Anti-EMMPRIN antibody treatment of head and neck squamous cell carcinoma in an ex-vivo model

Abstract

Targeting the molecular pathways associated with carcinogenesis remains the greatest opportunity to reduce treatment-related morbidity and mortality. Extracellular matrix metalloproteinase inducer (EMMPRIN), also known as CD147, is a cell surface molecule known to promote tumor growth and angiogenesis in preclinical studies of head and neck carcinoma making it an excellent therapeutic target. To evaluate the feasibility of anti-EMMPRIN therapy, an ex-vivo human head and neck cancer model was established using specimens obtained at the time of surgery (n=22). Tumor slices were exposed to varying concentrations of anti-EMMPRIN monoclonal antibody and cetuximab for comparison purposes. Cetuximab is the only monoclonal antibody currently approved for the treatment of head and neck carcinoma. After treatment, tumor slices were assessed by immunohistochemistry and western blot analysis for apoptosis (TUNEL) and EMMPRIN expression. Of the tumor specimens 33% showed a significant reduction in mean ATP levels after treatment with cetuximab compared with untreated controls, whereas 58% of the patients responded to anti-EMMPRIN therapy (P<0.05). Samples, which showed reactivity to anti-EMMPRIN, also had greater EMMPRIN expression based on immunohistochemistry staining (49%) when compared with nonresponders (25%, P=0.06). In addition, TUNEL analysis showed a larger number of cells undergoing apoptosis in antibody-treated tumor slices (77%) compared with controls (30%, P<0.001) with activation of apoptotic proteins, caspase 3 and caspase 8. This study shows the potential of anti-EMMPRIN to inhibit proliferation and promote apoptosis and suggests its future role in the targeted treatment of head and neck carcinoma.

Fig. 1

Viability of ex-vivo control tissue slices (n = 6) at 24, 48, and 72h compared to mean ATP level at time zero.

Fig. 2

Tissue slice cytotoxicity assays in head and neck squamous cell carcinoma patient specimens cultured with varying concentrations of CNTO3899 (0, 50, 100, and 200 µg/ml). Six replicate slices were prepared per treatment group. A significant reduction in ATP levels was obtained with the treatment of 100 µg/ml anti-extracellular matrix metalloproteinase inducer monoclonal antibody in patients 12, 13, and 15.

Fig. 3

Comparison of anti-extracellular matrix metalloproteinase inducer antibody and cetuximab-induced cytotoxicity in head and neck squamous cell carcinoma patient specimens. (a) Cytotoxicity curve for head and neck cancer tissue slices (n = 6 per treatment group) treated with varying concentrations of cetuximab (C225) (0, 5, 10, and 20 µg/ml). A significant reduction in ATP levels was obtained with 10 µg/ml. (b) Side-by-side comparison of CNTO3899 (100 µg/ml) and C225 (10 µg/ml)-treated tissue specimens for all patients as a percentage of control. No significant difference was observed between mean ATP levels for anti-extracellular matrix metalloproteinase inducer (57%) and cetuximab (45%)-treated tumor specimens (P = 0.13).

Fig. 4

Anti-extracellular matrix metalloproteinase inducer (EMMPRIN) monoclonal antibody induces caspase-mediated apoptosis; treatment response correlates with EMMPRIN expression. (a) Tissue specimens maintain intact tumor cells with surrounding vasculature and stroma on hematoxylin and eosin (H&E) staining. An increase in apoptosis in treated (100 µg/ml CNTO3899) (77%) versus untreated slices (30%, P < 0.001) was confirmed through TUNEL analysis. (b) EMMPRIN staining correlates with treatment response. Patient specimens classified as responders showed greater EMMPRIN expression (49%) than nonresponders (25%, P = 0.06) following treatment with anti-EMMPRIN monoclonal antibody. (c) CNTO3899 stimulates caspase-mediated apoptosis. Control and CNTO3899 treated tissue specimens were analyzed for caspase-3 and caspase-8 expression. An increase in caspase-3 expression was observed in both patients following treatment (P<0.01). Caspase-8 expression was significantly greater for anti-EMMPRIN treated tumor tissue from patient No. 15 (responder) when compared with control (P < 0.001).