Macrophage inflammatory protein-3alpha (mip-3alpha) promotes pancreatic cancer cell invasion by upregulation of matrix metalloproteinase-9 (MMP-9) production

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ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS

vasomotor response in wildtype mice. However, ApoE -/- mice have significantly altered vasomotor function at baseline and following injury. Early ATH renders the vessels more susceptible to injuryinduced IH and progression of ATH and stress the impact of ATH on vascular function as well as on vascular responses to injury. Potential therapies for IH should also be tested in models of ATH for histologic and physiologic improvement.

make DC/Apo vaccine. C-57/BL-6 mice (n ⫽ 6) were injected with 2.5 x 10 5 B-16 SQ and treated with 1 x 10 6 DC/Apo vaccine cells SQ or saline control weekly x4 starting 5 days after tumor implantation. In other experiments, splenocytes were harvested from control and immunized tumor-bearing animals at day 30 and stimulated weekly x2 with IFN-gamma treated, irradiated B-16 cells. Cytotoxic T lymphocyte (CTL) assays were performed with B-16 targets for 48 hr and % specific lysis calculated. Results: Summarized in table 1.

ONCOLOGY PARALLEL SESSION I TABLE—ABSTRACT 35 34. Predictors of Breast Conservation Therapy: A Statistical Analysis of 29,000 Patients. K. L. Hiotis, M.D., W. Ye, M.S., R. Sposto, Ph.D., K. A. Skinner, M.D. New York University, Department of Surgery , New York, NY. Background: Despite the National Institutes of Health Consensus statement in 1991 that breast conservation surgery followed by radiation is an appropriate approach to treatment of early-stage breast cancer, studies have shown a relatively low rate of breast conservation therapy (BCT) in the United States. This study investigates predictors of breast conservation therapy in a large, diverse patient population. Methods: Between 1990 and 1998, 43,111 patients underwent surgery for breast cancer and were entered into the Cancer Surveillance Program database for Los Angeles County. Of these, 29,642 (68.3%) had complete data on patient demographics, staging, surgeon and hospital. Data was collected regarding extent of disease, lymph node status, tumor size, age, race, socioeconomic status, doctor specialization, doctor volume, hospital specialization, and hospital volume. Univariate and multivariate analyses were performed. Results: On univariate analysis, extent of disease, lymph node status, tumor size, age, race, socioeconomic status, doctor and hospital specialization, and doctor and hospital volume all were significantly associated with surgery type (p ⬍ 0.0001). On multivariate analysis, not only does extent of disease impact choice of surgery, but race (white women are 12.7 times more likely to undergo BCT than Asian women, p ⬍ 0.0001), socioeconomic status (women of high socioeconomic status are 7.6 times more likely to undergo BCT than women of average socioeconomic status, p ⬍ 0.0001), hospital volume (women operated on at hospitals performing ⬎71 cases per year are 5 times more likely to undergo BCT than those at hospitals performing less cases per year, p ⬍ 0.0001), doctor volume (women operated on by surgeons who perform ⬎10 breast cancer cases per year are 5.4 times more likely to undergo BCT than those who are operated on by surgeons who perform less cases, p ⬍ 0.0001), and doctor specialization (women who undergo operations by surgeons who specialize in breast surgery are 5.56 times more likely to undergo BCT than those who undergo operation by surgeons who are not specialists, p ⬍ 0.0001). Conclusions: These results suggest that not only does the extent of locoregional disease play a role in the likelihood of a women undergoing breast conservation therapy, but socioeconomic status, racial/ethnic factors, and doctor and hospital experience have an effect. 35. Dendritic Cells (DC) Loaded With Apoptotic, Allogeneic Melanoma Cells Reduce Tumor Progression and Improve Survival in a Murine Immunotherapy Model. M. C. Kelley, M.D., S. Knobel, B.S., D. P. Carbone, M.D., Ph.D. Vanderbilt University Medical Center. Introduction: DC loaded with apoptotic, syngeneic melanoma cells slow tumor growth and improve survival in a murine immunotherapy model. The use of allogeneic cells as a source of tumor antigen could increase the clinical applicability of this therapy, so we investigated the efficacy of DC loaded with apoptotic, allogeneic cells in this model. Methods: DC were cultured from bone marrow from C-57/BL-6 mice with GMCSF (10 ng/ml), IL-4 (5 ng/ml). Syngeneic B-16 or allogeneic K-1735 melanoma cells were irradiated (20 Gy) to induce apoptosis and cultured with DC at a 1:1 ratio for 48 hr to

Control DC/ApoB-16 DC/ApoK-1735

Tumor size

Median survival

11 ⫾ 6 mm 3 ⫾ 3 mm* 4 ⫾ 3 mm*

31 days ⬎60 days* 45 days*

60d survival

Specific lysis# 9 ⫾ 12% 63 ⫾ 20%* 51 ⫾ 21%*

0% 67%* 33%*

* P ⬍ 0.05 vs control. # At E:T ratio of 100:1. Conclusions: Mice treated with DC loaded with apoptotic, syngeneic melanoma cells (DC/ApoB-16) or apoptotic, allogeneic melanoma cells (DC/ApoK-1735) had delayed tumor progression and improved survival compared to controls. CTL responses to B-16 cells were seen in both treatment groups, and there was no difference in tumor size, survival, or CTL responses among the treatment groups. We conclude that DC loaded with apoptotic allogeneic melanoma cells may be a practical and effective approach to polyvalent immunotherapy of melanoma. We are currently designing a clinical trial to evaluate the efficacy of this treatment in patients with high-risk disease. 36. Macrophage Inflammatory Protein-3alpha (mip-3alpha) Promotes Pancreatic Cancer Cell Invasion by Upregulation of Matrix Metalloproteinase-9 (MMP-9) Production. A. S. Campbell, M.D., D. Albo, M.D., Ph.D., T. F. Kimsey, M.D., S. L. White, T. N. Wang, M.D., Ph.D. Medical College of Georgia. Introduction: MIP-3␣ has been shown to promote tumor cell migration by upregulation of MMPs. We hypothesize that MIP-3␣, by binding to the transmembrane receptor CCR6, promotes pancreatic cancer invasion through the upregulation of MMP-9, a type IV collagenase that plays a crucial role in tumor progression. Methods: RT-PCR for MIP-3␣ was performed on PANC-1, human pancreatic adenocarcinoma cells. The effect of MIP-3␣ on MMP-9 production by PANC-1 cells was evaluated by Western analysis. Tumor cell invasion was evaluated using a modified Boyden chamber invasion assay. Results: RT-PCR confirmed the presence of MIP-3␣ in PANC-1 cells. MIP-3␣ stimulated the production of both latent and active forms of MMP-9 as demonstrated by Western analysis (see Figure). MIP-3␣ promoted a dose-dependent increase in pancreatic cancer cell invasion (see Table). Anti-CCR6 Ab and anti-MMP-9 Ab inhibited MIP-3␣ stimulated PANC-1 invasion of collagen to 37% and 35% of control, respectively (p ⬍ 0.05). Conclusion: MIP-3␣, through its CCR6 receptor, causes a dose-dependent increase in tumor cell invasion by the upregulation of MMP-9. Molecular based therapy aimed at the inhibition of MIP-3␣ through the CCR6 receptor may

TABLE—ABSTRACT 36 Summation of Invasive Cells in Five [MIP-3␣] ng/ml Invasive cells

0

10

50

100

9.0 ⫾ 6.1

13.0 ⫾ 2.6

16.7 ⫾ 4.5

23.3 ⫾ 5.0*

ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS serve as a future target to prevent tumor cell invasion in pancreatic adenocarcinoma.

37. Soluble Receptor for Advanced Glycation Endproducts (sRAGE) Effectively Reduces Tumor Growth as a Single Agent and in Combination with Doxorubicin in a Spontaneous Mammary Tumor Model. K. P. Joseph, M.D., M.P.H., L. Liu, M.D., Ph.D., W. Qu, M.D., F. R. Schnabel, M.D., A. Schmidt, M.D. Columbia-Presbyterian Medical Center, New York, NY. Introduction: The receptor for advanced glycation endproducts (RAGE), a member of the immunoglobulin family interacts with distinct ligands that have been implicated in various pathophysiologic states such as diabetes, atherosclerosis, inflammation as well as tumor growth. We have previously demonstrated that blockade of RAGE in tumors raised in mice from rat C6 glioma cells or murine Lewis lung carcinoma effectively reduced tumor growth, invasion and metastases. Blockade of RAGE has also previously been shown to halt the progression of breast tumors in a murine model that spontaneously produces breast tumors. We hypothesize that the addition of RAGE antagonists to standard chemotherapeutic agents for breast cancer may enhance the effectiveness of monotherapy. Methods: Female MMTV transgenic mice (5-8 weeks) were divided into four groups: (1) Control group-no treatment, (2) doxorubicin (DOX) 2 mg/kg IP weekly x 4 weeks (3) sRAGE 20 ␮g IP daily x 4 weeks and (4) DOX 2 mg/kg IP weekly x 4 weeks and sRAGE 20 ␮g IP daily x 4 weeks. The mice were sacrificed and tumors harvested on treatment day 35 and were weighed and measured. Results: There was no difference in tumor weight between the control mice (n ⫽ 9) and DOX-treated mice (n ⫽ 4) (0.192 g v. 0.193). The administration of sRAGE (n ⫽ 7) decreased tumor weight to 0.12 g and the combination of sRAGE and DOX (n ⫽ 8) decreased tumor weight by a third (0.192 g v. 0.061 g, p ⫽ 0.11). While the mean tumor volume in the control mice was 316.7 mm 3, sRAGE decreased tumor volume to 89.65 mm 3 and the combination of sRAGE and DOX decreased tumor volume by 6.5 fold (316.7 mm 3 vs. 48.08 mm 3, p ⫽ 0.006). The mean tumor volume in the DOX treated group was 500.9 mm 3. Conclusions: RAGE antagonists suppress local tumor growth in transgenic mice with spontaneously occurring cancer in an early intervention model. The addition of sRAGE, a low molecular weight RAGE antagonist, to doxorubicin enhanced the effectiveness of this standard chemotherapy agent. 38. Genetic Heterogeneity of Colorectal Cancer Liver Metastases. J. C. Sung, M.D., J.D., D. Boulware, M.S., S. Eschrich, Ph.D., F. Gonzalez, B.S., T. J. Yeatman, M.D. H. Lee Moffitt Cancer Center. Introduction: Although liver metastases are a leading cause of colorectal cancer, the molecular genetic basis of the advanced disease stages remains poorly understood. Whether the metastatic lesions are genetically homogeneous or heterogeneous may determine the response to therapy. We investigated whether synchronouslyoccurring, multi-focal colon cancer liver metastases were of multiclonal origin by using genome-scale microarray analysis. Methods: Five distinct colon cancer liver metastases were biopsied from a single patient harboring 15 or more lesions. All specimens were microdissected to ensure ⬎90% tumor cells and total RNA was

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extracted and prepared for hybridization. Each specimen was analyzed using the Affymetrix U133 GeneChip TM containing approximately 22,000 gene elements. The gene expression profiles of the five specimens were then compared using two independent methods: unsupervised hierarchical clustering to determine the relatedness of the samples and the chi-square test of independence to assess whether the distribution of ranks was different across the samples. Results: No histological difference was observed among the microdissected samples. Each specimen appeared to have equivalent numbers of tumor cells versus stromal cells. However, unsupervised hierarchical clustering of all 22,000 genes by five samples yielded five distinct clusters. Moreover, ␹ 2 analysis identified 5 statistically significant (p ⬍ 0.001) groups. Conclusions: Genetic profiling of multiple liver metastases using genome scale profiling suggests that colon cancer metastases are muti-clonal in origin. These results have negative implications for the effective application of single agent systemic therapy. 39. Endocytosis of VEGFR-2 on Endothelial Progenitor Cells by Dopamine Attenuates Tumour Growth and Vasculogenesis. B. D. Barry, M.D., E. Condon, M.D., J. H. Wang, Ph.D., H. P. Redmond, M.D. Cork University Hospital, Cork, Ireland. Introduction: Endothelial progenitor cells (EPC’s) andvasculogenesis are intricately involved in the initial neovascularisation of tumours. EPC’s are characterised by the expression of VEGFR-2 and AC133. Vasculogenesis offers a novel possibility for the targeting of anti-cancer therapies. Methods: Ex-vivo expanded EPC’s were cultured from peripheral blood samples from healthy male volunteers. To endocytose VEFGR-2 on EPC’s, cells were treated with incremental doses of dopamine and VEGFR-2 expression was analysed using anti-VEGFR-2 mAb on flow cytometry. Dil-labelled ex-vivo expanded EPC’s were transplanted into tumour bearing (3LL tumour cell line) MF1-nude mice (n⫽12). The mice received a daily i.p. injection of PBS or Dopamine (50 mg/kg). Tumour volume was assessed on alternate days. Vasculogenesis and angiogenesis were assessed using fluorescent microscopy and CD31 staining. To determine if Dopamine has a direct effect on 3LL cells, cells were cultured with incremental doses of dopamine for 48 h and cell apoptosis (PIstaining), proliferation (BrdU-Assay) and Cytotoxicity (LDH-Assay) were analysed. Statistical analysis was performed on SigmaStat using ANOVA. Results: Ex-vivo expanded EPC cultures were 80 – 85% pure. Dopamine caused an endocytosis of VEGFR-2 on EPC’s with the maximal effect seen at 1␮M (p ⬍ 0.05). Dopamine had no effect on 3LL cell apoptosis, proliferation or cytotoxicity in all doses performed. Dopamine, by endocytosing VEGFR-2 on EPC’s, attenuated tumour growth by decreasing both tumour volume (p ⬍ 0.05; 241.8 ⫹/- 106.8 v’s 59.7 ⫹/- 20.5 at day 10) and tumour weight (p ⬍ 0.05). Absolute and relative levels of vasculogenesis were decreased in the dopamine group as compared to controls (p ⬍ 0.05). Conclusion: Induction of VEGFR-2 endocytosis on EPC’s decreases tumour growth and vasculogenesis. This data suggests that VEGFR-2 is a target for attenuating tumour vasculogenesis. 40. Doxycycline and Indomethacin Synergistically Downregulate Beta-catenin Signaling and Inhibit Colon Cancer Cell Growth. N. K. Veeramachaneni, M.D., L. Lin, M.D., J. A. Pippin, B.A., E. R. Winslow, M.D., J. A. Drebin, M.D., Ph.D. Washington University. Introduction: Aberrant beta-catenin signaling, due to loss of the APC tumor suppressor gene, or mutation of beta-catenin, is found in the majority of colon cancers. We have recently demonstrated that doxycycline (DOX) decreases beta-catenin expression and transcriptional activity in colon cancer cells. NSAIDS also downregulate betacatenin by an undefined mechanism. We demonstrate synergistic inhibition of beta-catenin signaling and colon cancer cell growth by DOX and indomethacin (INDO). Methods: APC-mutant SW480 hu-