Cetuximab-based immunotherapy and radioimmunotherapy of head and neck squamous cell carcinoma

Abstract

Purpose: To show the relationship between antibody delivery and therapeutic efficacy in head and neck cancers, in this study we evaluated the pharmacokinetics and pharmacodynamics of epidermal growth factor receptor (EGFR)-targeted immunotherapy and radioimmunotherapy by quantitative positron emission tomography (PET) imaging.

Experimental design: EGFR expression on UM-SCC-22B and SCC1 human head and neck squamous cell cancer (HNSCC) cells were determined by flow cytometry and immunostaining. Tumor delivery and distribution of cetuximab in tumor-bearing nude mice were evaluated with small animal PET using (64)Cu-DOTA-cetuximab. The in vitro toxicity of cetuximab to HNSCC cells was evaluated by MTT assay. The tumor-bearing mice were then treated with four doses of cetuximab at 10 mg/kg per dose, and tumor growth was evaluated by caliper measurement. FDG PET was done after the third dose of antibody administration to evaluate tumor response. Apoptosis and tumor cell proliferation after cetuximab treatment were analyzed by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and Ki-67 staining. Radioimmunotherapy was done with (90)Y-DOTA-cetuximab.

Results: EGFR expression on UM-SCC-22B cells is lower than that on SCC1 cells. However, the UM-SCC-22B tumors showed much higher (64)Cu-DOTA-cetuximab accumulation than the SCC1 tumors. Cetuximab-induced apoptosis in SCC1 tumors and tumor growth was significantly inhibited, whereas an agonistic effect of cetuximab on UM-SCC-22B tumor growth was observed. After cetuximab treatment, the SCC1 tumors showed decreased FDG uptake, and the UM-SCC-22B tumors had increased FDG uptake. UM-SCC-22B tumors are more responsive to (90)Y-DOTA-cetuximab treatment than SCC1 tumors, partially due to the high tumor accumulation of the injected antibody.

Conclusion: Cetuximab has an agonistic effect on the growth of UM-SCC-22B tumors, indicating that tumor response to cetuximab treatment is not necessarily related to EGFR expression and antibody delivery efficiency, as determined by PET imaging. Although PET imaging with antibodies as tracers has limited function in patient screening, it can provide guidance for targeted therapy using antibodies as delivery vehicles.

FIGURE 1

a Flow cytometric analysis of EGFR expression on HNSCC cells. Cetuximab was used as the primary antibody and FITC-conjugated donkey anti-human IgG was used as secondary antibody. The mean values of FITC signal intensity (MFI) of the three measurements are shown (mean ± SD) (22B stands for UM-SCC-22B). b & c, Immunofluorescence examination of EGFR expression. Images were obtained under the same conditions and are displayed at the same magnification and scale. Tumor sections were directly stained with cetuximab as primary antibody, and FITC-conjugated donkey anti-human IgG as secondary antibody. Murine CD31 was stained with Cy3-conjugated IgG to visualize tumor vasculature (b). Thirty hours after DOTA-cetuximab injection, tumors were harvested and tumor sections were stained with FITC-conjugated donkey anti-human IgG (c). (red color from Cy3 for CD31; green color from FITC for EGFR and cetuximab; blue color from DAPI for nuclei visualization). Representative photos were taken under ×200 magnification and scale bar equals to 50 μm. MicroPET images of HNSCC UM-SCC-22B (d) and SCC1 (e) tumor-bearing nude mice at different time points after intravenous injection of ⁶⁴Cu-DOTA-cetuximab (n = 4 per group). Decay-corrected coronal images at different time points are shown and the tumors are indicated by white arrows.

FIGURE 2

Anti-tumor activity of cetuximab in established HNSCC xenografts. a, The cytotoxic effect of cetuximab on SCC1 (left panel) and UM-SCC-22B (right panel) cells. Cells were treated with serial concentrations of cetuximab. At 72 and 120 hr after inoculation, the cell proliferation was determined by MTT assay. b, Comparison of SCC1 (left panel) and UM-SCC-22B (right panel) tumor growth in nude mice treated with cetuximab vs. control animals. c, Comparison of body mass of nude mice bearing SCC1 (left panel) and UM-SCC-22B (right panel) treated with cetuximab vs. control animals. Animals were injected through tail vein with 50 mg/kg cetuximab for 4 doses. *, P < 0.05; **, P < 0.01.

FIGURE 3

Small animal PET images of SCC1 (a) or UM-SCC-22B (b) tumor-bearing nude mice with ¹⁸F-FDG at day 5 after treatment with cetuximab (n = 4 to 7). Decay-corrected whole-body coronal images that contain the tumor were shown and the tumors are indicated by white arrows. SCC1 (c) and UM-SCC-22B (d) tumor uptake of ¹⁸F-FDG as quantified from small animal FDG PET scans (n= 4 to 7).

FIGURE 4

Double staining of TUNEL and CD31 of SCC1 (a) and UM-SCC-22B (b) tumor section after treatment with 100mg/kg of cetuximab. Vasculature is shown in green and apoptotic nuclei are shown in red. Normal cell nuclei are shown in blue, stained by DAPI. Representative photos were taken under ×200 magnification and scale bar equals 50 μm. c and d, Microvascular Density (MVD) measurement in SCC1 (c) and UM-SCC-22B (d) tumors. *, P < 0.05; **, P < 0.01.

FIGURE 5

Double staining of Ki67 and CD31 of SCC1 (a) and UM-SCC-22B (b) tumor section after treatment with 100mg/kg of cetuximab. Vasculature is shown in green and Ki67 positive nuclei are shown in red. Representative photos were taken under ×200 magnification and scale bar equals 50 μm. c (SSC1) and d (UM-SCC-22B), Ki-67 staining index (SI) calculated based on staining. *, P < 0.05; **, P < 0.01.

FIGURE 6

⁹⁰Y-centuximab inhibition of SCC1 (a) and UM-SCC-22B (b) tumor growth in vivo. Female athymic nude mice bearing tumors were injected with a one-time dose of saline and 3.7 MBq of ⁹⁰Y-cetuximab. The growth inhibition of experimental groups was monitored via serial caliper measurements.