Figure 4

Combined therapy with PAF-receptor antagonist and dacarbazine targets both tumour and microenvironmental elements. B16F10 melanoma cells (5 × 10⁵) were injected s.c. into C57BL/6 mice and after 12 days the tumours were excised and processed for immunohistochemistry analysis with antibodies to caspase-3 (A), COX-2 (B), CD34 (C) or galectin-3 (D). Alternatively, spleens of animals of the very same groups and of naïve mice were analysed for the presence of galectin-3-expressing cells in the white pulp (E). WEB2170 (5 mg/Kg) was given 30 min before the tumour followed by daily injections for 12 days. DTIC (40 μg/animal) was injected i.p. every 3 days after tumour implantation. Morphometric analyses were performed using Eclipse Net software (Nikon). Grids were projected onto tissue sections and the number of grid intersections that overlaid an immunoreactive cell was counted. Results are expressed as the % of area occupied by the cells expressing a given marker (frequency). Data represent the mean ± SEM of positive cells (n = 10). Statistical analyses were performed using ANOVA and SNK (Student Neumans-Keuls test) and differences were considered significant at p < 0.05. (*) p < 0.05 comparing treated with non-treated (PBS) groups.