Intratumor (i.t.) injection of 35 mg/kg/day NAMI-A for six consecutive days to CBA mice bearing i.m. implants of MCa mammary carcinoma reduces primary tumor growth and particularly lung metastasis formation, causing 60% of animals to be free of macroscopically detectable metastases. The i.t. treatment allows study of the effects of NAMI-A on in vivo tumor cells exposed to millimolar concentrations for a relatively prolonged time. Under these conditions, NAMI-A reduces the number of CD44ϩ tumor cells and changes tumor cell phenotype to a lower aggressive behavior, as shown by scanning electron microscopy analysis. On primary tumor site, NAMI-A causes unbalance between 2n and aneuploid cells in favor of lymphocytes. Furthermore, in tumor tissue, nitric oxide production is increased and active matrix metalloproteinase 9 is decreased, and these effects are accompanied by a reduced hemoglobin concentration. These data are in agreement with the reduction of tumor invasion and metastasis and suggest the therapeutic usefulness of NAMI-A in neoadjuvant or tumor reduction treatments for preventing metastasis formation. These data further stress the usefulness of intratumor treatments as experimental preclinical model for studying in vivo the mechanism of tumor cell interactions after prolonged exposure to ruthenium-based compounds to be developed for metastasis inhibition.ImH[trans-RuCl 4 (DMSO)Im] (Im, imidazole; DMSO, dimethyl sulfoxide) (NAMI-A) is a metal-based compound effective against lungs metastases of solid tumors in murine models . This effect was always dissociated from a concomitant similar reduction of primary tumor growth and to any significant cytotoxic effect in vitro on human and on murine tumor cell lines up to millimolar concentrations (Bergamo et al., 2000;Pacor et al., 2001;Sava et al., 2003). These peculiarities make NAMI-A an interesting compound for treating human disseminated tumors with an as yet unknown mechanism of action but different from that of "classical" cytotoxic chemotherapeutic drugs. Recently, NAMI-A has completed a phase I clinical trial at the Netherlands Cancer Institute (Amsterdam, The Netherlands) on 24 patients, without any unexpected toxicity and a maximum tolerated dose at doses compatible with those active on metastases in preclinical models (J.H.M. Schellens, personal communication to G.S.).Chemical studies pointed out the propensity of NAMI-A to give hydrolyzed species in physiological conditions, but only the unmodified molecule is capable to bind to DNA (Bacac et al., 2004). To expose tumor cells to the unmodified molecule of NAMI-A, and to highlight the role of DNA as preferential target for the selective antimetastatic activity, we performed an intratumoral (i.t.) treatment in a gel-carrier system with the aim of achieving in situ a concentration higher and for prolonged times than that obtained with systemic routes.Recently, local administration (i.t.) of drugs has lead to a proliferation of research with the double aim of improving therapeutic outcomes and o...