Journal: International Journal of Nanomedicine

Article Title: Cetuximab-conjugated iron oxide nanoparticles for cancer imaging and therapy

doi: 10.2147/IJN.S80134

Figure Lengend Snippet: EGFRvIII-ECD-hFc binding determined by enzyme-linked immunosorbent assay. Notes: Soluble EGFRvIII-ECD-hFc was coated on enzyme-linked immunosorbent assay plates, and binding of cetuximab ( A ) or cetuximab-functionalized and non-functionalized iron oxide nanoparticles ( B ) was detected using a horseradish peroxidase-conjugated goat anti-human kappa light chain antibody. The concentrations tested ranged from 1.34×10 −4 to 10 μg/mL for the anti-EGFR monoclonal antibody cetuximab and from 6.4×10 −4 to 120 μg Fe/mL for cet-PEG-dexSPIONs and PEG-dexSPIONs. The EC 50 was 0.0242 and 0.0646 μg/mL for cetuximab and cet-PEG-dexSPIONs, respectively. Each point represents the mean ± standard deviation of three determinations. Abbreviations: cet, cetuximab; dex, dextran; ECD, extracellular domain; EGFR, epithelial growth factor receptor; OD, optical density; PEG, polyethylene glycol; SPIONs, superparamagnetic iron oxide nanoparticles.

Article Snippet: After acid strip with ice-cold 0.2 M acetic acid and 0.5 M NaCl (pH 2.5), on ice for 5 minutes, the cells were washed once with ice-cold 1× PBS prior to incubation on ice for one hour with phycoerythrin-conjugated mouse anti-human EGFR monoclonal antibody (BD Bio-science Pharmingen) to label EGFR remained on the cell surface.

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: International Journal of Nanomedicine

Article Title: Cetuximab-conjugated iron oxide nanoparticles for cancer imaging and therapy

doi: 10.2147/IJN.S80134

Figure Lengend Snippet: Iron content of differential EGFR-expressing cells after a 2-hour incubation at 37°C with the synthesized iron oxide nanoparticles

Article Snippet: After acid strip with ice-cold 0.2 M acetic acid and 0.5 M NaCl (pH 2.5), on ice for 5 minutes, the cells were washed once with ice-cold 1× PBS prior to incubation on ice for one hour with phycoerythrin-conjugated mouse anti-human EGFR monoclonal antibody (BD Bio-science Pharmingen) to label EGFR remained on the cell surface.

Techniques: Incubation, Synthesized, Concentration Assay

Journal: International Journal of Nanomedicine

Article Title: Cetuximab-conjugated iron oxide nanoparticles for cancer imaging and therapy

doi: 10.2147/IJN.S80134

Figure Lengend Snippet: Iron uptake among the cell lines with differential EGFR expression levels. The A431, 32D, and 32D/EGFR cell lines were incubated with 25 μg Fe/mL of PEG-dexSPIONs or cet-PEG-dexSPIONs at 37°C for 2 hours. The excess iron was removed by washing with 1× phosphate-buffered saline, and the iron concentration in 4×10 6 cells from each cell line was determined using a thiocyanate-based spectrophotometric assay. The results are presented as the mean ± standard deviation of four determinations. Abbreviations: cet, cetuximab; dex, dextran; EGFR, epidermal growth factor receptor; PEG, polyethylene glycol; SPIONs, superparamagnetic iron oxide nanoparticles.

Article Snippet: After acid strip with ice-cold 0.2 M acetic acid and 0.5 M NaCl (pH 2.5), on ice for 5 minutes, the cells were washed once with ice-cold 1× PBS prior to incubation on ice for one hour with phycoerythrin-conjugated mouse anti-human EGFR monoclonal antibody (BD Bio-science Pharmingen) to label EGFR remained on the cell surface.

Techniques: Expressing, Incubation, Concentration Assay, Spectrophotometric Assay, Standard Deviation

Journal: International Journal of Nanomedicine

Article Title: Cetuximab-conjugated iron oxide nanoparticles for cancer imaging and therapy

doi: 10.2147/IJN.S80134

Figure Lengend Snippet: Therapeutic effect of cet-PEG-SPIONs. Notes: ( A ) Inhibition of epidermal growth factor-induced tyrosine phosphorylation of EGFR by cet-PEG-dexSPIONs. A431 cells were serum-starved overnight and then treated with cetuximab, cet-PEG-dexSPIONs, or PEG-dexSPIONs at varying doses for 2 hours at 37°C. A431 cells were further stimulated with 30 ng/mL EGF for 30 minutes at 37°C. Cell lysates were separated by electrophoresis, and immunoblotting was performed to detect the p-EGFR (Tyr1173) and total EGFR levels. β-actin was used as a loading control. A representative of three blots is shown. ( B ) EGFR internalization in response to cet-PEG-dexSPIONs treatment in A431 cells. Cells were incubated for the indicated times in the presence of cetuximab (2.5 μg/mL), cet-PEG-dexSPIONs (50 μg Fe/mL), or PEG-dexSPIONs (50 μg Fe/mL). After stripping off the surface-bound cetuximab or nanoparticles, the cells were stained on ice for one hour with PE-conjugated mouse anti-human EGFR monoclonal antibody to determine the EGFR level remaining on the cell surface by flow cytometry. The expression level of EGFR on untreated A431 cells at 0 hours was set at 100%. ( C ) Apoptosis-inducing activity of cet-PEG-dexSPIONs in EGFR-expressing cell lines analyzed by an Annexin V-fluorescein isothiocyanate/propidium iodide assay. The stacked bar graph shows the proportion of different cell populations among the EGFR-null 32D cells, EGFR-overexpressing 32D/EGFR cells, and A431 cells. Abbreviations: cet, cetuximab; dex, dextran; EGF, epidermal growth factor; PEG, polyethylene glycol; PE, phycoerythrin; SPIONs, superparamagnetic iron oxide nanoparticles; p-EGFR, phosphorylated epidermal growth factor receptor.

Article Snippet: After acid strip with ice-cold 0.2 M acetic acid and 0.5 M NaCl (pH 2.5), on ice for 5 minutes, the cells were washed once with ice-cold 1× PBS prior to incubation on ice for one hour with phycoerythrin-conjugated mouse anti-human EGFR monoclonal antibody (BD Bio-science Pharmingen) to label EGFR remained on the cell surface.

Techniques: Inhibition, Electrophoresis, Western Blot, Incubation, Stripping Membranes, Staining, Flow Cytometry, Expressing, Activity Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: In Pancreatic Carcinoma, Dual EGFR/HER2 Targeting with Cetuximab/Trastuzumab Is More Effective than Treatment with Trastuzumab/Erlotinib or Lapatinib Alone: Implication of Receptors' Down-regulation and Dimers' Disruption 1

doi:

Figure Lengend Snippet: Comparison of the antitumor activity of the 2mAbs therapy (cetuximab/trastuzumab) and of the erlotinib/trastuzumab combination in nude mice xenografted subcutaneously with Capan-1 and BxPC-3 human pancreatic carcinoma cells. The weight of the animals was followed up during treatment in the Capan-1 (A) and BxPC-3 (B) xenograft models. At days 14 (Capan-1 cells) (C) and 26 (BxPC-3 cells) (D) after graft, groups of 10 mice were treated with the two mAbs (ratio 1:1; 2 mg/kg of each mAb, twice a week), erlotinib (100 mg/kg; daily) plus trastuzumab (2 mg/kg, twice a week), or sterile PBS (daily). Results are presented as the mean tumor volume for each group. Bars, SD of the mean. C + T indicates cetuximab/trastuzumab; E + T, erlotinib/trastuzumab.

Article Snippet: The anti-EGFR mAbs cetuximab (human) and m425 (mouse) were purchased from Merck KGaA (Darmstadt, Germany) and Merck AG (Frankfurt, Germany), respectively.

Techniques: Comparison, Activity Assay, Sterility

Journal: Neoplasia (New York, N.Y.)

Article Title: In Pancreatic Carcinoma, Dual EGFR/HER2 Targeting with Cetuximab/Trastuzumab Is More Effective than Treatment with Trastuzumab/Erlotinib or Lapatinib Alone: Implication of Receptors' Down-regulation and Dimers' Disruption 1

doi:

Figure Lengend Snippet: Median Survival and Therapeutic Benefit of BxPC3 and Capan-1 Xenografted Mice Treated with TKI and/or Monoclonal Antibodies.

Article Snippet: The anti-EGFR mAbs cetuximab (human) and m425 (mouse) were purchased from Merck KGaA (Darmstadt, Germany) and Merck AG (Frankfurt, Germany), respectively.

Techniques: Control

Journal: Neoplasia (New York, N.Y.)

Article Title: In Pancreatic Carcinoma, Dual EGFR/HER2 Targeting with Cetuximab/Trastuzumab Is More Effective than Treatment with Trastuzumab/Erlotinib or Lapatinib Alone: Implication of Receptors' Down-regulation and Dimers' Disruption 1

doi:

Figure Lengend Snippet: Comparison of the effect of lapatinib (dual HER2/EGFR TKI) and of the 2mAbs therapy on tumor growth in nude mice xenografted subcutaneously with BxPC-3 (A), Capan-1 (B), or SKOV-3 (C) cells and randomized in different groups (n = 10 per group). At days 14 (Capan-1), 20 (BxPC-3), and 11 (SKOV-3 cells) after graft, mice were treated with lapatinib (100, 200, or 300 mg/kg, daily), the 2mAbs therapy (ratio 1:1; 2 mg/kg of each mAb, twice per week) or sterile PBS (daily). Results are presented as the mean tumor volume of each group. Bars, SD of the mean. C indicates cetuximab; Lap, lapatinib; T, trastuzumab.

Article Snippet: The anti-EGFR mAbs cetuximab (human) and m425 (mouse) were purchased from Merck KGaA (Darmstadt, Germany) and Merck AG (Frankfurt, Germany), respectively.

Techniques: Comparison, Sterility

Journal: Neoplasia (New York, N.Y.)

Article Title: In Pancreatic Carcinoma, Dual EGFR/HER2 Targeting with Cetuximab/Trastuzumab Is More Effective than Treatment with Trastuzumab/Erlotinib or Lapatinib Alone: Implication of Receptors' Down-regulation and Dimers' Disruption 1

doi:

Figure Lengend Snippet: Comparison of the antitumor effect, in vitro and in vivo, of the 2mAbs therapy (cetuximab and trastuzumab) using intact mAbs or the F(ab′)2 fragments. (A) ADCC was assessed in vitro by incubating 51Cr-labeled BxPC-3 target cells with peripheral blood mononuclear cells (effector cells) at different concentrations in the presence of cetuximab and/or trastuzumab (whole mAbs) or their F(ab′)2 fragments. ADCC was determined by measuring the 51Cr released in the supernatant. (B) SCID/Beige mice bearing BxPC-3 pancreatic carcinoma xenografts were treated at day 20 after graft by daily coinjection of F(ab′)2 fragments of the two mAbs or twice per week with the intact mAbs. Results are presented as the mean tumor volume of each treated group. Bars, SD of the mean. C indicates cetuximab; double-head arrow, period of treatment; T, trastuzumab.

Article Snippet: The anti-EGFR mAbs cetuximab (human) and m425 (mouse) were purchased from Merck KGaA (Darmstadt, Germany) and Merck AG (Frankfurt, Germany), respectively.

Techniques: Comparison, In Vitro, In Vivo, Labeling

Journal: Neoplasia (New York, N.Y.)

Article Title: In Pancreatic Carcinoma, Dual EGFR/HER2 Targeting with Cetuximab/Trastuzumab Is More Effective than Treatment with Trastuzumab/Erlotinib or Lapatinib Alone: Implication of Receptors' Down-regulation and Dimers' Disruption 1

doi:

Figure Lengend Snippet: (A) The 2mAbs therapy induces long-term EGFR and HER2 down-regulation and inhibition of AKT phosphorylation in Capan-1 pancreatic carcinoma cells. Mice bearing Capan-1 xenografts were treated with the 2mAbs (cetuximab/trastuzumab) therapy (ratio 1:1; 2 mg/kg of each mAb, twice a week), erlotinib (100 mg/kg, daily) plus trastuzumab (2 mg/kg, twice per week), or lapatinib (300 mg/kg, daily). Tumors were resected at day 2, 7, or 15 after the beginning of treatment. Cell lysates were analyzed by Western blot analysis for EGFR and HER2, total AKT, phosphorylated AKT, total MAPK, and phosphorylated MAPK expression. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as loading control. C + T indicates 2mAbs. (B) The 2mAbs therapy induces long-term inhibition of the overall tyrosine phosphorylation profile in Capan-1 tumor cells, whereas the tested TKIs did not. Mice xenografted with Capan-1 pancreatic carcinoma cells were treated with either the two mAbs therapy (ratio 1:1; 2 mg/kg of each mAb, twice per week), erlotinib (100 mg/kg, daily) plus trastuzumab (2 mg/kg, twice per day), or lapatinib (300 mg/kg, daily). Tumors were resected at day 2, 3, or 7 after beginning the mAbs therapy and at 1 hour, 6 hours, 7 days, and 15 days after the beginning of the treatment with erlotinib/trastuzumab or lapatinib. Cell lysates were analyzed by Western blot analysis for tyrosine phosphorylation (P-Tyr). C + T indicates cetuximab/trastuzumab; E + T, erlotinib/trastuzumab.

Article Snippet: The anti-EGFR mAbs cetuximab (human) and m425 (mouse) were purchased from Merck KGaA (Darmstadt, Germany) and Merck AG (Frankfurt, Germany), respectively.

Techniques: Inhibition, Phospho-proteomics, Western Blot, Expressing, Control

Journal: Neoplasia (New York, N.Y.)

Article Title: In Pancreatic Carcinoma, Dual EGFR/HER2 Targeting with Cetuximab/Trastuzumab Is More Effective than Treatment with Trastuzumab/Erlotinib or Lapatinib Alone: Implication of Receptors' Down-regulation and Dimers' Disruption 1

doi:

Figure Lengend Snippet: Effect of the 2mAbs (cetuximab/trastuzumab) therapy, of erlotinib plus trastuzumab, or of lapatinib alone on receptor heterodimerization and homodimerization using the antibody-based TR-FRET assay. (A) Quantification of EGFR/HER2 heterodimers in Capan-1 and BxPC-3 cells. (B) BxPC-3 cells were treated with increasing concentrations of cetuximab/trastuzumab (ratio 1:1), erlotinib and/or trastuzumab, or lapatinib for 10 minutes and then fixed in 10% formalin. Fixed cells were then incubated with d2-labeled anti-EGFR and lumi4-terbium-labeled anti-HER2 mAbs (directed against different epitopes than the ones recognized by cetuximab and trastuzumab) for 6 hours. Cells were washed, and the TR-FRET signals were measured at 665 nm in the TRF mode (60-µs delay, 400-µs integration) on 337 nm excitation to estimate the dimer level. The TR-FRET signal was expressed as ΔF665 (%) and then as the dimer percentage (see Materials and Methods). (C) Effect of the different therapies on EGFR or (D) HER2 homodimer formation. Data are mean ± SEM of three independent experiments performed in triplicate. ****P < .0001; ***P < .001, by two-way analysis of variance, with Bonferroni multiple-comparison post test (each treatment was compared with the irrelevant mAb treatment).

Article Snippet: The anti-EGFR mAbs cetuximab (human) and m425 (mouse) were purchased from Merck KGaA (Darmstadt, Germany) and Merck AG (Frankfurt, Germany), respectively.

Techniques: Incubation, Labeling, Comparison