510 Inhibition of cyclooxygenase-2 suppresses the growth of human hepatocellular carcinoma implants in vivo

HEPATOLOGY, Vol. 38, No. 4, Suppl. 1, 2003

AASLD ABSTRACTS

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INHIBITION OF CYCLOOXYGENASE-2 SUPPRESSES THE GROWTH OF HUMAN HEPATOCELLULAR CARCINOMA IMPLANTS IN VIVO. Michael Andrd Kern, Miua Mareike

ADENOVIRUS-MEDIATED FLT3L-GENE THERAPY PROTECTS AGAINST COLON CANCER METASTASISIN A BALB/C MOUSE MODEL. Carina Riediger, Dept. of Internal

Sch6neweiss, Dina Sahi, Maryam Bahlo, AnkeMaria Haugg, Hans Peter Dienes, Inst#ute of Pathology, University of Cologne, Cologne, Germany; Herbert Kiiferstein, Institute of Forensic Medicine, University of Cologne, Cologne, Germany; Kai Breuhahn, Inst#ute of Pathology, University of Cologne, Cologne, Germany; Peter Schirmacher, Institute of Pathology and Center of Molecular Medicine (ZMMK), University of Cologne, Cologne, Germany

Medicine IV, Heidelberg, Germany; Gerhard Wingeuder, Percy Knolle, ZMBH Heidelberg, Heidelberg, Germany; Wolfgang Stremmel, Jens ncke, Dept. of Internal Medicine IV, Heidelberg, Germany

COX-2, an inducible enzyme, catalyzes the formation of prostaglandins from arachidonic acid. We have demonstrated that COX-2 inhibition suppresses growth of HCC cells in vitro by inducing apoptosis associated with early cleavage of the caspases-9, -3, and - 6 and reducing proliferation. To evaluate the in vivo efficacy, selective COX-2- and non- selective COX-inhibition were administered to HCCs grown in nude mice. 4x106 human HuH7 cells were injected s.c. Mice (n-40) were given standard diet with constant supplementation of meloxicam (Mobec) (selective COX-2 inhibitor) (group 1, 54 p p m (n-10); group 2, pretreatment with 162 ppm, after tumor cell injection 54 p p m (n-10)) or sulindac (non-selective COX-inhibitor) (group 3; 400 ppm; (n-10)) starting 5 days prior to tumor cell injection. Untreated mice served as control (group 4 (n-10)). Tumor growth was monitored constantly. After 35-days mice were euthanized and tumors were analysed morphologically and assayed immunohistochemically for proliferation (Ki67), apoptosis (M30), and COX-2. COX-2 expression was maintained in implant tumors at levels comparable to parental ceils. Selective COX-2 inhibition led to a sustained reduction of HuH7-tumor size and weight which was significant in group 2 (p<0.05). Average tumor surface in control mice was 0,62 cm 2 (weight 0.35 g) as compared with 0,19 cm 2 (weight 0.13 g) in treated mice (69% reduction). Treatment with sulindac (group 3) did not significantly reduce tumor growth under the selected conditions. COX-2 inhibition had a significant antimitogenic (37.4 % (control) versus 2 0 , 1 % (group 2) Ki-67 positive cells) and proapoptotic effect (12,8 % (control) versus 41,7 % (group 2) M30- positive cells) on tumor cells (p<0.05). These results demonstrate that under experimental conditions selective COX-2 inhibition suppresses solid HCC growth in vivo and may have preventive and therapeutic potential for h u m a n HCCs. Disclosures: Maryam Bahlo - No relationships to disclose Kai Breuhahn - No relationships to disclose Hans Peter Dienes - No relationships to disclose AnkeMaria Haugg - No relationships to disclose Herbert K~ferstein - No relationships to disclose Michael Andr6 Kern - No relationships to disclose Dina Sahi - No relationships to disclose Peter Schirmacher - No relationships to disclose Mirja Mareike SchSneweiss - No relationships to disclose

Flt3(fms-like tyrosine kinase 3)ligand (FIt3L) is a potent hematopoetic cytokine that effects growth and differentiation of progenitor and stem ceils. It also augments the numbers of dendritic ceils (DC) and natural killer (NK) ceils in mice and humans. DC are the key mediators in antigen presentation and in the induction and regulation of immune responses. Therefore they are thought to play a major role in the anti-tumor activity. In this study we describe a gene-therapeutic approach to stimulate antitumor immunity by a novel adenoviral-mediated transfer of FIt3L to treat liver metastasis. We generated and produced an adenovirus expressing the FIt3L gene (pAdFlt3L) and confirmed expression by Western Blot and ELISA technique: in vitro infection of a mouse colon carcinoma cell line (CT26) with adenoviral vector expressing FIt3L (pAdFlt3L) induced high levels of FIt3L in the supernatants as well as in the cell lysates. We injected CT26 cells subcutaneously into the flank of 4-week-old, female BALB/c mice as a model of colon carcinoma liver metastasis. After 13 days tumor nodules were palpable. FIt3L immunotherapy was initiated 13 days after tumor inoculation by injecting 109 pAdflt3L i.v. into the tail vein or directly into the tumor. High levels of FIt3L in the serum of pAdflt3L-treated mice during the first 3 days after i.v. as well as i.t. injection were detected by ELISA with a maximum FIt3L level after 24hours, but with an approximately 1000 to 10000 fold higher FIt3L level after i.v.-treatment. Interestingly animals with a second injection 7 days after the first showed a second peak of Flt3L-levels showing the low immunogenicity of the adenoviral vector. Spleen size and weight was strongly augmented in mice treated with pAdFlt3L. Flowcytometric analysis showed that therapy with pAdFlt3L caused a remarkable increase of DC (CDllc+/CDllb+). Furthermore we vaccinated a group of mice with CT26-ceil-lysate and coinjected the Flt3L-adenovirus s.c. Mice in the vaccinated group were challenged with the CT26 ceil line; while in the control group all mice died, in the vaccination group a 100 % survival was observed, demonstrating the potential of costimulation with FIt3L. Our results indicate that immunostimulatory anti-tumor effects against colon carcinoma liver metastasis are provided by FIt3L adenoviral therapy both by direct application and by coimmunization through stimulation and proliferation of DCs. Disclosures: Jens Encke - No relationships to disclose Percy Knolle - No relationships to disclose Carina Riediger - No relationships to disclose Wolfgang Stremmel - No relationships to disclose Gerhard Wingeuder - No relationships to disclose