EradicationofOvarianTumorXenograftsbyLocoregional AdministrationofTargeted Immunotherapy MichelandreaDeCesare, 1 ClaudiaCalcaterra, 3 GraziellaPratesi, 1 LauraGatti, 1 FrancoZunino, 1 SylvieMe'nard, 2 andAndreaBalsari 2,3 Abstract Purpose: Oligodeoxynucleotides containing unmethylated CpGmotifs (CpG-ODN) are potent activatorsofinnateandadaptiveimmunity.RecognitionofCpG-ODNismediatedbyToll-likere- ceptor9expressedbyimmunecells,endothelialandepithelialcells,andfibroblasts.Weexamined theantitumoreffectofCpG-ODNandtheroleofadministrationrouteonhumanovariancancers growingintheperitonealcavityofnudemice. ExperimentalDesign: Miceimplantedi.p.withhumanovariancarcinomacellsweretreatedi.p., s.c.,ori.v.andassessedforsurvivalandtumor-freeincidence.Peritonealwashingswereanalyzed forkeratinocytechemokineproductionandforfunctionalandphenotypicprofilesasindicatorsof thecelltypesinvolvedinmediatingtheantitumoreffects. Results: IGROV-1-bearingmicetreatedi.p.survivedsignificantlylongerthanthosetreatedi.v.or s.c.( P =0.0005),andnearlyhalfofthem(8of17)weretumor-freebytheendoftheexperiment, a rate never achieved using a variety of chemotherapeutic drugs. High rates of tumor-free mice were observed in three other ovarian tumor xenografts treated i.p. Compared with peritoneal washingsofmicetreateds.c.ori.v.,thosefrommicetreatedi.p.showedthehighestlevelofserum andtissuekeratinocytechemokine,thehighestnumberofnaturalkillercellsandneutrophils,and thehighestantiproliferativeactivity in vitro. Conclusions: The superior antitumor effectobtainedby locoregionaladministrationof CpG- ODNini.p.tumor-bearingmicewithalimitedadaptiveimmuneresponsepointstotheimportance ofinnateeffectorcellsamplificationatthesiteoftumorgrowthandsuggeststhepromiseofi.p. CpG-ODNinclinicaltrialsforovariancancer. Oligodeoxynucleotides containing dinucleotides with unme- thylated CpG motifs (CpG-ODN) are strong activators of both innate and adaptive immune systems (1, 2). Recognition of CpG-ODN is mediated by Toll-like receptor 9 (TLR9), a member of the TLR family, which is critically important in detecting microbial pathogens. TLRs, initially identified on cells of the immune system, are also expressed by nonprofessional immune cells such as endothelial cells, fibroblasts, and epithelial cells (3, 4). Both bone marrow and non-bone marrow-derived cells are thought to be involved in the response induced by TLR agonists. Successes in preclinical studies using CpG-ODN and early indications of its safe use in humans have led to considerable interest in the clinical development of these agents in the treatment of cancer patients (1, 5). Preclinical studies (6 – 8) have shown superior antitumor effects after intratumor administration of CpG-ODN; thus far, a mechanistic explana- tion of the route-dependent effects remains to be elucidated. The critical role ascribed to the adaptive immunity in CpG- ODN antitumor effects have led to clinical trials conducted using systemic administrations (9 – 11). In clinical practice, ovarian cancer is among the few tumor types suitable for intratumor treatment, because its growth is mostly confined to the peritoneal cavity. In such patients, locoregional delivery of therapeutic agents may be a suitable option. Ovarian cancer is the fifth most frequent cancer, accounting for about 6% of all cancer death in females. Progress in the treatment of the disease has been made in recent years, with the current 5-year survival rate around 45% (12). Cytoreductive surgery and systemic combination chemotherapy with a platinum drug and a taxane represent the standard of care for ovarian cancer patients (13). The role of immunotherapy for ovarian cancer patients has been addressed in an early clinical trial (14), but to our knowledge the efficacy of CpG-ODN against ovarian tumors has not been addressed clinically. Here, we examined the effects of CpG-ODN on human ovarian cancers in the peritoneal cavity of nude mice and the role of the administration route in determining these effects. The results indicate the superior antitumor activity after i.p. administration compared with other routes of delivery, with increased mouse survival time and tumor-free rates. Analyses of the cellular and immunologic bases of this effect point to the Cancer Therapy: Preclinical Authors’Affiliations: 1 Preclinical Chemotherapy and Pharmacology Unit and 2 MolecularTargeting Unit, Fondazione IRCCSIstituto Nazionale deiTumori; 3 InstituteofPathology,UniversityofMilan,Italy Received2/19/08;revised5/8/08;accepted5/13/08. Grantsupport: AssociazioneItalianaperlaRicercasulCancro(Milan,Italy). Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentofpage charges.Thisarticlemustthereforebeherebymarked advertisement inaccordance with18U.S.C.Section1734solelytoindicatethisfact. Requestsforreprints: GraziellaPratesi,FondazioneIRCCSIstitutoNazionaledei Tumori,ViaVenezian1, 20133 Milan, Italy. Phone: 39-2-23902626; Fax: 39-2- 23902692;E-mail:graziella.pratesi@istitutotumori.mi.it. 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