Figure 3. Antitumoral effect of H₄R agonists in 1205Lu xenografted tumor induced in nude mice.

(A) Evaluation of relative tumor volume. Daily sc. treatment 1 mg/kg of histamine (HA), clozapine (CLZ) or JNJ28610244 (JNJ28) significantly diminished the tumor volume, evidencing this effect at the end of the experiment. Non-linear regression fit was performed to evaluate the exponential growth, (Repeated Measures ANOVA, ***P < 0.001 HA, CLZ and JNJ28 vs. control; Two-way ANOVA and Bonferroni post-test ^#P < 0.05 JNJ28 vs. control). (B) 1205Lu tumors showed vertical growth (arrow), with invasion of reticular dermis (H&E staining, 200X-fold magnification). (C) 1205Lu tumors demonstrated intratumoral neutrophils (green arrow). (D) Dilated lymphatic vessels (red arrows, Masson´s trichromic staining, 200X-fold magnification). (E) Lymphatic emboli (arrow, H&E staining, 400X-fold magnification). Presence of tumor cells in mitosis. (F) HMB-45 positive and (G) Tyrosinase positive immunostaining. Scale bars = 100 and 20 μm. (H) Tumors of the control group showed significant anisocytosis and anisokaryosis, nuclear pleomorphism and atypical mitosis (yellow arrows). Tumors of mice treated with histamine, clozapine or JNJ28610244 presented cell homogeneity, with rounded and uniform nuclei with typical mitosis, and presence of inflammatory infiltrates (white arrows), (H&E staining, 400X-fold magnification). Immunohistochemistry of 1205Lu xenografted mice. Formalin-fixed paraffin embedded tissue sections of control, histamine, clozapine, and JNJ28610244 mice were stained to evaluate intracellular levels of histamine, HDC, H₄R expression, proliferation and vascular and connective tissue morphology. Pictures were taken at a 400X-fold magnification for immunostaining (Scale bar = 20 μm) and 50X-fold magnification for trichromic stain (Scale bar = 100 μm).