EPR Oximetry of Cetuximab-Treated Head-and-Neck Tumours in a Mouse Model

Abstract

Head and neck squamous cell carcinoma (HNSCC) tumours are associated with high mortality despite advances in therapy. The monoclonal antibody cetuximab (Erbitux^®) has been approved for the treatment of advanced HNSCC. However, only a subset of HNSC patients receiving cetuximab actually responds to treatment, underlining the need for a means to tailor treatments of individual patients. The aim of the present study was to investigate the effect of cetuximab treatment on tumour growth, on tumour partial oxygen pressure as measured by LiPc electron paramagnetic resonance oximetry and on the expression of proteins involved in tumour growth, metabolism and hypoxia. Two HNSCC cell lines, UT-SCC-2 and UT-SCC-14, were used to generate xenografts on female BALB/c (nu/nu) nude mice. Mice with xenografts were given three injections of intraperitoneal cetuximab or phosphate-buffered saline, and the tumour volume was recorded continuously. After treatment the tumour partial oxygen pressure was measured by LiPc electron paramagnetic resonance oximetry and the expression of epidermal growth factor receptor (EGFR), phosphorylated EGFR, Ki-67, MCT1, MCT4, GLUT1, CAIX and HIF-1α were investigated by immunohistochemistry. In xenografts from both cell lines (UT-SCC-2 and UT-SCC-14) cetuximab had effect on the tumour volume but the effect was more pronounced on UT-SCC-14 xenografts. A higher tumour oxygenation was measured in cetuximab-treated tumours from both cell lines compared to untreated controls. Immunocytochemical staining after cetuximab treatment shows a significantly decreased expression of EGFR, pEGFR, Ki67, CAIX and nuclear HIF-1α in UT-SCC-14 tumours compared to untreated controls. MCT1 and GLUT1 were significantly decreased in tumours from both cell lines but more pronounced in UT-SCC-14 tumours. Taken together, our results show that cetuximab treatment decreases the tumour growth and increases the tumour partial oxygen pressure of HNSCC xenografts. Furthermore we found a potential connection between the partial oxygen pressure of the tumours and the expression of proteins involved in tumour growth, metabolism and hypoxia.

Keywords: Cetuximab; EPR oximetry; Head and neck cancer; Hypoxia; Metabolism; Tumour oxygen pressure.

Fig. 1

Tumour growth. Xenografts were established in female nude mice (BALB c[nu/nu]) by subcutaneous injection of head and neck squamous cell carcinoma cell lines a UT-SCC-14 and b UT-SCC-2. Cetuximab (1 mg/injection) was administered by intraperitoneal injection at day 10, 13 and 16. The tumour size was recorded at an interval of 2–3 days, n = 10-14. Letter a indicates that the tumour volume of untreated controls is significantly different than that of cetuximab-treated group (P < 0.05)

Fig. 2

Partial oxygen pressure in response to cetuximab treatment. Partial oxygen pressure in response to cetuximab treatment analysed by EPR measurements in untreated controls and cetuximab-treated UT-SCC-2 (UT2) and UT-SCC-14 (UT14) xenografts at day 21. Asterisk shows that the partial oxygen pressure values of untreated controls are significantly lower compared to the cetuximab-treated group (P < 0.05) tested with one way ANOVA and post hoc Tukey’s HSD test, n = 10–14

Fig. 3

EGFR and pEGFR protein expression. Xenografts were established in female nude mice (BALB c[nu/nu]) by subcutaneous injection of head and neck squamous cell carcinoma cell lines UT-SCC-2 (UT-2) and UT-SCC-14 (UT-14). Cetuximab (1 mg/injection) or PBS was administered by intraperitoneal injection at day 10, 13, and 16. A tissue microarray was constructed from tumours harvested at day 21, and the expression of epidermal growth factor receptor (EGFR) and active phosphorylated EGFR (pEGFR) was evaluated by immunohistochemistry (IHC) in untreated controls and cetuximab-treated tumour specimens. Bar graphs showing the IHC staining score for EGFR and pEGFR, and one way ANOVA and post hoc Tukey’s HSD test were used to test differences between treated and untreated groups (*p ≤ 0.05), n = 10–14

Fig. 4

Ki67 and MCT1 protein expression. Xenografts were established in female nude mice (BALB c[nu/nu]) by subcutaneous injection of head and neck squamous cell carcinoma cell lines UT-SCC-2 (UT-2) and UT-SCC-14 (UT-14). Cetuximab (1 mg/injection) or PBS was administered by intraperitoneal injection at day 10, 14, and 17. A tissue microarray was constructed from tumours harvested at day 21, and the expression Ki67 (proliferation marker) and MCT1 was evaluated by immunohistochemistry (IHC) in untreated controls and cetuximab-treated tumour specimens. Bar graphs showing the IHC staining score for Ki67 and MCT1, and one way ANOVA and post hoc Tukey’s HSD test were used to test differences between treated and untreated groups (*p ≤ 0.05), n = 10–14

Fig. 5

GLUT1 and CAIX expression. Xenografts were established in female nude mice (BALB c[nu/nu]) by subcutaneous injection of head and neck squamous cell carcinoma cell lines UT-SCC-2 (UT-2) and UT-SCC-14 (UT-14). Cetuximab (1 mg/injection) or PBS was administered by intraperitoneal injection at day 10, 14, and 17. A tissue microarray was constructed from tumours harvested at day 21, and the expression of GLUT 1 and CAIX was evaluated by immunohistochemistry (IHC) in untreated controls and cetuximab-treated tumour specimens. Bar graphs showing the IHC staining score for GLUT1 and CAIX, and one way ANOVA and post hoc Tukey’s HSD test were used to test differences between treated and untreated groups (*p ≤ 0.05), n = 10–14

Fig. 6

Nuclear HIF-1α protein expression. Xenografts were established in female nude mice (BALB c[nu/nu]) by subcutaneous injection of head and neck squamous cell carcinoma cell lines UT-SCC-2 (UT-2) and UT-SCC-14 (UT-14). Cetuximab (1 mg/injection) or PBS was administered by intraperitoneal injection at day 10, 14, and 17. A tissue microarray was constructed from tumours harvested at day 21, and the expression of nuclear HIF-1α was evaluated by immunohistochemistry (IHC) in untreated controls and cetuximab-treated tumour specimens. Bar graphs showing the IHC staining score for HIF-1α. One way ANOVA and post hoc Tukey’s HSD test were used to test differences between treated and untreated groups (*p ≤ 0.05), n = 10–14