Epidermal growth factor receptor blockade with C225 plus gemcitabine results in regression of human pancreatic carcinoma growing orthotopically in nude mice by antiangiogenic mechanisms

Both epidermal growth factor receptor (EGF-R) signaling mechanisms and angi ogcnesis have been evaluated as independent targets for therapy of human pa ncreatic carcinoma, but a link between the two processes has been identifie d only recently. This study evaluated whether EGF-R blockade therapy with a nti-EGF-R antibody C225 inhibits pancreatic carcinoma growth and metastasis in an orthotopic nude mouse model via tumor-mediated angiogenesis and whet her gemcitabine potentiates this effect. Irt vitro treatment of human pancr eatic carcinoma L3.6pl cells with C225 inhibited EC;F-R autophosphorylation , producing a maximum of 20% cytostasis. Treatment with C225 plus gemcitabi ne resulted in additive cytotoxic effects that increased with increasing ge mcitabine concentrations. Dose-dependent decreases in expression of the ang iogenic factors vascular endothelial growth factor and interleukin 8 (but n ot basic fibroblast growth factor) were observed in the C225-treated cells (mRNA and protein levels), In L3.6pl tumors established in the pancreas of nude mice, systemic therapy with C225 alone and C225 in combination with ge mcitabine resulted in growth inhibition, tumor regression, and abrogation o f metastasis; median tumor volume was reduced from 538 to 0.3 and to 0 mm(3 ), respectively. Gemcitabine treatment alone reduced median tumor volume fr om 538 to 152 mm(3), Liver metastases were present in 50% of the controls, 30% of the gemcitabine-treated animals, and 20% of C225-treated animals, No macroscopically visible liver metastases were observed in the combination treatment group. iis early as 11 days after C225 treatment, the median perc entage of proliferating cell nuclear antigen-positive cells was substantial ly reduced compared with gemcitabine treatment alone (26% versus 73%, respe ctively) versus controls (92%), correlating with in vivo blockade of EGF-R activation. Similarly after II days treatment, production of vascular endot helial growth factor and interleukin 8 was significantly lower in C225 and C225 plus gemcitabine-treated tumors versus gemcitabine-treated and control tumors. Significant differences in microvessel density were observed 18 da ys after C225 or combination treatments (but not gemcitabine alone) in dire ct correlation with the difference in percentage of apoptotic endothelial c ells, as visualized by double immunofluorescence microscopy, These experime nts indicate that therapeutic strategies targeting EGF-R have a significant antitumor effect on human L3.6pl pancreatic carcinoma growing in nude mice which is mediated in part by inhibition of tumor-induced angiogenesis, lea ding to tumor cell apoptosis and regression. Furthermore, this effect is po tentiated in combination with gemcitabine.