Analysis of G55 tumors treated with ES, Tum and ES + Tum. Effect on tumor growth and tumor vessel density. (A) Subcutaneously grafted tumors were treated with ES, Tum and ES + Tum for 10 days, tumors were excised and tumor weights determined. Bars represent the mean values ± SE (n = 5); * on bars indicates significant differences vs. WT; * on connecting lines indicates significant differences between respective groups (p < 0.05). (B) Microvessel density was analysed by counting CD31-positive vessels in tumor tissue. Note that microvessel density is significantly reduced in ES- and ES + Tum-treated tumors, but not in Tum-treated tumors. Bars represent the means ± SE (n = 4-5); * significant differences vs. WT (p < 0.05). (C) Tumor tissue was analysed by H&E, TUNEL and immunohistochemical stainings for Ki67 and CD31. ES-, Tum- and ES + Tum-treated tumors showed large necrotic areas containing TUNEL-positive cells. Vital cells were restricted to thin cell layers around vessels (v) and at the outer margins of tumors. Control tumors (WT), in comparison, were highly proliferative and lacked large necrotic areas.