect that was greater than with cetuximab alone due to more efficient, CD133-independent cellular targeting and uptake, EGFR signaling alterations, EGFR internalization, and apoptosis induction in EGFR-expressing GSCs and neurospheres. A significant increase in survival was found after cetuximab-IONP convection-enhanced delivery treatment of 3 intracranial rodent GBM models employing human EGFR-expressing GBM xenografts.

No MeSH data available.


Related in: MedlinePlus