Tumor Cells In Pancreatic Cancer Research Articles

Polymer-Mediated Inhibition of Pro-invasive Nucleic Acid DAMPs and Microvesicles Limits Pancreatic Cancer Metastasis.

Nucleic acid binding polymers (NABPs) have been extensively used as vehicles for DNA and RNA delivery. More recently, we discovered that a subset of these NABPs can also serve as anti-inflammatoryagents by capturing pro-inflammatory extracellular nucleic acids and associated protein complexes that promote activation of toll-like receptors (TLRs) in diseases such as lupuserythematosus. Nucleic-acid-mediated TLR signaling also facilitates tumor progression and metastasis in several cancers, including pancreatic cancer (PC). In addition, extracellular DNA and RNA circulate on or within lipid microvesicles, such as microparticles or exosomes, which also promote metastasis by inducing pro-tumorigenic signaling in cancer cells and pre-conditioning secondary sites for metastatic establishment. Here, we explore the use of an NABP, the 3rd generation polyamidoamine dendrimer (PAMAM-G3), as an anti-metastatic agent. We show that PAMAM-G3 not only inhibits nucleic-acid-mediated activation of TLRs and invasion of PC tumor cells invitro, but can also directly bind extracellular microvesicles to neutralize their pro-invasive effects as well. Moreover, we demonstrate that PAMAM-G3 dramatically reduces liver metastases in a syngeneic murine model of PC. Our findings identify a promising therapeutic application of NABPs for combating metastatic disease in PC and potentially other malignancies.

Circulating and disseminated tumor cells in pancreatic cancer and their role in patient prognosis: a systematic review and meta-analysis.

BackgroundDisseminated tumor cells (DTCs) and circulating tumor cells (CTCs) have been postulated to seed metastases and contribute to poorer patient outcomes in many types of solid cancer. To date, no systematic reviews have examined the role of both DTCs and CTCs in pancreatic cancer. We aimed to determine the prognostic value of DTCs/CTCs in pancreatic cancer using a systematic review and meta-analysis.Materials and MethodsA comprehensive literature search identified studies examining DTCs and CTCs in the bone marrow and blood of pancreatic cancer patients at diagnosis with follow-up to determine disease-free/progression-free survival (DFS/PFS) and overall survival (OS). Statistical analyses were performed to determine the hazard ratio (HR) of DTCs/CTCs on DFS/PFS and OS.ResultsThe literature search identified 16 articles meeting the inclusion criteria. The meta-analysis demonstrated statistically significant HR differences in DFS/PFS (HR = 1.93, 95% CI 1.19–3.11, P = 0.007) and OS (HR = 1.84, 95% CI 1.37–2.45, P =< 0.0001), indicating patients with detectable DTCs/CTCs at diagnosis have worse prognoses. Subgroup analyses suggested CTCs in the peripheral blood (HR =2.03) were more indicative of poor OS prognosis than DTCs in the bone marrow (HR = 1.91), although the difference between these was not statistically significant. Positivity of the CellSearch detection method for DTC/CTC had the highest correlation with decreased OS (HR = 2.79) while immunodetection (HR = 1.91) and RT-PCR (HR = 1.25) were less effective in determining prognosis.ConclusionThe detection of DTCs/CTCs at diagnosis is associated with poorer DFS/PFS and OS in pancreatic cancer.

Abstract A10: Biomarker independant microfluidic Labyrinth for the high throughput enrichment and characterization of circulating tumor cells in pancreatic cancer

Abstract Introduction and Objective: Pancreatic cancer is a devastating disease with a 5-year survival rate of less than 6%. By the time of diagnosis, less than 15% of patients have surgically resectable tumors, which is one reason why it has the highest mortality rate among all cancer types. Emerging evidence has pointed the importance of circulating tumor cells (CTCs) in the spread of cancers and metastasis. CTCs are believed to be the most promising cancer biomarkers available in blood. Recent reports also show that these cells could be a good surrogate biomarker for not only prognosis, but also for cancer detection and development of personalized treatment. We have developed an inertial microfluidic-based separation technique for high throughput and label-free isolation of CTCs. Methods: To investigate the isolation efficiency of our technology before running cancer patients’ samples, PANC-1 cells labeled with cell tracker dye were spiked into 1 mL of whole blood. Using this optimized platform we isolate CTCs from pancreatic cancer patients, followed by downstream analysis . CTCs are characterized using immunocytochemistry for heterogeneity studies among CTC population. Results: Our microfluidic device consists of a size based separation platform in which CTCs are separated from other blood components. This high throughput label free separation device was designed and developed by our lab. After more than 20 generations of device revision, an optimized flow rate of 2.5 ml/min for PANC-1 pancreatic cancer cell line has yielded a 95% recovery of these cells from whole blood, with cancer cells traveling to the second outlet and 80% of the White Blood Cells (WBC) to the first outlet. We have successfully isolated CTCs from over 100 pancreatic cancer patients. Among several patients we were able to characterize different subpopulations using the epithelial marker CK-19 and EpCAM, and the EMT-like marker ZEB1. Conclusion: The implementation of our engineering technology to the study of pancreatic cancer can present novel ways to confront major hurdles in cancer research, such as early detection and the lack of effective therapeutics. The isolation and expansion of CTCs, the heterogeneous population of cells that promote metastasis, can provide meaningful information to elucidate the process of pancreatic tumorigenesis to preempt its fatal result. Citation Format: Lianette Rivera-Baez, Eric Lin, Meghna Waghray, Shamileh Fouladdel, Ebrahim Azizi, Max Wicha, Vaibhav Sahai, Diane Simeone, Sunitha Nagrath.{Authors}. Biomarker independant microfluidic Labyrinth for the high throughput enrichment and characterization of circulating tumor cells in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A10.

Clinical Significance of Circulating Tumor Cells in Pancreatic Cancer

Pancreatic cancer (PC) is a highly aggressive malignancy with limited therapeutic options. Early and accurate detection is critically important for successful cancer treatment. Currently, the most widely used and best validated marker for pancreatic cancer is carbohydrate antigen 19-9 (CA 19-9) and carcinoembryonic antigen (CEA). However, inadequate sensitivity and specificity limit the use of CA 19-9 and CEA in the early screening and diagnosis of pancreatic cancer. During past ten years, circulating tumor cells (CTCs) as a new liquid biomarker have received enormous attention in cancer diagnosis and management. Although CTCs are extremely rare and challenging to be isolated, their clinical significance has extensively been investigated in numerous clinical trials. In this review, we summarize the CTC application in translational pancreatic cancer research and its outlook in the future, specifically evaluating its clinical potential in pancreatic cancer diagnosis, prognosis, and treatments. Development of sensitive, specific, and reliable methods is expected towards a major impact on our ability in cancer management.

Abstract 1594: Application of label-free microfluidic technologies for the enrichment, expansion and characterization of circulating tumor cells in pancreatic cancer

Abstract Introduction and Objective: Pancreatic cancer is a devastating disease with a 5-year survival rate of less than 6%. By the time of diagnosis, less than 15% of patients have surgically resectable tumors, which is one reason why it has the highest mortality rate among all cancer types. Emerging evidence has pointed the importance of circulating tumor cells (CTCs) in the spread of cancers and metastasis. CTCs are believed to be the most promising cancer biomarkers available in blood. Recent reports also show that these cells could be a good surrogate biomarker for not only prognosis, but also for cancer detection and development of personalized treatment. We have developed an inertial microfluidic-based separation technique for high throughput and label-free isolation of CTCs. Methods: To investigate the isolation efficiency of our technology before running cancer patients’ samples, PANC-1 cells labeled with cell tracker dye were spiked into 1 mL of whole blood. Using this optimized platform we isolate CTCs from pancreatic cancer patients, followed by downstream analysis. CTCs are characterized using immunocytochemistry for heterogeneity studies among CTC population. Results: Our microfluidic device consists of a size based separation platform in which CTCs are separated from other blood components. This high throughput label free separation device was designed and developed by our lab. After more than 20 generations of device revision, an optimized flow rate of 2.5 ml/min for PANC-1 pancreatic cancer cell line has yielded a 95% recovery of these cells from whole blood, with cancer cells traveling to the second outlet and 80% of the White Blood Cells (WBC) to the first outlet. We have successfully isolated CTCs from over 100 pancreatic cancer patients. Among several patients we were able to characterize different subpopulations using the epithelial marker CK-19 and EpCAM, and the EMT-like marker ZEB1. Conclusion: The implementation of our engineering technology to the study of pancreatic cancer can present novel ways to confront major hurdles in cancer research, such as early detection and the lack of effective therapeutics. The isolation and expansion of CTCs, the heterogeneous population of cells that promote metastasis, can provide meaningful information to elucidate the process of pancreatic tumorigenesis to preempt its fatal result. Citation Format: Lianette Rivera-Baez, Eric Lin, Hyeun Joong Yoon, Meghna Waghray, Shamileh Fouladdel, Ebrahim Azizi, Max Wicha, Diane Simeone, Sunitha Nagrath. Application of label-free microfluidic technologies for the enrichment, expansion and characterization of circulating tumor cells in pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1594. doi:10.1158/1538-7445.AM2015-1594

Abstract 1481: Development of theranostic mesoporous silica nanoparticles for pancreatic cancer

Abstract Purpose: Modern methods of pancreatic cancer diagnosis and treatment are severely lacking and have failed to provide effectual treatment options for patients. The root cause of this inadequacy stems from the hypovascularized nature of pancreatic cancer, making traditional chemotherapeutics and cancer detecting contrast agents nearly obsolete. A potential solution for tumor-targeting difficulties is through the implementation of nanotechnology, specifically targeting ligand capped, theranostic nanoparticles. We hypothesize that silica based, mesoporous nanoparticles capped with the targeting ligand, urokinase plasminogen activator (UPA) will serve as theranostic agents for treatment and diagnosis of pancreatic cancer. Methods: Mesoporous, silica nanoparticles (MSNs) were synthesized through the covering of acetyl trimethylammonium bromide (CTAB) scaffolding with tetraethyl orthosilicate (TEOS) in an aqueous environment. Refluxing was later used to remove the CTAB scaffolding and construct the desired mesoporous structure of the nanoparticles. MSNs were chemically engineered to be sensitive to acidic pH, contain fluorescent dye, and utilize a protein targeting ligand UPA. Multiple characterization techniques were employed to analyze the synthesized MSNs including UV-Vis spectroscopy, Zeta Potential, and Transmission Electron Microscopy (TEM). Pancreatic cancer tumor cells, S2CP9 and S2VP10, were treated with UPA-targeted or no-ligand encapsulated MSNs at pH 7.4 and 6.5. Following treatment, cells were washed to remove unbound particles followed by particle visualization based upon fluorescent dyes rhodamine or indocyanine green. Alternatively, cells treated with indocyanine green (ICG) loaded MSNs were placed into a tissue phantom and viewed using the Multispectral Optoacoustic Tomography system (MSOT). Mice with S2CP9 orthotopic pancreatic tumors were i.v. injected with 100 μL of UPA-ICG loaded MSNs. Particle localization was conducted using MSOT after 4h post injection. Results: Zeta potential and UV-Vis analysis confirmed the MSNs chemical conjugation, attributing the MSNs properties of pH sensitivity and fluorescence. pH sensitivity tests on the MSNs displayed drug release quantities at pH 6.5 to be ∼x2 the quantities of pH 6.8 and 7.4. Fluorescence microscopy showed the MSN uptake into S2VP10 and S2CP9 cell lines, while NIH3T3 cells (negative control) showed less binding. Use of UPAR antibody on S2VP10 and S2CP9 cell lines (positive control) blocked cellular binding. In tissue phantoms, UPA-ICG loaded MSNs at pH 6.5 demonstrated 20X and 7X more cell signal than without ligand or at pH 7.4, respectively. In-vivo, UPA-ICG loaded MSNs were detected within the orthotopic pancreatic tumor. Conclusion: UPA targeted, pH sensitive MSNs were found to possess substantial tumor cell binding against multiple cell lines and demonstrate potential as a theranostic nanoparticle for pancreatic cancer. Citation Format: Dillon S. Pender, Anil Khanal, Michael E. Egger, Lacey R. McNally. Development of theranostic mesoporous silica nanoparticles for pancreatic cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1481. doi:10.1158/1538-7445.AM2015-1481

Polymeric Micelles of PEG-PLA Copolymer as a Carrier for Salinomycin Against Gemcitabine-Resistant Pancreatic Cancer.

Resistance to gemcitabine in pancreatic cancer (PC) may account for the failure of conventional treatments. Recently, salinomycin (SAL) has been identified as selective inhibitor of cancer stem cells (CSCs). In our study, we aimed to deliver SAL to gemcitabine-resistant PC by the aid of poly ethylene glycol-b-poly lactic acid (PEG-b-PLA) polymeric micelles (PMs). SAL-loaded PMs were prepared and investigated in terms of pharmaceutical properties. MTT and Annexin V/PI assays were used to study cell proliferation and apoptosis in AsPC-1 cells in response to treatment with SAL micellar formulations. Alterations in CSC phenotype, invasion strength, and mRNA expression of epithelial mesenchymal transition (EMT) markers were also determined in the treated cells. In vivo antitumor study was performed in Balb/c AsPC-1 xenograft mice. PM formulations of SAL were prepared in suitable size and loading traits. In gemcitabine-resistant AsPC-1 cells, SAL was found to significantly increase cell mortality and apoptosis. It was also observed that SAL micellar formulations inhibited invasion and harnessed EMT in spite of induced expression of Snail. The in vivo antitumor experiment showed significant tumor eradication and the highest survival probability in mice treated with SAL PMs. The obtained results showed the efficacy of SAL nano-formulation against PC tumor cells.

Abstract B18: MicroRNA-21 is involved in pancreatic cancer fibroblast tumor supportiveness

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by poor survival despite current medical and surgical therapies and has a propensity towards subclinical metastasis even with resectable disease. The tumor microenvironment consists of a network of supporting cells, including tumor-associated fibroblasts (TAFs), which promote tumor invasion and metastasis, and contribute to PDAC’s extremely poor prognosis. MicroRNAs (miRs) have recently emerged as critical players in post-transcriptional regulation of pathways in cancer biology. In this study, we identified miRs in fibroblasts induced by PDAC tumor cells (TCs). Further, we study the stable suppression of one of these miRs in TAFs and the effects upon fibroblast migration and invasion, as well as tumor cell proliferation. Methods: Primary pancreatic fibroblasts were cultured with MIA PaCa-2 TC conditioned media and RNA was analyzed by the NanoString MicroRNA Expression Assay. Stable miR knockdown (KD) was achieved with a stably expressed shRNA anti-microRNA by lentiviral vector. Migration was determined by an 18 hour scratch assay and 24 hour invasion by modified Boyden chamber. TC proliferation was measured by MTT assay. In vivo tumor growth was evaluated by subcutaneous TC and TAF co-implantation in NSG mice. Results: In primary pancreatic fibroblasts, MIA PaCa-2 tumor cells induced 62 miRs &amp;gt;50% increase or 30% decrease in expression. The most prevalent induced miR with 2-fold increase in expression was miR-21. miR-21 KD in TAFs was achieved by introducing stable expression of anti-miR-21, which resulted in a 75% decrease in expression (P&amp;lt;0.001). miR-21 KD TAFs had a &amp;gt;50% decreased ability to migrate (p&amp;lt;0.001) and were 67% less invasive (p&amp;lt;0.001). miR-21 KD TAF conditioned media induced less rapid TC proliferation (p&amp;lt;0.05). Further, in an in vivo model of tumor proliferation, TCs co-implanted with miR-21 KD TAFs showed 45% decreased tumor size (P&amp;lt;0.05), compared to controls. Conclusions: Pancreatic cancer TCs can induce tumor supportive miRs in TAFs. miR-21 KD in PDAC TAFs results in decreased TAF migration and invasion, as well as reduced ability to support in vitro and in vivo tumor growth. Citation Format: Andrew Hieu Nguyen, Luyi Li, Paul Toste, Nanping Wu, Timothy R. Donahue. MicroRNA-21 is involved in pancreatic cancer fibroblast tumor supportiveness. [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 B18.

“Tissue is the Issue”: Circulating Tumor Cells in Pancreatic Cancer

Pancreatic cancer (PCa) is the fourth most common cause of all cancer-related deaths in the USA. Currently, the overall median survival is less than 1 year and 5-year survival rate is about 5 % [1]. The poor outcome for PCa is largely due to the fact that the vast majority of patients (pts) are diagnosed at an advanced disease stage or with occult metastases that soon reveal themselves despite no clinical or radiographic clues. Therefore, early and accurate diagnosis is critical to appropriately risk stratify and intervene to improve the clinical outcomes of pts with PCa. Diagnostic modalities in current use include cross-sectional imaging and the use of the non-specific serum biomarker carbohydrate antigen 19-9 (CA-19-9) and occasionally carcinoembryonic antigen (CEA). Radiographic imaging of PCa can be challenging due to the potential absence of a discrete pancreatic mass or presence of alternate symptom etiologies including pancreatic inflammation. Even in the setting of an appropriate clinical context with supportive imaging and serum biomarkers, assurance of histological or cytological confirmation of PCa is directly related to adequate tissue acquisition [2]. Methodologies for pancreatic tissue acquisition are invasive, of relative low diagnostic yield, and associated with potential complications such as pancreatitis, bleeding, duodenal perforations, and infections [3]. Therefore, novel non-invasive or minimally invasive methods of accurate PCa cell acquisition represent a clinical unmet need. Progression of most solid cancers is associated with intravasation of cancer cells into the pt’s circulatory system with dissemination to metastatic sites. These circulating tumor cells (CTCs), measured by collection and detection of epithelial cells in the peripheral circulation, are known to be detectable in pts with solid tumors from early through advanced disease [4]. In addition to diagnostic sampling, CTCs have many potential clinical applications in the management of patients with solid tumors. These include, but are not limited to, risk stratification/prognostication, monitoring of response to therapy, characterizing the tumor’s molecular alterations, screening for early relapse, and other potential ways to personalize therapy [5–9]. In this brief report, we describe the successful isolation and molecular characterization of CTCs from a pt with PCa in whom traditional acquisition of tissue for diagnosis and management was unsuccessful despite multiple traditional attempts.

Low cytokeratin- and low EpCAM-expressing circulating tumor cells in pancreatic cancer.

11046 Background: To date, detecting circulating tumor cells (CTCs) in the peripheral blood of pancreatic patients using standard immuno-capture techniques has met with limited success. As pancreatic cancer is prone to metastasize at distant sites, and therefore should have high numbers of CTCs, it is possible that immuno-capture methods are not suitable for this disease. Using a microfiltration approach, we show that CTCs are present in the peripheral blood in over 75% of pancreatic cancer patients, and that two distinct subtypes can be identified. Methods: Pancreatic patient samples were provided by Medical College of Wisconsin, Milwaukee, WI. CellSieve microfilters, with precision 7 micron diameter pores distributed in uniform arrays were employed. 7.5 mL of whole blood was diluted in pre-fixation solution and filtered through CellSieve microfilters. CTCs collected by this size exclusion technique were fixed, permeabilized, and stained with DAPI, an antibody cocktail against cytokeratin 8, 18 and 19 (FITC), EpCAM (PE), and CD45 (Cy5). CTCs, defined as cytokeratin positive and CD45 negative, were found in two distinct subtypes. One subtype had the “classic” characteristics of a CTC, with high EpCAM and cytokeratin expression, identifiable cytokeratin filamentation, and a cancer-like nuclear structure. The second subtype is indicative of a CTC undergoing epithelial-mesenchymal transition (EMT), with low or no EpCAM, weak cytokeratin expression, and a smooth oval nuclear structure. Results: The “classic” CTCs were found in ~20% (n=40) of patient samples. The EMT-like CTCs were found in ~75% of the same patient cohort. Neither cell was present in any healthy subjects (n=30). EMT-like CTCs consistently lacked EpCAM expression and commonly presented as multi-cell clusters, or microemboli, in ~40% of the cases. Conclusions: We show that two distinct CTC subtypes circulate in the blood of most pancreatic patients. The low expression of cytokeratin and EpCAM of the EMT-like subtype implies that immuno-capture based CTC isolation methods have limited utility for pancreatic cancer. Further, this subtype provides a useful strategy for tracking pancreatic CTCs over the course of treatment.

Silencing of MBD1 reverses pancreatic cancer therapy resistance through inhibition of DNA damage repair

High resistance to traditional chemo- and radiotherapies contributes to the poor prognosis of pancreatic cancer (PC). Methyl-CpG binding domain protein 1 (MBD1), which plays an important role in disease progression, contributes to the drug resistance of PC cells; however, the mechanism underlying the drug resistance endowed by MBD1 remains unknown. In this study, we found that MBD1 was recruited to DNA damage sites under DNA damage conditions. Silencing of MBD1 significantly impaired activation of the DNA damage checkpoint response and inhibited DNA repair capacity. MBD1 binds mediator of DNA damage checkpoint protein 1 (MDC1), which is induced by radiation and regulates NBS1 activation in the presence of DNA damage repair. Knockdown of MBD1 significantly increased the sensitivity of cells to radiation and cisplatin (diamindichloridoplatin, DDP) in vitro. Importantly, the function of MBD1 in regulating chemoradioresistance is also partially dependent on DNA damage repair. Thus, we hypothesize that MBD1 may promote PC chemoradioresistance by regulating PC cell fate in the presence of DNA damage. Collectively, these findings reveal an important function of MBD1 in DNA repair and mediation of chemoradioresistance of cancer cells. Moreover, this study suggests that MBD1 is a promising molecular target for sensitizing resistant PC tumor cells to chemoradiotherapy.

A pilot study to explore circulating tumour cells in pancreatic cancer as a novel biomarker

Background:Obtaining tissue for pancreatic carcinoma diagnosis and biomarker assessment to aid drug development is challenging. Circulating tumour cells (CTCs) may represent a potential biomarker to address these unmet needs. We compared prospectively the utility of two platforms for CTC enumeration and characterisation in pancreatic cancer patients in a pilot exploratory study.Patients and methods:Blood samples were obtained prospectively from 54 consenting patients and analysed by CellSearch and isolation by size of epithelial tumour cells (ISET). CellSearch exploits immunomagnetic capture of CTCs-expressing epithelial markers, whereas ISET is a marker independent, blood filtration device. Circulating tumour cell expression of epithelial and mesenchymal markers was assessed to explore any discrepancy in CTC number between the two platforms.Results:ISET detected CTCs in more patients than CellSearch (93% vs 40%) and in higher numbers (median CTCs/7.5 ml, 9 (range 0–240) vs 0 (range 0–144)). Heterogeneity observed for epithelial cell adhesion molecule, pan-cytokeratin (CK), E-Cadherin, Vimentin and CK 7 expression in CTCs may account for discrepancy in CTC number between platforms.Conclusion:ISET detects more CTCs than CellSearch and offers flexible CTC characterisation with potential to investigate CTC biology and develop biomarkers for pancreatic cancer patient management.

Disseminated tumor cells in pancreatic cancer—an independent prognosticator of disease progression and survival

Pancreatic cancer is one of the most devastating cancers with a 6-month median survival and a 5-year survival rate of 3-5%. Still important aspects of its aggressive biology remain elusive and advanced therapeutic regimens have not been substantially successful. We investigated the prognostic role of disseminated tumor cells (DTC) in bone marrow, a reservoir for early DTC potentially contributing to metastatic progression, of pancreatic cancer patients. After exclusion of patients with different postsurgery diagnosis or missing DTC status (n = 40) a total of 175 patients remained for final analyses. One-hundred and nineteen patients were male and 96 female with a median age of 67 years, 96 patients underwent complete resection. Bone marrow aspirates taken at primary surgery were analyzed for DTC by an immunocytochemical cytokeratin assay and correlated to survival data. Overall 13.7% of patient samples (24/175) harbored DTC in their bone marrow. Histopathological parameters did not correlate significantly. Univariate survival analysis revealed a borderline significant correlation between DTC and decreased progression-free survival (p = 0.069), and was significant for overall survival (p = 0.036). Regarding patients with resected tumors, the respective p-values were 0.058 for progression-free and 0.016 for overall survival. Importantly, the prognostic influence was independent from other risk factors as shown by multivariate analyses for progression-free (p = 0.030, HR: 2.057; CI (95%): 1.073-3.943) and overall survival (p = 0.006, HR: 2.283; CI (95%): 1.260-4.135). The presence of DTC in bone marrow is a strong and independent prognostic factor of survival in patients with pancreatic cancer. Thus, bone-targeting may be a new future therapeutic option for DTC-positive patients.

Abstract 149: Antisense inhibition of microRNA-21 and -221 in tumor-initiating stem-like cells modulates biological functions of pancreatic cancer including tumorigenesis, metastasis, and chemoresistance

Abstract Introduction. Our preliminary studies identified a small population of pancreatic cancer cells with stem-like properties. These cells, called Side Population (SP) cells, were able to induce fast and aggressive tumor formation in nude mice. Cultured SP were able to differentiate into daughter SP or non-SP cells and were found to be highly chemoresistant. Furthermore, performed gene expression analysis showed a significant difference in the expression of more than 1300 genes in SP cells, among which the difference in microRNA expression between SP and non-SP cells was identified as the most interesting candidate for our further studies. Methods. Pancreatic cancer stem-like cells from highly metastatic cell line L3.6pl were identified and characterized by flow cytometry using Hoechst 33342 dye staining. The gene expression was assessed by Affymetrix and the results were further confirmed by quantitative RT-PCR. The antagomir transfection was performed using microRNA-21 and -221 oligonucleotide antisense. Tumor cell apoptosis, cell cycle progression, chemoresistance, and invasion were quantitated by propidium iodide staining and Boyden chamber assay, respectively. Results. In our study, some microRNAs, including miR-21, miR-221, miR-211, and miR-30c-2 were significantly upregulated in stem-like SP from L3.6pl cells. Interestingly, in these cells both miR-21 and miR-221 were involved in the modulation of expression of more than 200 genes, including RASSF6, RAB2B, TP63, TP53INP1, TP53INP2, TET1, MAPK10, MAP2K6, CDK6, TNFRSF11B, SOCS6, STK33, and SMAD7. The administration of antagomir-21 and -221 significantly reduced the SP fracture, decreased SP cell differentiation, positively affected L3.6pl cell proliferation, invasion, and chemoresistance against gemcitabine and 5-Fluorouracil. Conclusion. Our results demonstrate the significance of both microRNA-21 and -221 in tumor-initiating capability of stem-like tumor cells in pancreatic cancer. Both microRNAs contribute to the most important biological functions of pancreatic cancer including apoptosis, metastasis, and chemoresistance, and may further serve as a potential target for pancreatic cancer therapy. 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 149. doi:10.1158/1538-7445.AM2011-149

A phase I/II study of an antitumor vaccination using α (1,3) galactosyltransferase expressing allogeneic tumor cells in pancreatic cancer

13512 Background: Prognosis of pancreatic cancer remains poor despite surgical resection. In a phase I trial, we examined the safety and feasibility of antitumor vaccination in Pts with surgically resected pancreatic cancer with two irradiated genetically altered human pancreatic cancer cell lines engineered to express xenotransplantation antigens by retroviral transfer of the murine a(1,3) galactosyltrasferase gene {HyperAcute Pancreatic Cancer Vaccine (HAPa)}. Methods: Pts had undergone complete surgical resection for stage IA to IIB pancreatic adenocarcinoma, ECOG PS≤2, no immunosuppressive drug use and an adequate organ function were eligible. Various adjuvant treatments were allowed prior to the start of HAPa according to the institutional preferences (5FU/radiation, cisplatin/gemicitabine followed by 5FU/radiation, 5FU/radiation followed by gemicitabine or gemicitabine/capecitabine/bevasizumab/radiation). Six Pts were scheduled to receive 12 every two week-intradermal injections (initial priming dose followed by 11 boost doses). The first cohort (Pts 1–3) was to receive at lower doses (2 × 108 cells priming and 1 x 108 cells boost). The second cohort (Pts 4–6) was to receive at higher doses (5 x 10^8 cells priming and 3 x 10^8 cells boost). Toxicity was assessed using the CTC v3.0. Results: To date, 6 Pts, 3 males, 3 females, median age 59 (range 50–66) were treated. Two Pts completed all 12 injections. Others received 9, 6, 6 and 4 injections, respectively. To date no serious adverse events can be attributed to the vaccine. Adverse events (≤CTC grade 2) attributable to the vaccine include injection site pain/discomfort and local skin reaction in all Pts. Other adverse events (≤grade 2) include hyperkalemia, fever and increased LDH levels in one Pt. One Pt with non-malignant hepatic lesions at the time of initial surgery was removed from the study after confirmation of metastatic disease in the liver. At this point the median 6.5 (range 9–4) months in the study all remaining (5/6) patients have no evidence of recurrent disease. Conclusions: Preliminary data indicate that antitumor vaccination with genetically altered allogeneic human pancreatic cancer cells expressing a(1, 3) galactosyltrasferase is safe and feasible [Table: see text]

Tumor-associated stroma cell therapy in patients with pancreatic cancer potentiates therapeutic effect of tumor B-cell hybrid (TBH) auto-vaccines

4569 Background: Pancreatic Cancer (PC) tumor-associated stroma cells play an important role in PC’s immune surveillance escape. Secretion of Transforming Growth Factor β (TGFβ) and IL10 by PC tumor cells disturb the antigen presenting function of the stroma cells. It may explain the poor results of most immunotherapeutic approaches. Granger et al. reported a successful approach for treating small size pancreatic tumors, namely Mixed Lymphocyte Culture (MLC) cytoimplant. MLC cytoimplants, acting as a Th1 cytokine pump inside the tumor, are thought to revert the tumor-associated stroma cell dysfunction, and facilitate an immune attack. In view that TBH auto-vaccines have modest activity in advanced PC, we considered the possibility that a combination of MLC cytoimplant and TBH vaccines might potentiate the immune response. In this study we compared the response of MLC cytoimplant, TBH vaccines, and combinations of both therapies. Methods: 40 patients with advanced PC were treated: Group 1 MLC cytoimplan (10); Group 2 TBH vaccines (3); Group 3 one MLC cytoimplant and three TBH vaccines (9); Group 4 one MLC cytoimplant and six TBH vaccines (12) and Group 5 one MLC cytoimplant followed by two TBH immunizations, then second MLC cytoimplant followed by 4 TBH vaccines (6). Anti-tumor immune response was measured by Lymphocyte proliferation assay against autologous PC. Results: MLC cytoimplant combined with TBH vaccines appears to have synergistic and effective anti-tumor activity in advanced PC. Kaplan Meyer analysis showed a significant difference in the survival of group 4 as compared with the other four groups (P&lt;0.001). After cytoimplant administration, 1/12 patients in Group 4 and 1/6 patients in Group 5 experienced transient bleeding episodes (lasting less than 12 hours). After the second MLC cytoimplant, all patients in Group 5 developed significant Graft Vs Host Disease (GVHD), an occurrence that probably contributed to the Group’s shorter survival when compared to Group 4’s survival. Conclusions: The observation that a second MLC cytoimplant leads to GVHD is consistent with a pro-inflammatory change in the tumor-associated stroma cells. MLC cytoimplant followed by TBH vaccines appear to prolong survival. No significant financial relationships to disclose.

Enhanced levels of Hsulf-1 interfere with heparin-binding growth factor signaling in pancreatic cancer

Hsulf-1 is a newly identified enzyme, which has the ability to decrease the growth of hepatocellular, ovarian, and head and neck squamous cell carcinoma cells by interfering with heparin-binding growth factor signaling. Since pancreatic cancers over-express a number of heparin-binding growth factors and their receptors, the expression and function of this enzyme in pancreatic cancer was analyzed.ResultsPancreatic cancer samples expressed significantly (22.5-fold) increased Hsulf-1 mRNA levels compared to normal controls, and Hsulf-1 mRNA was localized in the cancer cells themselves as well as in peritumoral fibroblasts. 4 out of 8 examined pancreatic cancer cell lines expressed Hsulf-1, whereas its expression was below the level of detection in the other cell lines. Stable transfection of the Hsulf-1 negative Panc-1 pancreatic cancer cell line with a full length Hsulf-1 expression vector resulted in increased sulfatase activity and decreased cell-surface heparan-sulfate proteoglycan (HSPG) sulfation. Hsulf-1 expression reduced both anchorage-dependent and -independent cell growth and decreased FGF-2 mediated cell growth and invasion in this cell line.ConclusionHigh expression of Hsulf-1 occurs in the stromal elements as well as in the tumor cells in pancreatic cancer and interferes with heparin-binding growth factor signaling.