activation of tumor-associated macrophages is required for hepatocyte growth factor enhanced...

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protease inhibitor, inhibits gemcitabine-induced NF-kB activation in pancreatic cancer. We hypothesized that combination chemotherapy of gemcitabine and nafamostat mesilate improves therapeutic out- come of gallbladder cancer. Methods: In vitro, we assessed NF-kB ac- tivity, cell viability, and induction of caspase cascade of a human gallbladder cancer cell line (NOZ) treated with nafamostat mesilate alone, gemcitabine alone, combination of nafamostat mesilate and gemcitabine, or medium as control. In vivo, we established xenograft gallbladder cancer mice model by subctenous injection of NOZ cells. Five weeks after injection, the animals were treated with nafamostat mesilate 3 times a week as nafamostat mesilate group, with gemcita- bine once a week as gemcitabine group, or with combination of nafa- mostat mesilate 3 times a week and gemcitabine once a week as combination group. In control group, only vehicle of gemcitabine and nafamostat mesilate were injected at the same time course. Results: In vitro, gemcitabine-induced NF-kB activation in combina- tion group was significantly inhibited in comparison with gemcitabine alone (p¼0.0003). Cell viability in combination group was lower than that in gemcitabine group. Cleaved caspase-8 level in combination group was the greatest in the 4 experimental groups. In vivo, tumor growth in combination group was significantly slower than those in other groups (p<0.0001). NF-kB activation of the excised tumor in combination group was significantly inhibited in comparison with that in gemcitabine group. Conclusions: Combination chemotherapy of gemcitabine with nafamostat mesilate exerts enhanced anti-tumor effect against xenograft gallbladder cancer model in mice. 42.5. Role of Stearoyl-CoA Desaturase (SCD), Key Enzyme in Obesity, in Development of Human Hepatocellular Car- cinoma (HCC): Is HCC Expected to Rise With Obesity Ep- idemic? S. Bansal, 1,2 M. Berk, 2 N. Alkhouri, 2 J. J. Fung, 2 A. Feldstein 2,3 ; 1 Children’s Hospital Colorado, Aurora, CO; 2 Cleveland Clinic Foundation, Cleveland, OH; 3 University of California, San Diego, La Jolla, CA Introduction: Hepatocellular carcinoma (HCC), the third leading cause of cancer death worldwide, is often diagnosed at an advanced stage, when it is not amenable for radical therapies such as surgical resection or liver transplantation. Current therapeutic options achieve clinical responses in only a small percentage of cases. As a consequence, effective approaches for prevention and treatment are greatly needed. Altered lipid metabolism characterized by an increase in endogenous fatty acid synthesis has been recently linked to HCC pathogenesis. Methods: The aims of this study were to define the cellular and molecular mechanisms linking stearoyl-CoA desaturase (SCD), the rate limiting enzyme in the bio- synthesis of monounsaturated fats and an essential regulator of lipid homeostasis in liver cells to carcinogenesis in HCC. Results: SCD was strongly expressed in surgically resected HCC from a well characterized group of HCC patient (n¼64) as well as various human HCC cell lines (HepG2, Hep3B, PLC/PLF/5) when exam- ined by Western blot analysis and immunohistochemistry. The levels of SCD negatively correlated with degree of tumor differenti- ation (p < 0.01). Treatment of these HCC cell lines with a panel of chemotherapeutic drugs results in a time-dependent phosphatidy- linositol 3-kinase (PI3K)- and JNK1/2-mediated up-regulation of SCD expression which paralleled the degree of resistance to drug- induced apoptosis. Specific genetic or pharmacological SCD sup- pression resulted in inhibition of cell proliferation (p < 0.001) and significant increase sensitivity to chemotherapy induced apoptosis. Conclusions: Our data suggest that increased SCD expression plays an important role in HCC development and resistance to chemo- therapy induced apoptosis and this is in part mediated by PI3K/ JNK activation. Specific targeted interruption of this pathway in HCC could be a desirable approach in designing novel therapeutic strategies and potentially plays an important role in prevention of HCC development. 42.6. Activation of Tumor-Associated Macrophages is Re- quired for Hepatocyte Growth Factor Enhanced Inva- siveness and Predicts Survival in Hepatocellular Carcinoma. M. Kundu, Z. Mi, C. Weber, N. Li, T. Lynch, P. C. Kuo, P. Y. Wai; Loyola University Medical Center, Maywood, IL Introduction: Hepatocellular cancer (HCC) progression requires activation of the tumor-stromal microenvironment. Polarized tu- mor-associated macrophages with M2-like phenotype (TAM2) represent a critical pro-tumor component in this niche. The mechanisms that regulate TAM2 in HCC remain poorly charac- terized. Recently, tumor-derived osteopontin (OPN) has been shown to be significantly expressed in HCC. We hypothesize that TAM2 are critical to OPN-dependent signaling and they significantly enhance HCC invasion. Methods: Macrophage (MAC) polarization into M1 vs. M2 phenotype was assessed by mea- suring cytokines using RT-PCR and ELISA in (i) MAC cultured with recombinant OPN and (ii) MAC/HCC transwell co-culture. HCC cells expressing high OPN (HepG2 or SK-Hep1) or low OPN (Hep3B) were alternatively co-cultured with MAC. Antibodies against OPN or CD44/avb-integrin, OPN-aptamer, or RGD compet- itive inhibitor were used as loss-of-function controls. Tumor re- sponse to OPN-stimulated MAC was assessed by isolating tumor RNA from our co-culture system and analyzing expression in an 84 gene microarray. Up-regulation of identified target genes was confirmed with RT-PCR and ELISA and assessed for OPN- and/or TAM2-dependence using inhibitors. The functional effect of TAM2 on tumor phenotype was measured using proliferation, mi- gration, and 3-D invasion assays. Immunohistochemical analysis of OPN expression, TAM2 activation, and tumor target gene ex- pression was performed on human HCC tissue microarrays for clin- ical correlation. Results: MAC polarization with up-regulated expression of the M2 markers Arg-1 (w5-fold), TGF-ß(w5.5-fold) and IL-10 (w11-fold) occurred in both MAC exposed to recombinant OPN and in MAC/HepG2 co-culture compared to controls (un- treated MAC; MAC/Hep3B co-culture; and MAC/HepG2 co-culture ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS 309

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Page 1: Activation of Tumor-Associated Macrophages is Required for Hepatocyte Growth Factor Enhanced Invasiveness and Predicts Survival in Hepatocellular Carcinoma

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS 309

protease inhibitor, inhibits gemcitabine-induced NF-kB activation inpancreatic cancer. We hypothesized that combination chemotherapyof gemcitabine and nafamostat mesilate improves therapeutic out-come of gallbladder cancer. Methods: In vitro, we assessed NF-kB ac-tivity, cell viability, and induction of caspase cascade of a humangallbladder cancer cell line (NOZ) treated with nafamostat mesilatealone, gemcitabine alone, combination of nafamostat mesilate andgemcitabine, or medium as control. In vivo, we established xenograftgallbladder cancer mice model by subctenous injection of NOZ cells.Five weeks after injection, the animals were treated with nafamostatmesilate 3 times a week as nafamostat mesilate group, with gemcita-bine once a week as gemcitabine group, or with combination of nafa-mostat mesilate 3 times a week and gemcitabine once a week ascombination group. In control group, only vehicle of gemcitabineand nafamostat mesilate were injected at the same time course.Results: In vitro, gemcitabine-induced NF-kB activation in combina-tion groupwas significantly inhibited in comparisonwith gemcitabinealone (p¼0.0003). Cell viability in combination group was lower thanthat in gemcitabine group. Cleaved caspase-8 level in combinationgroup was the greatest in the 4 experimental groups. In vivo, tumorgrowth in combination group was significantly slower than those inother groups (p<0.0001). NF-kB activation of the excised tumor incombination group was significantly inhibited in comparison withthat in gemcitabine group. Conclusions: Combination chemotherapyof gemcitabine with nafamostat mesilate exerts enhanced anti-tumoreffect against xenograft gallbladder cancer model in mice.

42.5. Role of Stearoyl-CoA Desaturase (SCD), Key Enzyme inObesity, in Development of Human Hepatocellular Car-cinoma (HCC): IsHCCExpected toRiseWithObesity Ep-idemic? S. Bansal,1,2 M. Berk,2 N. Alkhouri,2 J. J. Fung,2 A.Feldstein2,3; 1Children’s Hospital Colorado, Aurora, CO;2Cleveland Clinic Foundation, Cleveland, OH; 3University ofCalifornia, San Diego, La Jolla, CA

Introduction: Hepatocellular carcinoma (HCC), the third leadingcause of cancer death worldwide, is often diagnosed at an advancedstage, when it is not amenable for radical therapies such as surgicalresection or liver transplantation. Current therapeutic optionsachieve clinical responses in only a small percentage of cases. Asa consequence, effective approaches for prevention and treatmentare greatly needed. Altered lipid metabolism characterized by anincrease in endogenous fatty acid synthesis has been recentlylinked to HCC pathogenesis. Methods: The aims of this studywere to define the cellular and molecular mechanisms linkingstearoyl-CoA desaturase (SCD), the rate limiting enzyme in the bio-synthesis of monounsaturated fats and an essential regulator oflipid homeostasis in liver cells to carcinogenesis in HCC. Results:SCD was strongly expressed in surgically resected HCC froma well characterized group of HCC patient (n¼64) as well as varioushuman HCC cell lines (HepG2, Hep3B, PLC/PLF/5) when exam-ined by Western blot analysis and immunohistochemistry. Thelevels of SCD negatively correlated with degree of tumor differenti-ation (p < 0.01). Treatment of these HCC cell lines with a panel ofchemotherapeutic drugs results in a time-dependent phosphatidy-linositol 3-kinase (PI3K)- and JNK1/2-mediated up-regulation ofSCD expression which paralleled the degree of resistance to drug-induced apoptosis. Specific genetic or pharmacological SCD sup-pression resulted in inhibition of cell proliferation (p < 0.001) andsignificant increase sensitivity to chemotherapy induced apoptosis.Conclusions:Our data suggest that increased SCD expression playsan important role in HCC development and resistance to chemo-therapy induced apoptosis and this is in part mediated by PI3K/JNK activation. Specific targeted interruption of this pathway inHCC could be a desirable approach in designing novel therapeuticstrategies and potentially plays an important role in prevention ofHCC development.

42.6. Activation of Tumor-Associated Macrophages is Re-quired for Hepatocyte Growth Factor Enhanced Inva-siveness and Predicts Survival in HepatocellularCarcinoma. M. Kundu, Z. Mi, C. Weber, N. Li, T. Lynch,P. C. Kuo, P. Y. Wai; Loyola University Medical Center,Maywood, IL

Introduction: Hepatocellular cancer (HCC) progression requiresactivation of the tumor-stromal microenvironment. Polarized tu-mor-associated macrophages with M2-like phenotype (TAM2)represent a critical pro-tumor component in this niche. Themechanisms that regulate TAM2 in HCC remain poorly charac-terized. Recently, tumor-derived osteopontin (OPN) has beenshown to be significantly expressed in HCC. We hypothesizethat TAM2 are critical to OPN-dependent signaling and theysignificantly enhance HCC invasion. Methods: Macrophage(MAC) polarization intoM1 vs. M2 phenotype was assessed bymea-suring cytokines using RT-PCR and ELISA in (i) MAC culturedwith recombinant OPN and (ii) MAC/HCC transwell co-culture.HCC cells expressing high OPN (HepG2 or SK-Hep1) or low OPN(Hep3B) were alternatively co-cultured with MAC. Antibodiesagainst OPN or CD44/avb-integrin, OPN-aptamer, or RGD compet-itive inhibitor were used as loss-of-function controls. Tumor re-sponse to OPN-stimulated MAC was assessed by isolating tumorRNA from our co-culture system and analyzing expression in an84 gene microarray. Up-regulation of identified target genes wasconfirmed with RT-PCR and ELISA and assessed for OPN- and/orTAM2-dependence using inhibitors. The functional effect ofTAM2 on tumor phenotype was measured using proliferation, mi-gration, and 3-D invasion assays. Immunohistochemical analysisof OPN expression, TAM2 activation, and tumor target gene ex-pression was performed on humanHCC tissue microarrays for clin-ical correlation. Results: MAC polarization with up-regulatedexpression of the M2 markers Arg-1 (w5-fold), TGF-ß(w5.5-fold)and IL-10 (w11-fold) occurred in bothMAC exposed to recombinantOPN and in MAC/HepG2 co-culture compared to controls (un-treated MAC; MAC/Hep3B co-culture; and MAC/HepG2 co-culture

Page 2: Activation of Tumor-Associated Macrophages is Required for Hepatocyte Growth Factor Enhanced Invasiveness and Predicts Survival in Hepatocellular Carcinoma

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS310

with OPN inhibitors). Addition of recombinant OPN to MAC/Hep3B co-culture restored TAM2 polarization. Gene expressionmi-croarray and RT-PCR demonstrated that OPN-stimulated MAC in-duced HepG2 expression of key pro-tumor genes including HGF(w8-fold), CDH11 (w5-fold), and K-RAS (w8-fold) compared to con-trols. Tumor target gene expression decreased w4-6 fold in MAC/HepG2 co-culture treated with OPN inhibitors. OPN-activatedMAC increased HepG2 migration (>10-fold) and proliferation(>2-fold) vs. controls. Inhibition of OPN or M2 cytokines in MAC/HEPG2 co-culture reduced TAM2-mediated expression of HepG2HGF w6-fold. Analysis of 96 human HCC tissue microarrayspecimens demonstrated that increased tumor-OPN expressioncorrelates with increased MAC IL-10 expression, increased tu-mor-HGF expression, and increased HCC TNM stage (P<0.01).Conclusions: Tumor-derived OPN activates TAM2 to elaboratepro-tumor cytokines that induce HCC HGF secretion and invasive-ness. This OPN-HGF axis is TAM2-dependent and represents thefirst report correlating with relevant HCC TNM-stage and clinicalsurvival. TAM2 targeting and inhibition represents a novel adju-vant strategy to decrease HCC metastasis.

42.7. Combination Treatment Using Adenovirus-mediatedTumor Necrosis Factor-Alpha Gene Transfer and NF-kappaB Inhibitor for Hepatocellular Carcinoma inMice. K. Haruki,1,2 H. Shiba,1 R. Iwase,1,2 Y. Fujiwara,1 K.Furukawa,1 T. Iida,1 T. Uwagawa,1 T. Misawa,1 T. Ohashi,2

K. Yanaga1; 1Department of Surgery, The Jikei UniversitySchool of Medicine, Tokyo, Japan; 2Department of GeneTherapy, Institute of DNA Medicine, The Jikei UniversitySchool of Medicine, Tokyo, Japan

Introduction: Hepatocellular carcinoma (HCC) is often resistant tochemotherapy. Gene therapy using an adenoviral vector express-ing tumor necrosis factor-alpha (TNF-a) is a new therapeutic ap-proach for chemoresistant malignancies. However, the efficacy ofTNF-a is limited because of the activation of nuclear factor kB(NF-kB). We previously reported that nafamostat mesilate(FUT175), a synthetic serine protease inhibitor, inhibits NF-kB ac-tivation and promotes caspase-8-mediated apoptosis in pancreaticcancer. We hypothesized that addition of FUT175 may enhanceanti-tumor effect of adenovirus vector-mediated TNF-a gene ther-apy for HCC. Methods: In vitro, we assessed if FUT175 inhibitedTNF-a-induced NF-kB activation and enhanced apoptosis in a hu-man hepatocellular carcinoma cell line (Huh-7). In vivo, we estab-lished a xenograft hepatocellular carcinoma model in mice bysubcutaneous injection of Huh-7. When the implanted tumor sizereached 8.0 mm, control group was injected AxCALacZ intratumor-ally once a week and distilled water (vehicle of FUT175) intraperi-toneally thrice a week, while FUT175 group was injected FUT175intraperitoneally thrice a week, TNF-a group was injected Ax-CAhTNF-a intratumorally once a week, and combination groupwas injected AxCAhTNF-a intratumorally once a week as well asFUT175 intraperitoneally thrice a week. Results: In vitro,Huh-7 in-fected with AxCAhTNF-a expressed TNF-a in a dose-dependentmanner. NF-kB activity in combination group was significantlylower than that in TNF-a group (p¼0.0001). Cleaved caspase-8,cleaved caspase-3 and cleaved PARP levels in combination groupwere greater than those in other groups. Changes in cell prolifera-tion rates in combination group after treatment were significantlylower than those in TNF-a group (p<0.0001). In vivo, tumor growthin the combination group was significantly slower than those ofFUT175 or TNF-a groups (p<0.0001). At the end of study, volumeand weight of excised tumor in the combination group was signifi-cantly less than those in the TNF-a groups (p<0.0001 andp<0.0001, respectively). Conclusions: Inhibition of NF-kB byFUT175 enhances the anti-tumor effect of TNF-a gene therapyfor HCC.

42.8. The Bioflavonoid Baicalein Inhibits Human PancreaticCancer Cell Migration. G. W. Donald, M. Chen, A. Moro, H.Pham, K. M. Hertzer, H. A. Reber, O. J. Hines, G. Eibl;Department of Surgery, David Geffen School of Medicine AtUCLA, Los Angeles, CA

Introduction: Baicalein, a bioflavonoid component of Scutellariabaicalensis, has been shown in our laboratory to decrease hu-man pancreatic cancer cell proliferation by inducing apoptosisvia inhibition of lipoxygenase pathways and via the down-regu-lation of anti-apoptotic Mcl-1. Since the oral bioavailability offlavonoids is limited we sought to determine the impact of lowmicromolar concentrations of baicalein on pancreatic cancer cel-lular migration -a process critical for tumor growth and meta-static progression. Methods: The human pancreatic cancer celllines MiaPaCa-2 and Panc-1 were grown in DMEM with fetal bo-vine serum. After a period of starvation, cells were treated with bai-calein (100nM, 500nM, 1uM, 5 uM) or 0.1% DMSO as control.Scratch assays were performed with images taken at 24, 48, and72 hours of treatment to document the influence of baicalein oncell migration. The number of migrating cells in the wound wascounted on a grid with three counts per time point. MTT assaywas used to determine the impact of baicalein on cellular prolifera-tion. Finally, conformational changes in cytoskeletal structurewere assessed with fluorescent phalloidin staining at 24, 48, and72 hours of treatment to assess the qualitative effect of baicaleinon cytoskeletal disruption. Results: Scratch assays demonstratedclear inhibition of wound closure at 72 hours in the baicalein treat-ment groups for both MiaPaCa-2 and Panc-1 (Figure). Quantifica-tion of the number of migrating cells revealed a dose-dependentresponse with increasing concentrations of baicalein. 1uM baica-lein most effectively inhibited migration vs. control (60616 vs.115627; p<0.05). MTT assay showed no difference in proliferationcompared to control at 48 hours of treatment at any baicalein dos-age for both cell lines (0.3360.1 vs 0.3260.09 control vs. 5uM;p¼0.96). Finally, actin staining in the MiaPaCa-2 cell line sug-gested that there were fewer stress fibers after 72 hours of treat-ment at 1uM and 5uM of baicalein (Figure inset). Conclusions:Baicalein at low micromolar concentrations disrupts pancreaticcancer cell migration in vitro after 72 hours of treatment in two hu-man cell lines. We found this effect on migration at concentrationsthat do not affect proliferation. Since baicalein tissue levels in hu-mans can reliably be achieved at the concentrations tested in thisstudy, these data suggest that baicalein may serve as a clinical ad-junct against pancreatic cancer progression.