Murine Model Of Pancreatic Adenocarcinoma Research Articles

Nanoparticles use magnetoelectricity to target and eradicate cancer cells.

We investigated the theranostic capabilities of magnetoelectric nanoparticles (MENPs) combined with MRI via a murine model of pancreatic adenocarcinoma. MENPs leverage the magnetoelectric effect to convert an applied magnetic field into local electric fields, which can induce irreversible electroporation of tumor cell membranes when activated by MRI. Additionally, MENPs modulate MRI relaxivity, which can be used to predict the degree of tumor ablation. Through a pilot study (n=21) and a confirmatory study (n=27), we demonstrated that, ≥300 µg of MRI-activated MENPs significantly reduced tumor volumes, averaging a three-fold decrease as compared to controls. Furthermore, there was a direct correlation between the reduction in tumor T 2 relaxation times and tumor volume reduction, highlighting the predictive prognostic value of MENPs. Six of 17 mice in the confirmatory study's experimental arms achieved a durable complete response, showcasing the potential for durable treatment outcomes. Importantly, the administration of MENPs was not associated with any evident toxicities. This study presents the first in vivo evidence of an externally controlled, MRI-based, theranostic agent that effectively targets and treats solid tumors via irreversible electroporation while sparing normal tissues, offering a new and promising approach to cancer therapy.

UPF1 deficiency enhances mitochondrial ROS which promotes an immunosuppressive microenvironment in pancreatic ductal adenocarcinoma

Upstream frameshift 1 (UPF1) is an RNA helicase involved in a number of mRNA regulatory processes including nonsense-mediated decay. Mutations in the UPF1 locus that reduce its expression have been associated with adenosquamous carcinoma of the pancreas, a particularly aggressive form of the disease. To determine the effect of Upf1 suppression in a murine model of pancreatic adenocarcinoma, we silenced with shRNA Upf1 in cells derived from an autochthonous tumor in an LSL-Kras G12D/+ ; Trp53 R172H/+ ; Pdx-1 Cre/+ mouse (KPC) and orthotopically implanted these cells in the pancreas of C57BL/6 mice. Tumors derived from Upf1-deficient cells were markedly larger than those derived from control cells, a difference observed only in immunocompetent mice. The immune infiltrate of Upf1-deficient tumors was enriched in myeloid-derived suppressor cells (MDSCs) and depleted of CD8 + cells compared to control KPC tumors. Upf1-deficient KPC cells secreted inflammatory cytokines including G-CSF and CXCL2, known to recruit MDSCs. Cytokine secretion from Upf1-deficient KPC cells was induced by increased levels of mitochondrial reactive oxygen species (ROS), which in turn were due to an increase in complex I activity in the electron transport chain. Thus, Upf1 helicase deficiency leads to increased mitochondrial complex I activity which produces ROS that signals for cytokine release that drives immune suppression and enhanced tumor growth.

Trapping all ERBB ligands decreases pancreatic lesions in a murine model of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest of cancers. Attempts to develop targeted therapies still need to be established. Some oncogenic mechanisms in PDAC carcinogenesis harness the EGFR/ERBB receptor family. To explore the effects on pancreatic lesions, we attempted simultaneous blockade of all ERBB ligands in a PDAC mouse model. To this end, we engineered a molecular decoy, TRAP-FC , comprising the ligand-binding domains of both EGFR and ERBB4 and able to trap all ERBB ligands. Next, we generated a transgenic mouse model (CBATRAP/0 ) expressing TRAP-FC ubiquitously under the control of the chicken beta-actin promoter and crossed these mice with KRASG12D/+ mice (Kras) to generate Trap/Kras mice. The resulting mice displayed decreased emergence of spontaneous pancreatic lesion areas and exhibited reduced RAS activity and decreased activities of ERBBs, with the exception of ERBB4, which showed increased activity. To identify the involved receptor(s), we employed CRISPR/Cas9 DNA editing to singly delete each ERBB receptor in the human pancreatic carcinoma cell line Panc1. Ablation of each ERBB family member, especially loss of EGFR or ERBB2/HER2, altered signaling downstream of the other three ERBB receptors and decreased cell proliferation, migration and tumor growth. We conclude that simultaneously blocking the entire ERBB receptor family is therapeutically more effective than individually inhibiting only one receptor or ligand in terms of reducing pancreatic tumor burden. In summary, trapping all ERBB ligands can reduce pancreatic lesion area and RAS activity in a murine model of pancreatic adenocarcinoma; hence, it might represent a promising approach to treat PDAC in patients.

Visceral Surgery Profoundly Affects the Cellular and Humoral Components of the Anti-Tumour Immune Response in a Murine Pancreatic Adenocarcinoma Model.

Simple SummarySurgery is a fundamental part of the multimodal therapy concepts in oncological patients, especially in the early stage of pancreas tumour. There are numerous studies on the impact of primary tumour resection on the immune status, but to our knowledge, the impact of tumour-unrelated surgery on the anti-tumour immune response to the primary tumour it is not yet understood. Therefore, we used a murine model combining an orthotopically implanted adenocarcinoma of the pancreas and the model of surgically-induced immune dysfunction to assess the impact of postoperative immunosuppression on the growth of the primary tumour, on mortality and on the most important immune cell compartments in tumour defence. This knowledge might contribute to a basic understanding of the interaction of the primary tumour and the immune system and could guide new approaches to therapeutic strategies.(1) Background: Surgery is the most important element of multimodal treatment concepts in oncological patients, especially in the early stages of pancreatic tumours. While the influence of primary tumour resection on the immune status was analysed in several studies, the impact of tumour-unrelated visceral surgery on the tumour-bearing organism and on the primary tumour itself is not yet fully understood. (2) Methods: We combined a murine model of orthotopically implanted adenocarcinoma of the pancreas with the model of surgically-induced immune dysfunction (SID). Mortality and general condition including body weight were observed over a period of 28 days. Tumour growth was analysed by MRI scans on days 8 and 27 following tumour implantation. On day 28, the immune cell populations in the blood and spleen as well as the serum cytokines were quantified. (3) Results: SID results in a significant deterioration of the general condition and a reduced increase in the body weight of tumour-bearing mice compared to the control groups, while mortality and tumour growth rate were not influenced. The numbers of spleen macrophages and neutrophils were increased in tumour-bearing animals following SID. Furthermore, both macrophage and neutrophil levels were increased in the peripheral blood. (4) Conclusions: The presented results might contribute to the basic understanding of the interaction of tumour and immune system and could contribute to new approaches to immunotherapeutic strategies.

Abstract B48: Targeting the semaphorin 4D-plexin B axis to augment FOLFIRINOX in a murine model of pancreatic adenocarcinoma

Abstract Background: Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related mortality, with dismal 5-year prognosis of 8%, in part due to poor response to available therapies. Thus, new systemic cancer control therapies are in dire need. Semaphorin 4D (SEMA4D) is a soluble and membrane bound glycoprotein that binds its cognate Plexin B1/B2 receptors, expressed on monocytic and granulocytic leukocytes. Prior work has shown that increased expression of SEMA4D and Plexin B in resected PDAC patients is associated with lymph node and distant metastasis, as well as a worse prognosis. Additionally, SEMA4D blockade has conferred improved immune checkpoint blockade response in murine models of colorectal carcinoma and head and neck squamous cell carcinoma via abrogation of myeloid-derived suppressor cell recruitment and function, as well as enhanced T-cell recruitment and activity within the tumor microenvironment. Here we study the effects of SEMA4D blockade in a murine model of PDAC. Methods: C57b/6 mice were orthotopically injected with murine PDAC line (KP2) derived from KRASG12D,TP53Flox/Wt;P48-Cre autochthonous tumors. Mice were treated with Folfirinox (5-FU, irinotecan, oxaliplatin, weekly), immune checkpoint blockade (ICB) (anti-PD1, anti-CTLA-4 mAbs bi-weekly), and anti-SEMA4D mAB (biweekly). Peripheral blood and tumor-infiltrating leukocytes from patients with PDAC undergoing pancreaticoduodenectomy were assessed for Plexin B1 via flow cytometry. Results: Human PDAC demonstrates penetration of Plexin B1 positive leukocytes, most notably tumor-associated macrophages, neutrophils, and monocytes. Mice injected with KP2 developed tumors detectable via high-frequency ultrasound and exhibited longer survival when treated with the combination of Folfirinox, ICB, and anti-SEMA4D antibody, compared to Folfirinox alone, Folfirinox plus ICB, or Folfirinox plus anti-SEMA4D antibody. Conclusions: Plexin B1+ myeloid subsets penetrate human PDAC tumors, and treatment with SEMA4D-blocking antibody improved response to ICB in combination with standard-of-care Folfirinox in preclinical murine studies. Future work will focus on understanding the immune mechanism of improved therapeutic response, as well as further characterization of the prevalence and prognostic ramifications of the SEMA4D-Plexin B axis in PDAC. Citation Format: Luis I. Ruffolo, Katherine M. Jackson, Nicholas Ullman, Alexander Chacon, Alexa Melucci, Paul Burchard, Mary Georger, Rachel Jewell, Peter Prieto, Brian Belt, Crystal Mallow, Elizabeth Evans, Terrence Fisher, Maurice Zauderer, Christina Wu, Brian Olson, Gregory B. Lesinski, David C. Linehan. Targeting the semaphorin 4D-plexin B axis to augment FOLFIRINOX in a murine model of pancreatic adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B48.

Blockade of fibroblast activation protein in combination with radiation treatment in murine models of pancreatic adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a fibrotic stroma with a poor lymphocyte infiltrate, in part driven by cancer-associated fibroblasts (CAFs). CAFs, which express fibroblast activation protein (FAP), contribute to immune escape via exclusion of anti-tumor CD8+ T cells from cancer cells, upregulation of immune checkpoint ligand expression, immunosuppressive cytokine production, and polarization of tumor infiltrating inflammatory cells. FAP is a post-proline peptidase selectively expressed during tissue remodeling and repair, such as with wound healing, and in the tumor microenvironment by cancer-associated fibroblasts. We targeted FAP function using a novel small molecule inhibitor, UAMC-1110, and mice with germline knockout of FAP and concomitant knock-in of E. coli beta-galactosidase. We depleted CAFs by adoptive transfer of anti-βgal T cells into the FAP knockout animals. Established syngeneic pancreatic tumors in immune competent mice were targeted with these 3 strategies, followed by focal radiotherapy to the tumor. FAP loss was associated with improved antigen-specific tumor T cell infiltrate and enhanced collagen deposition. However, FAP targeting alone or with tumor-directed radiation did not improve survival even when combined with anti-PD1 therapy. Targeting of CAFs alone or in combination with radiation did not improve survival. We conclude that targeting FAP and CAFs in combination with radiation is capable of enhancing anti-tumor T cell infiltrate and function, but does not result in sufficient tumor clearance to extend survival.

Multiplex immunophenotyping reveals unique characteristics of pancreatic neoplasms

Introduction: The tumor microenvironment of pancreatic adenocarcinoma (PDA) is characterized by a dense stroma and infiltration with primarily immunosuppressive cells. While much is known about the immune cellular changes occurring during tumorigenesis in murine models of PDA, data in humans is lacking. We sought to identify difference in the immune microenvironment of neoplastic and pre-neoplastic lesions using multiplex immunohistochemistry. Method: Operative pancreatic tissue was collected from 289 patients undergoing pancreatectomy at the University of Michigan. Slides were prepared from formalin-fixed, paraffin embedded tissue which were subjected to serial rounds of primary and secondary antibody staining followed by tyramide signal amplification. Result: Analysis was performed on tissue from patients with normal pancreata (n=42), mucinous cystic neoplasms (MCN;n=22), intraductal papillary mucinous neoplasms (IPMN;n=80), pancreatic intraepithelial neoplasia (PanIN;n=37) and PDA (n=108). IPMNs and normal pancreata had significantly more epithelial cells when compared to MCN and PDA. In IPMNs, these cells were more frequently positive for the checkpoint inhibitor PD-L1 (mean = 16.4%; p<0.0001). Mucinous lesions (MCN and IPMN) had significantly more antigen presenting cells than other lesion types (mean = 4.6% and 3.2%, respectively; p=0.0048). While there were no differences in relative infiltration of cytotoxic T lymphocytes, PDA had a far greater overall and relative number of regulatory T lymphocytes (Treg) (mean = 17%; p<0.0001). The accumulation of Tregs paralleled progression of neoplasia with increasing infiltration in normal pancreata, PanIN and PDA (1%, 3%, 17%, respectively; p<0.0001). Conclusion: A better understanding of the immune microenvironment of PDA is needed for the development of novel immunotherapeutic approaches. These data demonstrate an accumulation of Tregs as tumors progress from preinvasive to invasive cancer, suggesting a potential avenue for intervention.

Abstract 4393: Electrochemotherapy with irreversible electroporation and FOLFIRINOX improves survival in murine pancreatic adenocarcinoma by increasing apoptosis and decreasing tumor cell proliferation

Abstract Introduction: Combining gemcitabine (gem) with irreversible electroporation (IRE) has been shown to result in increased tumor cell apoptosis in a murine model of pancreatic adenocarcinoma (PDAC). However, it remains unclear whether this effect can be augmented locally and systemically by utilizing more active chemotherapies. The objective of this study was to evaluate the efficacy of IRE+FOLFIRINOX (FOL: 5-FU, leucovorin, irinotecan, and oxaliplatin). Methods: Athymic nude mice underwent intrapancreatic injection with S2-013 PDAC cells. After 7-10 days, tumors were confirmed with ultrasound and mice were treated with chemotherapy (gem or FOL), IRE, or chemotherapy+IRE. Four hours after last chemotherapy, pancreas was evaluated histologically. Levels of irinotecan and its active metabolite (SN-38) were evaluated in organs of mice treated with FOL and IRE+FOL. Experiments were repeated and survival analyses performed to evaluate long-term efficacy. Pancreas was harvested for histology and evaluation of apoptotic machinery, cell cycle proteins, and tumor cell proliferation. Results: IRE+FOL (ECT) resulted in increased tumor cells apoptosis compared to gem, gem+IRE, or FOL (apoptotic index (AI) 34.6% vs. 3.8%, 7.3%, 5.8%, respectively, p&amp;lt;0.001). High performance liquid chromatography (HPLC) demonstrated a 1.5-2-fold increase in irinotecan and SN-38 in the pancreas and a 4-fold increase of both compounds in the liver in mice treated with ECT vs. FOL. ECT significantly improved overall survival when compared to mice treated with IRE or FOL (median 7 days ECT vs. 4 days IRE vs. 3 days FOL, p=0.03). Histology demonstrated increased tumor cell apoptosis at time of death (AI = 34.2% ECT vs. 13.2% IRE vs. 4.4% FOL, p=0.026). Western blot demonstrated an increase in active caspase 3 (0.14 ECT vs. 0.00 IRE vs. 0.03 FOL fold change/GAPDH, p=0.02). ECT resulted in lower cell proliferation compared to IRE or FOL (0.2% vs. 0.3% vs. 0.9, p&amp;lt;0.001). Conclusions: ECT with IRE and FOL causes significant tumor cell apoptosis, decreases tumor cell proliferation, enhances systemic FOL exposure, and improves overall survival. Human Phase 1 trials have been initiated to evaluate safety and efficacy of this therapeutic modality. Citation Format: Neal Bhutiani, Harshul Pandit, Qianqian Zheng, Suping Li, Yan Li, Robert C. Martin. Electrochemotherapy with irreversible electroporation and FOLFIRINOX improves survival in murine pancreatic adenocarcinoma by increasing apoptosis and decreasing tumor cell proliferation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4393.

Chloroquine reduces hypercoagulability in pancreatic cancer through inhibition of neutrophil extracellular traps

BackgroundThe hypercoagulable state associated with pancreatic adenocarcinoma (PDA) results in increased risk of venous thromboembolism, leading to substantial morbidity and mortality. Recently, neutrophil extracellular traps (NETs), whereby activated neutrophils release their intracellular contents containing DNA, histones, tissue factor, high mobility group box 1 (HMGB1) and other components have been implicated in PDA and in cancer-associated thrombosis.MethodsUtilizing an orthotopic murine PDA model in C57/Bl6 mice and patient correlative samples, we studied the role of NETs in PDA hypercoagulability and targeted this pathway through treatment with the NET inhibitor chloroquine. PAD4 and RAGE knockout mice, deficient in NET formation, were used to study the role of NETs in platelet aggregation, release of tissue factor and hypercoagulability. Platelet aggregation was assessed using collagen-activated impedance aggregometry. Levels of circulating tissue factor, the initiator of extrinsic coagulation, were measured using ELISA. Thromboelastograms (TEGs) were performed to assess hypercoagulability and changes associated with treatment. Correlative data and samples from a randomized clinical trial of preoperative gemcitabine/nab-paclitaxel with and without hydroxychloroquine were studied and the impact of treatment on venous thromboembolism (VTE) rate was evaluated.ResultsThe addition of NETs to whole blood stimulated platelet activation and aggregation. DNA and the receptor for advanced glycation end products (RAGE) were necessary for induction of NET associated platelet aggregation. PAD4 knockout tumor-burdened mice, unable to form NETs, had decreased aggregation and decreased circulating tissue factor. The NET inhibitor chloroquine reduces platelet aggregation, reduces circulating tissue factor and decreases hypercoagulability on TEG. Review of correlative data from patients treated on a randomized protocol of preoperative chemotherapy with and without hydroxychloroquine demonstrated a reduction in peri-operative VTE rate from 30 to 9.1% with hydroxychloroquine that neared statistical significance (p = 0.053) despite the trial not being designed to study VTE.ConclusionNETs promote hypercoagulability in murine PDA through stimulation of platelets and release of tissue factor. Chloroquine inhibits NETs and diminishes hypercoagulability. These findings support clinical study of chloroquine to lower rates of venous thromboembolism in patients with cancer.Trial registrationThis study reports correlative data from two clinical trials that registered with clinicaltrials.gov, NCT01128296 (May 21, 2010) and NCT01978184 (November 7, 2013).

Development of a Method to Implement Whole-Genome Bisulfite Sequencing of cfDNA from Cancer Patients and a Mouse Tumor Model.

The goal of this study was to develop a method for whole genome cell-free DNA (cfDNA) methylation analysis in humans and mice with the ultimate goal to facilitate the identification of tumor derived DNA methylation changes in the blood. Plasma or serum from patients with pancreatic neuroendocrine tumors or lung cancer, and plasma from a murine model of pancreatic adenocarcinoma was used to develop a protocol for cfDNA isolation, library preparation and whole-genome bisulfite sequencing of ultra low quantities of cfDNA, including tumor-specific DNA. The protocol developed produced high quality libraries consistently generating a conversion rate >98% that will be applicable for the analysis of human and mouse plasma or serum to detect tumor-derived changes in DNA methylation.

Pancreatic duct ligation in a Kras murine model of pancreatic adenocarcinoma reduces the number and the risk of appearance of premalignant lesions over time

Pancreatic duct ligation in a Kras murine model of pancreatic adenocarcinoma reduces the number and the risk of appearance of premalignant lesions over time

Abstract A30: AR20.5-based novel immunotherapy for pancreatic cancer

Abstract Pancreatic cancer is a fourth leading cause of cancer death in United States. Recent advances in tumor immunology have provided evidence for immune surveillance against pancreatic cancer. Previous studies have documented the presence of specific antibodies and cytotoxic T cell activities to Mucin 1 (MUC1), an antigen that is expressed in most pancreatic cancers. Hence, MUC1 remains a candidate for immunotherapeutic strategies for pancreatic cancer. Numerous attempts have been made to target this mucin glycoprotein; however, these efforts have shown only moderate success in part due to the complexity of the pancreatic tumor microenvironment. Previously, administration of murine monoclonal antibody BrevaRexAb-AR20.5 alone produced MUC1 specific immune response in a phase I study of advanced cancer patients, including increased human anti-MUC1 antibody levels and MUC1 specific T cell response in few patients. Though this was a phase I study, modest changes in tumor marker (CA15.3) but no anecdotal anti-tumor activity was observed in this small group of patients. Moving forward it is obvious that future immunization strategies should include other immune-modulators to amplify MUC1 specific immune responses of mAbAR20.5 and enhance other factors that contribute to tumor rejection. To that end, we investigated the anti-tumor effect of mAbAR20.5 in combination with anti-PD-L1 and poly (I:C) in murine models of pancreatic adenocarcinoma using human MUC1 expressing transgenic (hMUC1.Tg) mice, which are immunologically tolerant to MUC1. The therapeutic combination of mAb-AR20.5+anti-PD-L1+Poly (I:C) induced rejection or significant inhibition of tumor growth for two different MUC1 expressing pancreatic tumor cell lines, which was accompanied by persistent MUC1 specific memory immune response, which could be adoptively transferred to other mice and shown to protect against subsequent tumor challenge. We show that the anti-tumor response was effected by CD8 T cells, as their abrogation attenuated the anti-tumor response. Flow cytometric analysis of immunized mice demonstrated progressive increases in activated CD8 T cells in the peripheral circulation of combination treated mice. Together, these data support the hypothesis that targeting checkpoint induced immunosuppression (anti-PD-L1) together with the use of toll-like receptor 3 agonist as an adjuvant (poly (I:C) ) enhances the capacity of mAbAR20.5 to induce specific cell mediated immune responses to MUC1, which in turn provide long lasting anti-tumor response against pancreatic tumors. Our study supports the rapid translation of this strategy into clinical trials for pancreatic cancer patients. Citation Format: Kamiya Mehla, James A. Grunkemeyer, Kelly A. O'Connell, Maria M. Steele, Thomas Caffrey, Ragupathy Madiyalakan, Christopher F. Nicodemus, Michael A. Hollingsworth. AR20.5-based novel immunotherapy for pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A30.

Coadministration of a Gloriosa superba extract improves the in vivo antitumoural activity of gemcitabine in a murine pancreatic tumour model

BackgroundGloriosa superba L. (glory lily, Colchicaceae) contains colchicine, and related alkaloids such as 3-O-demethylcolchicine and its glycoside colchicoside. Previously the in vivo efficacy of a crude extract and a colchicine-poor / colchicoside-rich extract of G. superba seeds was shown in a murine model of pancreatic adenocarcinoma. Hypothesis/PurposeThe efficacy can be improved without obvious signs of toxicity by increasing the treatment dose; the efficacy of gemcitabine can be improved by coadministration of a Gloriosa superba extract. Study designA survival experiment was carried out in a murine model of pancreatic adenocarcinoma and the semi-long-term toxicity of both G. superba extracts was determined; a combination therapy with gemcitabine was evaluated. MethodsA crude ethanolic extract (GS) and a colchicine-poor / colchicoside-rich extract (GS2B) were prepared, containing 3.22% colchicine, 2.52% colchicoside and 1.52% 3-O-demethylcolchicine (GS), and 0.07%, 2.26% and 0.46% (m/m) (GS2B). They were evaluated in a murine model of pancreatic adenocarcinoma at a dose of 4.5mg/kg (p.o., daily) total content of colchicine and derivatives during 3 weeks, or at 3.0mg/kg (p.o., daily) combined with gemcitabine (60mg/kg, i.p., 3x/week) during 54 days. ResultsA significant effect in tumour growth over time was observed for gemcitabine and the combination therapy compared to the control group. No significant difference was observed for the groups treated with colchicine and both extracts. However, combination therapy was significantly better than the monotherapy with gemcitabine. Moreover, survival analysis showed a significant prolongation of the survival of the groups treated with gemcitabine and the combination therapy. A slight difference in survival was observed between gemcitabine and the combination therapy, the latter one being slightly better. No significant prolongation of survival was observed for the extracts and colchicine compared to the control group. ConclusionAlthough a relevant tumour growth inhibition and a difference of relative tumour volume compared to the control group were observed on day 11, and a slightly longer survival was noticed for GS2B, the most important conclusion from this study is that the crude G. superba extract (GS) might have an added value combined with gemcitabine in the treatment of pancreatic tumours.

Comparison of Two Syngeneic Orthotopic Murine Models of Pancreatic Adenocarcinoma

ABSTRACTBackground: Pancreatic adenocarcinoma has an extremely poor prognosis. The use of appropriate in vivo models is essential in devising methods to improve treatment outcomes. Methods: A pancreatic adenocarcinoma model based on tumor injection into the pancreatic head was compared with a pancreatic tail injection model in C57/BL6 mice. The murine pancreatic adenocarcinoma cell line PAN02, dispersed in MatrigelTM, was used for tumor induction. Results: Tumors developed in all animals in both models. Tumor size was more consistent within the pancreatic tail group at 20 days following induction, with no evidence of metastatic disease. Animals in the pancreatic head injection group showed signs of reduced health by 20 days following injection and developed jaundice. Microscopic liver metastases were noted in some of these animals at this time point. The overall survival of animals at 40 days following tumor induction was significantly lower in the pancreatic head injection group (0% vs. 35%; p < .001). Multiple liver metastases were noted in five of 10 (50%) animals in the head injection group, without evidence of peritoneal metastases. In the pancreatic tail injection group, 18 of 20 (90%) animals had multiple peritoneal metastases, and nine of 20 (45%) animals had evidence of isolated liver deposits. Tumors in both regions of the pancreas had similar histologic characteristics, with a dense fibrotic stroma at the interface between the tumor and the normal pancreas. Conclusion: Pancreatic head and tail orthotopic cancer models produce consistent tumors, but the patterns of tumor spread and survival differ according to the site of injection.

Abstract 4975: The role of the Ikaros transcription factor in regulatory T cell (Treg) development and function in a murine pancreatic adenocarcinoma model.

Abstract Pancreatic cancer (PC) is currently the only major form of cancer that still has survival rates in the single digits. The limited success of cancer therapies in treating pancreatic cancer has been primarily due to the immunosuppressive tumor microenvironment. In particular, there is an increased prevalence of regulatory T cells (Tregs) (CD4+CD25+CD127−), which suppress anti-tumor immune responses in PC tumor-bearing (TB) hosts. Tregs express the Forkhead BoxP3 (FoxP3) gene, which is critical for their suppressive function. Studies have shown that Ikaros, a zinc finger transcription factor, is an important regulator of T lymphocyte development and function. The regulation of Ikaros’ expression and function is controlled by post-translational modification events. Deficiencies in Ikaros are observed in various T cell leukemias and lymphomas. However, little is known about the possible role of Ikaros in regulating immune cell development in response to solid cancers. In this study, we aim to identify the role of Ikaros in regulating Treg homeostasis and function in a pancreatic tumor microenvironment. Therefore, using our murine model of pancreatic cancer, we isolated splenocytes from TB and control mice and performed flow cytometry and magnetic activated cell sorting (MACS) to immunophenotype and enrich T cell populations for in vivo and in vitro analyses. Also, we performed quantitative real-time PCR (qRT-PCR) and western blot analyses to evaluate Ikaros and FoxP3 mRNA and protein expression in whole and enriched CD3+ T cells from TB and control splenocytes. Our results showed that effector T cell percentages (CD4+ and CD8+) were significantly lower in splenocytes from TB mice compared to control. However, our results also showed a significant expansion of Tregs in splenocytes from TB mice compared to control. In addition, enriched TB Tregs (CD4+CD25+) suppressed antigen-specific CD8+T cell immune responses in a dose-dependent manner, in vitro. Preliminary qRT-PCR results revealed no significant difference in Ikaros mRNA expression; whereas, Ikaros protein expression was reduced in TB whole splenocytes compared to control. Also, Ikaros protein expression was reduced in TB enriched CD3+ T cells compared to control. Furthermore, our results showed an increase in FoxP3 protein expression in TB CD3+ T cells compared to control. These findings suggest that the pancreatic tumor microenvironment potentially down-regulates Ikaros’ protein expression, which may contribute to the expansion of Tregs and their suppression of CD8+T cell (anti-tumor) immune responses. Citation Format: Nadine Nelson, Maya Jerald, Laura Pendleton, Karoly Szekeres, Nasreen Vohra, Shari Pilon-Thomas, Tomar Ghansah. The role of the Ikaros transcription factor in regulatory T cell (Treg) development and function in a murine pancreatic adenocarcinoma model. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4975. doi:10.1158/1538-7445.AM2013-4975

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.

Activation state of stromal inflammatory cells in murine metastatic pancreatic adenocarcinoma

The histologic presence of macrophages (tumor-associated macrophages, TAMs) and neutrophils (tumor-associated neutrophils, TANs) has been linked to poor clinical outcomes for solid tumors. The exact mechanism for this association with worsened prognosis is unclear. It has been theorized that TAMs are immunomodulated to an alternatively activated state and promote tumor progression. Similarly, TANs have been shown to promote angiogenesis and tumor detachment. TAMs and TANs were characterized for activation state and production of prometastatic mediators in an immunocompetent murine model of pancreatic adenocarcinoma. Specimens from liver metastases were evaluated by immunofluorescence and immunoblotting. TAMS have upregulated expression of CD206 and CD163 markers of alternative activation, (4.14 ± 0.55-fold and 7.36 ± 1.13-fold over control, respectively, P < 0.001) but do not have increased expression of classically activated macrophage markers CCR2 and CCR5. TAMs also express oncostatin M (OSM). We found that TANs, not TAMs, predominantly produce matrix metalloproteinase-9 (MMP-9) in this metastatic tumor microenvironment, while MMP-2 production is pan-tumoral. Moreover, increased expression of VEGF colocalized with TAMs as opposed to TANs. TAMs and TANs may act as distinct effector cells, with TAMs phenotypically exhibiting alternative activation and releasing OSM and VEGF. TANs are localized at the invasive front of the metastasis, where they colocalize with MMP-9. Improved understanding of these interactions may lead to targeted therapies for pancreas adenocarcinoma.

A Syngeneic Orthotopic Murine Model of Pancreatic Adenocarcinoma in the C57/BL6 Mouse Using the Panc02 and 6606PDA Cell Lines

Background/Aims: To develop a clinically relevant immunocompetent murine model to study pancreatic cancer using two different syngeneic pancreatic cancer cell lines and to assess MRI for its applicability in this model. Methods: Two cell lines, 6606PDA and Panc02, were employed for the experiments. Cell proliferation and migration were monitored in vitro. Matrigel™ was tested for its role in tumor induction. Tumor cell growth was assessed after orthotopic injection of tumor cells into the pancreatic head of C57/BL6 mice by MRI and histology. Results: Proliferation and migration of Panc02 were significantly faster than those of 6606PDA. Matrigel did not affect tumor growth/migration but prevented tumor cell spread after injection thus avoiding undesired peritoneal tumor growth. MRI could reliably monitor longitudinal tumor growth in both cell lines: Panc02 had a more irregular finger-like growth, and 6606PDA grew more spherically. Both tumors showed local invasiveness. Histologically, Panc02 showed a sarcoma-like undifferentiated growth pattern, whereas 6606PDA displayed a moderately differentiated glandular tumor growth. Panc02 mice had a significantly shorter (28 days) survival than 6606PDA mice (50 days). Conclusion: This model closely mimics human pancreatic cancer. MRI was invaluable for longitudinal monitoring of tumor growth thus reducing the number of mice required. Employing two different cell lines, this model can be used for various treatment and imaging studies.

The Plasma Fraction of Stored Erythrocytes Augments Pancreatic Cancer Metastasis in Male Versus Female Mice

Males with pancreatic cancer have decreased survival compared with females. Interestingly, perioperative blood transfusions have been shown to reduce survival in patients with pancreatic adenocarcinoma. Recent evidence incriminates blood transfusions from female donors as a causative factor in acute lung injury. We therefore hypothesize that male mice with pancreatic cancer will have greater tumor progression than female mice in response to transfusion. Mice previously inoculated with pancreatic cancer cells received an intravenous injection of acellular plasma collected from single donor erythrocytes from either male or female donors. Control mice received an equal volume of intravenous saline. Necropsy to determine metastasis was performed in female mice at 4 wk status post-transfusion. The male group necessitated sacrifice at 3 wk post-transfusion due to clinical deterioration. Male mice developed more metastatic events than female mice, and this was accentuated when receiving blood from female donors. Male mice experienced weight loss within 2 wk of tail vein injection, and three mice in the male transfused groups died secondary to malignancy. Female mice did not manifest substantial weight loss, and did not die in the study time period. Male mice, compared with female, had significantly more metastatic events following transfusion of plasma from stored erythrocytes in an immunocompetent murine model of pancreatic adenocarcinoma. Moreover, the adverse effect of transfusion was augmented with female donor blood. These data are consistent with clinical outcomes from centers of excellence in treating pancreatic cancer and warrant further investigation.

EGF Receptor Antagonism Improves Survival in a Murine Model of Pancreatic Adenocarcinoma

The purpose of this study was to determine whether inhibition of the epidermal growth factor receptor (EGFR) is a plausible therapeutic strategy in pancreatic cancer. A human pancreatic cancer cell line (HPAC) was evaluated for the presence of EGFR with rtPCR and immunohistochemistry. Cells were grown in the presence of either 50 or 100 microM of erlotinib (EGFRI) for 72 hours and evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Eighty-six athymic nude/nude mice underwent orthotopic implantation of 10(7) HPAC cells and were blindly randomized into four groups: (1) Control; (2) Batimastat, a matrix metalloproteinase inhibitor (MMPI) at 400 ng/ml qod; (3) EGFRI at 100 mg/kg qd; and (4) MMPI and EGRRI (both). In vitro and in vivo effects of EGFRI with and without MMPI were compared. HPAC demonstrated high levels of expression of both the EGFR gene and the gene product. In vitro, both doses of EGFRI significantly reduced proliferation of HPAC at 48 (50 microM: 1.15 + 0.05 [st dev] versus 0.63 + 0.09 abs, P < 0.001) and 72 h (50 microM: 1.48 +/- 0.09 versus 0.73 +/- 0.05 abs, P < 0.001, paired Student's t-test). In vivo, each treatment group demonstrated a significant survival advantage (P = 0.0002 group 2, P = 0.0001 group 3, P = 0.012 group 4, log rank test) over controls. Mice treated with EGFRI showed reduced tumor implantation, size, weight, metastatic potential, and jaundice as compared to controls and MMPI-treated mice (all P < 0.05, Fisher's exact test). EGF receptor antagonism is not only a plausible therapy for treatment of ductal adenocarcinoma of the pancreas, but is also superior to matrix metalloproteinase inhibition alone or in combination.