Model Of Metastatic Pancreatic Cancer Research Articles

Efficacy of MDX-124, a novel anti-annexin-A1 antibody, in preclinical models of pancreatic cancer.

590 Background: Pancreatic cancer is a highly fatal disease with poor survival and response to both chemotherapy and immunotherapy. Novel approaches to treat this disease are urgently required. Annexin-A1 (ANXA1) is secreted in response to several physiological stimuli where it activates formyl peptide receptors (FPR1/2) triggering multiple oncogenic processes. High ANXA1 expression in pancreatic cancer patients is associated with poor overall survival, and influences cancer progression, drug sensitivity, migration and invasion. MDX-124 is a novel humanized antibody targeting ANXA1 and we have previously presented data demonstrating its significant antiproliferative activity. Here we present further data showing the efficacy of MDX-124 in several preclinical models of pancreatic cancer. Methods: In-vitro models utilized MIA PaCa-2, PANC-1 or BxPC-3 human pancreatic cancer cell lines. Cell cycle progression was evaluated by measuring changes in DNA content via flow cytometry. Pancreatic cancer cell viability following incubation with MDX-124 (0-10 µM) and 5FU (IC50) was assessed via MTT assay. A transwell migration assay was used to evaluate the effect of MDX-124 (0-50 µM) on pancreatic cancer cell migration. In-vivo efficacy was evaluated using an orthotopic mouse model of metastatic pancreatic cancer (FC1242luc/zsGreen; KPC-derived cell line) with bioluminescent imaging used to quantify the incidence and burden of lung metastases. Results: When compared to untreated MIA PaCa-2 pancreatic cancer cells, MDX-124 treatment decreased the proportion of cells in S-phase by 29% and G2 phase by 9.1%, with a concomitant increase in G1 of 38.1%. This occurred in a dose-dependent manner and is consistent with an MDX-124 mediated increase in cell cycle arrest. MDX-124 significantly reduced the viability of MIA PaCa-2 and PANC-1 cell lines versus an IgG control in a dose-dependent manner. Additionally in these two cell lines, combination of MDX-124 with 5FU (IC50) had a significant synergistic impact reducing cancer cell viability by 99.8% and 91.2% respectively. Furthermore, MDX-124 significantly reduced the migratory ability of MIA PaCa-2 and BxPC-3 pancreatic cancer cells. In the orthotopic model of metastatic pancreatic cancer, the murine analog of MDX-124 (MDX-001), markedly reduced both the incidence and size of lung metastases. Conclusions: MDX-124 demonstrated significant anti-tumor efficacy in several preclinical models of pancreatic cancer as a single agent, with increased potency observed when used in combination with 5FU. Medannex will initiate a First-In-Human study in Q4 2021 to evaluate MDX-124 in solid malignancies, including pancreatic cancer.

Intraperitoneal siRNA Nanoparticles for Augmentation of Gemcitabine Efficacy in the Treatment of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma is a deadly disease with limited treatment options due to late diagnosis and resistance to conventional chemotherapy. Among emerging therapeutic targets, the CXCR4 chemokine receptor and polo-like kinase 1 (PLK1) play critical roles in the progression, metastasis, and chemoresistance of pancreatic cancer. Here, we tested the hypothesis that combining CXCR4 inhibition by a polymeric CXCR4 antagonist PAMD-CHOL with PLK1 knockdown by siRNA will enhance the therapeutic effect of gemcitabine (GEM) in the orthotopic model of metastatic pancreatic cancer. We formulated nanoparticles with cholesterol-modified PAMD and siPLK1 and found strong synergism when combined with GEM treatment in vitro in both murine and human pancreatic cancer cell lines. The biodistribution of the nanoparticles in orthotopic pancreatic cancer models revealed strong accumulation in primary and metastatic tumors, with limited hepatic disposition. The cholesterol-containing nanoparticles showed not only increased tumor accumulation than the cholesterol-lacking control but also deeper penetration into the tumors. In a therapeutic study in vivo, the triple combination of PAMD-CHOL/siPLK1 and GEM showed superior anticancer activity when compared with single and dual combination controls. In conclusion, PAMD-CHOL/siPLK1 nanoparticles synergistically enhance anticancer activity of GEM in pancreatic cancer and represent a promising addition to the treatment arsenal.

Single-cell lineage tracing of metastatic cancer reveals selection of hybrid EMT states.

The underpinnings of cancer metastasis remain poorly understood, in part due to a lack of tools for probing their emergence at high resolution. Here we present macsGESTALT, an inducible CRISPR-Cas9-based lineage recorder with highly efficient single-cell capture of both transcriptional and phylogenetic information. Applying macsGESTALT to a mouse model of metastatic pancreatic cancer, we recover ∼380,000 CRISPR target sites and reconstruct dissemination of ∼28,000 single cells across multiple metastatic sites. We find that cells occupy a continuum of epithelial-to-mesenchymal transition (EMT) states. Metastatic potential peaks in rare, late-hybrid EMT states, which are aggressively selected from a predominately epithelial ancestral pool. The gene signatures of these late-hybrid EMT states are predictive of reduced survival in both human pancreatic and lung cancer patients, highlighting their relevance to clinical disease progression. Finally, we observe evidence for invivo propagation of S100 family gene expression across clonally distinct metastatic subpopulations.

H-scan, Shear Wave and Bioluminescent Assessment of the Progression of Pancreatic Cancer Metastases in the Liver

The non-invasive quantification of tumor burden and the response to therapies remain an important objective for imaging modalities. To characterize the performance of two newly optimized ultrasound-based analyses, we applied shear wave and H-scan scattering analyses to repeated trans-abdominal ultrasound scans of a murine model of metastatic pancreatic cancer. In addition, bioluminescence measurements were obtained as an alternative reference. The tumor metastases grow aggressively and result in death at approximately 4 wk if untreated, but longer for those treated with chemotherapy. We found that our three imaging methods (shear wave speed, H-scan, bioluminescence) trended toward increasing output measures with time during tumor growth, and these measures were delayed for the group receiving chemotherapy. The relative sensitivity of H-scan tracked closely with bioluminescence measurements, particularly in the early to mid-stages of tumor growth. The correlation between H-scan and bioluminescence was found to be strong, with a Spearman's rank correlation coefficient greater than 0.7 across the entire series. These preliminary results suggest that non-invasive ultrasound imaging analyses are capable of tracking the response of tumor models to therapeutic agents.

03:09 PM Abstract No. 219 Local immunotherapy: Intra-arterial liver tumor vaccination in a pig model of metastatic pancreatic cancer

Locoregional therapy lacks the ability to treat systemic disease. Systemic chemotherapy or immunotherapy has significant systemic side effects. Here, we present a proof-of-concept of intra-arterial immunotherapy using saponins (a vaccine adjuvant) in a pig tumor model. Liver and peripancreatic tumors were induced in an Oncopig (a transgenic pig with Cre-inducible p53 and Kras mutations) using an adenoviral vector carrying the Cre recombinase gene. A lipiodol/saponin emulsion was injected selectively into a branch of the hepatic artery through a 4 F catheter. Response to therapy was evaluated on contrast-enhanced CT and necropsy. As a negative control, selective hepatic artery embolization using 100-300 micron Embospheres was performed in a different pig. After selective hepatic artery immunoembolization using the lipiodol/saponin emulsion, CT performed 3 days later showed 19% decrease in size of the embolized liver tumors (n=2), 71% decrease in size of an unembolized liver tumor (n=1), and 25% decrease in size of peripancreatic tumors (n=2). On histology, tumor-infiltrating lymphocytes were seen in the necrotic liver tumors, and no viable tumor cells were seen in the liver or peripancreatic tumors. In the negative control pig (n=1), selective hepatic artery embolization using Embospheres resulted in tissue necrosis without a detectable local immune response on histology. In a pig model, intra-arterial tumor vaccination using lipiodol/saponin emulsion resulted in tumor-infiltrating lymphocytes, tumor necrosis, and regression of both intrahepatic and extrahepatic tumors. Additional experiments are necessary to confirm these encouraging preliminary results.

Dual role of Par-4 in abrogation of EMT and switching on Mesenchymal to Epithelial Transition (MET) in metastatic pancreatic cancer cells.

Epithelial-mesenchymal transition (EMT) is a critical event that occurs during the invasion and metastatic spread of cancer cells. Here, we conceive a dual mechanism of Par-4-mediated inhibition of EMT and induction of MET in metastatic pancreatic cancer cells. First, we demonstrate that 1,1'-β-D-glucopyranosyl-3,3'-bis(5-bromoindolyl)-octyl methane (NGD16), an N-glycosylated derivative of medicinally important phytochemical 3,3'-diindolylmethane (DIM) abrogates EMT by inducing pro-apoptotic protein Par-4. Induction of Par-4 (by NGD16 or ectopic overexpression) strongly impedes invasion with inhibition of major mesenchymal markers viz. Vimentin and Twist-1 epithelial marker- E-cadherin. Further, NGD16 triggers MET phenotypes in pancreatic cancer cells by augmenting ALK2/Smad4 signaling in a Par-4-dependent manner. Conversely, siRNA-mediated silencing of endogenous Par-4 unveil reversal of MET with diminished E-cadherin expression and invasive phenotypes. Additionally, we demonstrate that intact Smad4 is essential for Par-4-mediated maintenance of E-cadherin level in MET induced cells. Notably, we imply that Par-4 induction regulates E-cadherin levels in the pancreatic cancer cells via modulating Twist-1 promoter activity. Finally, in vivo studies with syngenic mouse metastatic pancreatic cancer model reveal that NGD16 strongly suppresses metastatic burden, ascites formation, and prolongs the overall survival of animals effectively.

Leiodermatolide, a novel marine natural product, has potent cytotoxic and antimitotic activity against cancer cells, appears to affect microtubule dynamics, and exhibits antitumor activity.

Pancreatic cancer, the fourth leading cause of cancer death in the United States, has a negative prognosis because metastasis occurs before symptoms manifest. Leiodermatolide, a polyketide macrolide with antimitotic activity isolated from a deep water sponge of the genus Leiodermatium, exhibits potent and selective cytotoxicity toward the pancreatic cancer cell lines AsPC-1, PANC-1, BxPC-3, and MIA PaCa-2, and potent cytotoxicity against skin, breast and colon cancer cell lines. Induction of apoptosis by leiodermatolide was confirmed in the AsPC-1, BxPC-3 and MIA PaCa-2 cells. Leiodermatolide induces cell cycle arrest but has no effects on in vitro polymerization or depolymerization of tubulin alone, while it enhances polymerization of tubulin containing microtubule associated proteins (MAPs). Observations through confocal microscopy show that leiodermatolide, at low concentrations, causes minimal effects on polymerization or depolymerization of the microtubule network in interphase cells, but disruption of spindle formation in mitotic cells. At higher concentrations, depolymerization of the microtubule network is observed. Visualization of the growing microtubule in HeLa cells expressing GFP-tagged plus end binding protein EB-1 showed that leiodermatolide stopped the polymerization of tubulin. These results suggest that leiodermatolide may affect tubulin dynamics without directly interacting with tubulin and hint at a unique mechanism of action. In a mouse model of metastatic pancreatic cancer, leiodermatolide exhibited significant tumor reduction when compared to gemcitabine and controls. The antitumor activities of leiodermatolide, as well as the proven utility of antimitotic compounds against cancer, make leiodermatolide an interesting compound with potential chemotherapeutic effects that may merit further research.

Su2073 Establishment of Metastatic Pancreatic Cancer Model That Can Monitor Tumor Burdens

Su2073 Establishment of Metastatic Pancreatic Cancer Model That Can Monitor Tumor Burdens

The CaSm (LSm1) oncogene promotes transformation, chemoresistance and metastasis of pancreatic cancer cells.

The cancer-associated Sm-like (CaSm) oncogene is overexpressed in 87% of human pancreatic tumor samples and CaSm knockdown has demonstrated therapeutic efficacy in murine models of pancreatic cancer. Evidence indicates that CaSm modulates messenger RNA degradation; however, its target genes and the mechanisms by which CaSm promotes pancreatic cancer remain largely unknown. Here, we demonstrate that the CaSm overexpression alters several hallmarks of cancer—including transformation, proliferation, chemoresistance and metastasis. Doxycycline-induced CaSm expression enhanced proliferation and both anchorage-dependent and -independent growth of the human Panc-1 cells in vitro. CaSm induction decreased gemcitabine-induced cytotoxicity and altered the expression of apoptotic regulation genes, including Bad, E2F1 and Bcl-XL. CaSm-overexpressing Panc-1 cells were twofold more migratory and fourfold more invasive than the driver controls and demonstrated characteristics of epithelial-to-mesenchymal transition such as morphological changes and decreased E-cadherin expression. CaSm induction resulted in changes in RNA expression of metastasis-associated genes such as MMP1, SerpinB5, uPAR and Slug. Using a murine model of metastatic pancreatic cancer, injection of CaSm-induced Panc-1 cells resulted in a higher abundance of hepatic metastatic lesions. Overall, CaSm overexpression contributed to a more aggressive cancer phenotype in Panc-1 cells, further supporting the use of CaSm as a therapeutic target against pancreatic cancer.

Abstract B60: Role of inflammatory monocyte mobilization in metastatic pancreatic cancer

Abstract Introduction: More than half of patients with Pancreatic Cancer (PC) present with metastasis. Our group and others have shown that PC induces cellular changes in the liver long before metastatic spread. These pre-metastatic changes include a significant recruitment of inflammatory monocytes (IM) by the chemokine CCL2 (produced by pre-metastatic liver) and its receptor CCR2 (expressed on IM). Based on our preliminary data, we hypothesized that IM mobilization to the liver is essential for growth of metastatic PC. We therefore optimized our model of metastatic PC to study the role of CCR2 inhibition on growth of metastatic PC. Methods: We implanted a spontaneously derived murine PC cell line (KCKO) orthotopically in the tail of the pancreas of C57BL/6 (WT) mice. We removed the primary tumor on day 10 and divided the spleen into two hemi-spleens. At this time, mice were injected with luciferase expressing tumor cells in the inferior pole of the spleen which was removed after injection and liver metastases were detected by bioluminescence (BLI). Results: Mice with established liver metastasis were randomized to treatment with CCR2 antagonist (CCR2i), FOLFIRINOX, CCR2i and FOLFIRINOX combination (CCR2i+FOLFIRINOX), or vehicle. Mice were treated for three weeks and were imaged biweekly in order to quantify tumor burden in the liver. Mice were sacrificed after 3 weeks and gene expression and flow cytometry studies were performed on peripheral blood, bone marrow, and liver. Liver metastases were detected by BLI in 70% of mice after 10 days and 100% of mice after 15 days following splenic injection with luciferase expressing tumor cells. Peripheral blood and liver inflammatory monocytes/macrophages were significantly increased in mice bearing liver metastasis. However, CCR2i efficiently blocked the recruitment of inflammatory monocyte/macrophage populations in the liver (p<0.01). Additionally mice treated with CCR2i alone and CCR2i and FOLFIRINOX combination exhibited a shift in the immune gene profile in metastatic liver from a pro-tumor (Type 2) to an anti-tumor (Type 1) immune response. Consequently, metastatic tumor burden was significantly decreased after treatment with CCR2i and FOLFIRINOX. Interestingly, the lowest tumor burden was found in livers of mice treated with combination of CCR2 inhibitor and FOLFIRINOX. This suggests that targeting CCR2 can decrease the growth of liver metastasis in PC. Conclusion: Our murine model of PC metastasis formation recapitulates the human form of the disease. We demonstrate that inflammatory monocyte recruitment to the liver is important in promoting the development of metastasis in liver. CCR2 blockade in combination with FOLFIRINOX decreases macrophages in the liver and impairs growth of liver metastasis. Citation Format: Roheena Z. Panni, Dominic E. Sanford, Brian A. Belt, Timothy M. Nywening, Peter Goedegebuure, David C. Linehan. Role of inflammatory monocyte mobilization in metastatic pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B60.

Establishment and study of highly metastatic pancreatic cancer model in nude mice

Objective To study a method of orthotopic transplantation model of pancreatic cancer with high success rate and high transfer rate. Methods This research used the MIA PaCa-2 cell lines to establish a nude mouse orthotopic transplantation model of pancreatic cancer.The cancer cells were subcutaneously injected into 30 nude mice, after being tumor, removed subcutaneous tumor with sterile conditions, grew into nude mice pancreas tail in situ.Morphological and biological features, and metastasis of the transplanted tumor were studied after 4, 6 and 8 weeks. Results In this study the rate of tumor was 100%,and there was no obvious difference from the cells morphology of pancreatic tumor between 4 weeks and 8 weeks with hematoxylin and eosin(HE) staining, there were poorly differentiated pancreatic cancer cells.The distant metastasis rate was 0% in postoperative 4 weeks by the microscopic observation, distant metastasis in nude mice was 100% in postoperative 8 weeks, including local lymph node metastasis and distant organ metastasis, there were extremely significant statistical differences(P< 0. 05).The hepatic metastasis rate was 10% only. Conclusion That showed tumor of 4 weeks belonged to pancreatic cancer early in the disease process, and tumor of 8 weeks belonged to the late stages of pancreatic cancer disease process, it is high success rate, high transfer rate and low hepatic metastasis rate of using this method to build the orthotopic transplantation model. Key words: Pancreatic cancer; Nude mouse; Orthotopic model; Highly metastatic

Role of inflammatory monocyte mobilization in growth of liver metastasis in a murine model of metastatic pancreatic cancer.

299 Background: Pancreatic cancer (PC) is currently the fourth leading cause of death and the mortality rate approaches 100% due to early metastatic spread. Liver is the most common site of metastasis in PC. Our group and others have shown that PC induces cellular changes in the liver which include increase in inflammatory monocyte and macrophage population, long before development of metastasis. Based on our preliminary data, we developed a murine model of metastatic PC to study the role of chemotherapy in addition to novel IM blocking agents in established PC metastasis. Methods: We implanted spontaneously derived murine PC cell line (KCKO) orthotopically in the tail of pancreas of WT mice. 10 days after tumor implantation, there was an increase in infiltrating IM and macrophages in the liver by flow-cytometry and IHC. There was also an upregulation of genes associated with development of metastasis and recruitment of myeloid cells i.e. CCL2, CSF1, s100a4, s100a8, CXCL12, LOX, VEGF-A (P&lt;0.01) by RT-PCR. However, there was no evidence of tumor metastasis at this stage in the liver. We removed the primary tumor on day 10. At this time, mice were injected with βluc-KCKO in the inferior pole of the spleen which was removed. Liver metastases were detectable by bioluminescence in 100% mice after 15 days of splenic injection. Results: Mice with established liver metastasis were randomized to treatment with CCR2i, FOLFIRINOX, CCR2i and CCR2i+FOLFIRINOX or vehicle. Mice were treated for 4 weeks and were imaged biweekly in-order to quantify tumor burden in the liver. Mice were sacrificed after 4 weeks and flow-cytometry studies were performed on peripheral blood, bone marrow and liver. The peripheral blood IM were significantly increased in mice bearing liver metastasis. However, CCR2i efficiently blocked the recruitment of IM and macrophages in liver (p&lt;0.01). The burden of liver metastasis was significantly decreased after four weeks of treatment. Interestingly, the lowest tumor burden was found in livers of mice treated with combination of CCR2i and FOLFIRINOX. Conclusions: We demonstrate that CCR2i in combination with FOLFIRINOX decreases macrophages in the liver and impairs growth of liver metastasis.

Abstract IA12: Oncogenic KRAS and the inflammatory microenvironment in pancreatic cancer

Abstract Pancreatic cancer -one of the deadliest human malignancies- is preceded by precursor lesions, most commonly Pancreatic Intraepithelial Neoplasia (PanIN). PanINs are characterized by changes in the epithelial architecture, and by the accumulation of a fibroinflammatory stroma. Oncogenic mutation in Kras are almost invariably associated with invasive pancreatic cancer (1-3) and often detected in PanIN lesions (4). Expression of oncogenic Kras in the mouse pancreas recapitulates the step-wise progression of the human disease (5). We have previously published a genetically engineered mouse model that allows inducible, tissue specific and reversible expression of oncogenic Kras in the pancreas, the iKras* mouse (6). By taking advantage of the reversible nature of oncogenic Kras expression in this model, we were able to address the role of this oncogene in PanIN and cancer maintenance. First, we showed that inactivation of oncogenic Kras at the PanIN stage leads to regression of the lesions and recovery of the pancreatic parenchyma. Then, by crossing iKras* mice with a mouse conditionally expressing a mutant form of the tumor suppressor p53 (7), we generated a model of metastatic pancreatic cancer, the iKras*p53* mouse(8). Inactivation of oncogenic Kras* in iKras*p53* mice bearing invasive tumors and metastases led to tumor regression, albeit without complete eradication of the malignant cells. Taking advantage of the reversible nature of oncogenic Kras expression in our model, we have undertaken to investigate the effect of modulating epithelial Kras expression on the tumor stroma. We have previously shown that the extensive myofibroblast population surrounding PanINs and invasive cancer depends on epithelial Kras for its proliferation, active status and maintenance. Recently, we have started characterizing the changes in immune cells infiltrating the pancreas during the PanIN stage. The induction of acute pancreatitis leads to extensive immune infiltration both in wild-type and iKras* mice. However, in wild type animals the immune infiltration is transient, and coincides with the dynamics of tissue repair. In contrast, in iKras* mice the immune infiltration is sustained over time. Moreover, the nature of the infiltrating cells is different in iKras* mice, with increased CD4+ T cells, and immature myeloid cells and fewer CD8+ T cells. This infiltration of immune cells is dependent on oncogenic Kras over time, as inactivation of Kras expression leads to a reduction in the number of inflammatory cells over time. While the nature of infiltrating immune cells over pancreatic carcinogenesis has been characterized before (9), the function of each specific subset is not fully understood. CD4 T cells are prevalent within the Kras-mutant pancreas, and include immune-suppressive cells such as regulator T cells and Th17 cells. Thus, we hypothesized that CD4+ T cells are required in pancreatic cancer. By genetically ablating CD4 T cell in iKras* mice (thus generating iKras*;CD4-/- animals) we determined that CD4 T cells are required for PanIN formation. Furthermore, we showed that CD4+ T cells promote pancreatic carcinogenesis by inhibiting the anti-tumor activity of CD8 + T cells. Thus, our results indicate that a potential anti-cancer immune response is inhibited by CD4+ T cells in response to signals derived from Kras mutant epithelial cells. Given the potential of immune-modulation as part of pancreatic cancer therapy, we plan to follow up on our initial observations by identifying the epithelial signals that modulate the immune response in pancreatic cancer.

Transferrin receptor targeting nanomedicine delivering wild-type p53 gene sensitizes pancreatic cancer to gemcitabine therapy

To overcome gene therapy barriers such as low transfection efficiency and nonspecific delivery, liposomal nanoparticles targeted by a single-chain antibody fragment to the transferrin receptor (TfRscFv) delivering wild-type (wt) human p53 (SGT-53) were developed for tumor-specific targeting. We hypothesize that SGT-53 in combination with gemcitabine will demonstrate enhanced therapeutic benefit in an in vivo metastatic pancreatic cancer model. Intrasplenic injection of 1 × 10(6) Panc02 murine pancreatic cancer cells was used to generate in vivo hepatic metastatic tumors. Nanoparticle localization was assessed by tail vein injection of TfRscFv with fluorescently labeled oligonucleotides (6-carboxyfluorescein phosphoramidite (6FAM) ODN) imaged by Xenogen IVIS 200 scan. SGT-53 (equivalent to 30 μg of p53 intravenously) and gemcitabine (20 mg/kg intraperitoneally) alone and in combination were administered biweekly and compared with untreated mice. Survival was determined by blinded daily assessment of morbidity. Human wtp53 expression and transferrin levels in the tumors were assessed by western blot analysis. Tumor burden was quantified by liver weight. Xenogen imaging demonstrated tumor-specific uptake of TfRscFv-6FAM ODN. Exogenous human wtp53 protein was detected in the SGT-53-treated tumors compared with control. Compared with untreated mice with metastatic tumors demonstrating median survival of 20 days, SGT-53, gemcitabine and the combination demonstrated improved median survival of 29, 30 and 37 days, respectively. The combination treatment prolonged median survival when compared with single drug treatment and decreased tumor burden. The tumor targeting liposomal-based SGT-53 nanoparticle is capable of sensitizing pancreatic cancer to conventional chemotherapy in pancreatic cancer models. This approach has the potential to be translated into a new, more effective therapy for pancreatic cancer. Further optimization is ongoing, moving towards a Phase 1B/2 clinical trial.

Transferrin Receptor Targeting Nanomedicine Delivering Wild Type P53 Gene Sensitizes Pancreatic Cancer to Gemcitabine Therapy

To overcome gene therapy barriers such as low transfection efficiency and non-specific delivery, liposomal nanoparticles targeted by a single chain antibody fragment to the transferrin receptor (TfRscFv) delivering wild type human p53 (SGT-53) were developed for tumor-specific targeting. We hypothesize that SGT-53 in combination with gemcitabine will demonstrate enhanced therapeutic benefit in an in vivo metastatic pancreatic cancer model.

Thrombelastography Delineates Hypercoagulability in an Immunocompetent Murine Model of Metastatic Pancreatic Cancer

Introduction: Pancreatic cancer has the highest risk for venous thrombosis of all gastrointestinal malignancies. Although there are recent consensus guidelines for anticoagulation in cancer patients, the implimentation of prophlaxis anticoagulation is still sub optimal. Current diagnostic tests are unreliable in predicting cancer related hypercoagulability, leading to interest in examining the kinetics of clot formation by thrombelastography. We hypothesize that thrombelastography will characterize hypercoagulability in a metastatic murine model of pancreatic adenocarcinoma. Methods: C57/BL6 mice, age 7-9 weeks, underwent splenic inoculation with 2.5×105 Pan02 murine pancreatic adenocarcinoma cells. At necropsy, (7 weeks) blood was collected with citrate (1:10 ratio) and TEG was obtained on Thrombelastograph® Analyzer. TEG was compared between mice with cancer and control mice. Data were analyzed using non-parametric methods because our n=5. Results: Mice with cancer were found to have significantly higher Maximum Amplitude (MA) and G than control mice. Median MA was 60.6 (IQR: 59.4-62) mm in control mice compared to 74.2 (IQR 71.2-76) mm in mice with cancer. Conclusions: Thrombelastography identifies hypercoagulability in an immunocompetent murine metastatic pancreatic cancer model. Further, as thromboelastography can identify abnormalities in blood coagulation, specific patient guided anti-coagulation treatment may be possible.

Abstract LB-205: Using cell-delivered nanoparticles to cause local hyperthermia increases survival in a murine metastatic pancreatic cancer model

Abstract Hyperthermia has been a method for cancer treatment for several decades now because cancer cells are slightly more susceptible to hyperthermia than healthy cells. Unfortunately, whole body hyperthermia has prohibitive side effects limiting its use or usefulness. Local hyperthermia, directed only to tumor tissue, could alleviate this problem and prove to be a potent cancer treatment. One method of generating local hyperthermia is to deliver magnetic nanoparticles to the tumor site and then generate heat using an alternating magnetic field (AMF). Here we demonstrate a system that uses tumor homing cells to actively carry iron/iron oxide nanoparticles into tumor tissue for AMF treatment. Paramagnetic iron/iron oxide nanoparticles were synthesized that absorb and convert AMF energy into heat very efficiently. These nanoparticles were loaded into Raw264.7 cells (mouse monocyte/macrophage like cells, Mo/Ma), that we have previously shown to be tumor homing cells. Test showed that the nanoparticles loaded at high concentration in the cells with very low toxicity. To test the system, a murine pancreatic cancer model was generated by injection of Pan02 cells i.p. After tumor development, Mo/Ma loaded with iron/iron oxide nanoparticles were injected i.p. and allowed to crawl into the tumor. Three days after injection, mice were exposed to an alternative magnetic field for twenty minutes to cause the cell-delivered nanoparticles to generate heat. This treatment regimen was repeated three times. A survival study demonstrated that this system can significantly increase survival in a murine pancreatic cancer model, with an average post-diagnosis life expectancy increase of 33%. Thus, for the first time, a cell-delivered nanoparticle system for generating localized hyperthermia has been demonstrated that can significantly prolong the life of i.p. pancreatic tumor bearing mice. This system has the potential to become a useful method for specifically and actively delivering nanoparticles for local hyperthermia treatment of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-205. doi:10.1158/1538-7445.AM2011-LB-205

Abstract LB-199: SN38-dextran prodrug synthesis and cell delivery in a murine metastatic pancreatic cancer model

Abstract Enzyme activated prodrugs have been investigated and sought after as highly-specific, low side effect treatments, especially for cancer therapy. We synthesized an SN38-dextran prodrug and utilized it in a cell delivery system that can carry both the prodrug and an activating enzyme to the cancer site. For delivery of our prodrug, we engineered Raw264.7 cells (mouse monocyte/macrophage like cells, Mo/Ma) to express intracellular rabbit carboxylesterase (InCE), which cleaves the prodrug to activate SN38. InCE expression was regulated by the TetOn® system, which silences the gene unless a tetracycline, such as doxycycline is present. To test the system, a murine pancreatic cancer model was generated by intraperitoneal (i.p.) injection of Pan02 cells. Engineered Mo/Ma cells were loaded with the SN38-dextran prodrug and were injected i.p. into the mice. Two days following injection, doxycycline was given i.p. to activate the InCE, which in turn activates the prodrug into active SN38. A survival study demonstrated that this system significantly increased survival in a murine pancreatic cancer model, with an average post-diagnosis life expectancy increase of 20%. Thus, for the first time, a prodrug/activating enzyme system self-contained within tumor-homing cells has been demonstrated that can significantly prolong the life of i.p. pancreatic tumor bearing mice. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-199. doi:10.1158/1538-7445.AM2011-LB-199

Transforming growth factor‐beta 2 gene silencing with trabedersen (AP 12009) in pancreatic cancer

Pancreatic cancer is one of the most aggressive human cancers with a 5-year survival rate of <5%. Overexpression of transforming growth factor-beta 2 (TGF-β2) in pancreatic malignancies is suggested to be a pivotal factor for malignant progression by inducing immunosuppression, metastasis, angiogenesis and proliferation. Trabedersen (AP 12009) is a phosphorothioate antisense oligodeoxynucleotide specific for human TGF-β2 mRNA and was successfully tested in a randomized, active-controlled phase IIb clinical study in patients with high-grade glioma. Here, we report on the antitumor activity of trabedersen in human pancreatic cancer cells and in an orthotopic xenograft mouse model of human metastatic pancreatic cancer. Trabedersen reduced TGF-β2 secretion in human pancreatic cell lines with an IC50 in the low μM range without transfection reagent, clearly inhibited cell proliferation, and completely blocked migration of pancreatic cancer cells. Additionally, trabedersen reversed TGF-β2-mediated immunosuppression of pancreatic cancer cells targeted by lymphokine activated killer (LAK) cells, resulting in considerably increased LAK cell-mediated cytotoxicity. Moreover, in an orthotopic mouse model of metastatic pancreatic cancer, intraperitoneal (i.p.) treatment with trabedersen significantly reduced tumor growth, lymph node metastasis and angiogenesis. These promising results warrant further clinical development of trabedersen.

LY2109761, a novel transforming growth factor β receptor type I and type II dual inhibitor, as a therapeutic approach to suppressing pancreatic cancer metastasis

Most pancreatic cancer patients present with inoperable disease or develop metastases after surgery. Conventional therapies are usually ineffective in treating metastatic disease. It is evident that novel therapies remain to be developed. Transforming growth factor beta (TGF-beta) plays a key role in cancer metastasis, signaling through the TGF-beta type I/II receptors (TbetaRI/II). We hypothesized that targeting TbetaRI/II kinase activity with the novel inhibitor LY2109761 would suppress pancreatic cancer metastatic processes. The effect of LY2109761 has been evaluated on soft agar growth, migration, invasion using a fibroblast coculture model, and detachment-induced apoptosis (anoikis) by Annexin V flow cytometric analysis. The efficacy of LY2109761 on tumor growth, survival, and reduction of spontaneous metastasis have been evaluated in an orthotopic murine model of metastatic pancreatic cancer expressing both luciferase and green fluorescence proteins (L3.6pl/GLT). To determine whether pancreatic cancer cells or the cells in the liver microenvironment were involved in LY2109761-mediated reduction of liver metastasis, we used a model of experimental liver metastasis. LY2109761 significantly inhibited the L3.6pl/GLT soft agar growth, suppressed both basal and TGF-beta1-induced cell migration and invasion, and induced anoikis. In vivo, LY2109761, in combination with gemcitabine, significantly reduced the tumor burden, prolonged survival, and reduced spontaneous abdominal metastases. Results from the experimental liver metastasis models indicate an important role for targeting TbetaRI/II kinase activity on tumor and liver microenvironment cells in suppressing liver metastasis. Targeting TbetaRI/II kinase activity on pancreatic cancer cells or the cells of the liver microenvironment represents a novel therapeutic approach to prevent pancreatic cancer metastasis.