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Evaluation of stromal quantification in pancreatic ductal adenocarcinoma using a tissue microarray
S22
Abstracts / Pancreatology 15 (2015) S1eS141
839. HuR mediated post-transcriptional regulation as a new potential therapeutic target in adjuvant treatment of pancreatic cancer
Light and fluorescent microscopy was used to visualise the monolayer and G-SPIONs, respectively. Results: Marked cell lysis occurred over areas of maximal magnetic field strength (MAG).
Zilvinas Dambrauskas, Aldona Jakstaite, Aurelija Maziukiene, Kristina Kmieliute, Antanas Gulbinas Institute for Digestive System Research, Lithuanian University of Health Sciences, Lithuania Introduction: HuR has been reported to regulate the expression of many proteins by different post-transcriptional mechanisms, including mRNA trafficking, mRNA stabilization, and protein translation. HuR is also involved in the post-transcriptional modification of cytoprotective molecules, such as cyclooxygenase-2 (COX-2) and heme oxygenase-1 (HO-1), which may be related to the increased resistance of pancreatic cancer to the chemotherapy. Aims: The aim of our study was to investigate the expression of HuR in pancreatic ductal adenocarcinoma (PDA). Assess the effects of HuR silencing and its role in chemoresistance of pancreatic cancer. Materials & methods: We compared the expression of HuR, COX-2 and HO-1 mRNA in PDA with normal pancreatic tissue. In addition, the HuR, COX-2 and HO-1 mRNA expression was analysed in four types of cancer cell lines that were treated with Gemcitabine (GEM). Cell viability and response to GEM were also analysed after HuR silencing. Results: HuR mRNA expression was 3.2 times lower in PDA tissue, while COX-2 and HO-1 mRNA expression were, accordingly, 2.3-fold and 7.2-fold higher, when compared to normal pancreatic tissue. Western blot analysis showed that COX-2 and HO-1 levels significantly decreased in cancer cells after HuR silencing. Furthermore, HuR silencing resulted in increased response to GEM treatment and 11.6 e 53.7 % decrease in cell viability, compared to control cell lines. Conclusion: HuR is associated with the upregulation of COX-2 and HO-1 expression in pancreatic cancer after treatment with GEM. HuR silencing significantly increases the effectiveness of GEM treatment in vitro and is a new important molecular target in treatment of human pancreatic cancer.
1095. Use of gemcitabine-loaded superparamagnetic iron oxide nanoparticles against pancreatic cancer cells in an artificial circulatory model Sumit Nandi 1, Paul Sykes 1, Erol Hasan 2, Michael Barrow 2, John Neoptolemos 1, Eithne Costello 1, Matthew Rosseinsky 2, John Hunt 3, Christopher Halloran 1 1
NIHR Pancreas Biomedical Research Unit, Department of Molecular and Clinical Cancer Medicine, University of Liverpool, United Kingdom 2 Department of Chemistry, University of Liverpool, United Kingdom 3 Institute of Ageing and Chronic Disease, University of Liverpool, United Kingdom
Introduction: Nanotechnology can provide novel and targeted therapy for pancreatic cancer. Gemcitabine-loaded superparamagnetic iron oxide nanoparticles (G-SPIONs) have proven cytotoxic effect against static pancreatic cancer cell lines in vitro. This model, however does not replicate how these particles could behave when administered intravenously. Aims: To determine: i) whether G-SPIONs can be removed from a dynamic artificial circulation by magnetic targeting and ii) whether they have a cytotoxic effect upon pancreatic cancer cell lines. Materials & methods: Culture media (RPMI) was continuously circulated through two specifically manufactured 25cm2 flasks containing monolayers of 2x106 MiaPaCa-2 cells within a 37 C/5%CO2 incubator. Monolayers were subjected to control media or 2% rhodamine-labelled GSPIONs, with/without a magnet under the flasks for 16/24/48/72 hours. LDH assay was performed as a measure of cell lysis following treatment.
Time (hr)
No MAG (% lysis)
MAG (%)
16 24 48 72
4.79 8.77 69.47 67.12
32.05 34.07 73.21 70.18
(p<0.001) (p<0.001) (p¼0.02) (p¼0.004)
Light microscopy confirmed destruction of monolayers, especially over MAG. Co-localisation fluorescent microscopy demonstrated uptake of GSPIONs by cells with increased signal seen over areas of MAG. Conclusion: G-SPIONs can successfully be drawn from circulation and destroy monolayers. The magnetic targeting mechanism has most effect at early time points and although has reducing effect, is still significant at 72 hours. Bio-magnetic targeting in custom-made nano-vehicles provides a potential novel theranostic solution to chemotherapy for pancreatic cancer.
1099. Evaluation of stromal quantification in pancreatic ductal adenocarcinoma using a tissue microarray Anthony Evans 1, Frances Oldfield 1, Katharine Hand 1, Christopher Halloran 1, Fiona Campbell 2, John Neoptolemos 1, William Greenhalf 1, Eithne Costello 1 1
University of Liverpool, Molecular and Clinical Cancer Medicine, United Kingdom 2 Royal Liverpool University Hospital, Department of Pathology, United Kingdom Introduction: Pancreatic ductal adenocarcinoma (PDAC) is characterised by dense desmoplastic stroma, which comprises cancer-associated fibroblasts, extracellular matrix proteins, immune cells, nerve fibres, lymphatic and blood vessels. Tissue microarrays (TMAs) are commonly used to quantify protein expression in PDAC, although their accuracy for quantifying stromal elements is not tested. Aims: To determine whether TMAs can accurately enable quantification of specific stromal elements in PDAC. Materials & methods: A TMA was constructed using tumour samples from 48 patients with PDAC, sampling from three regions per patient. The TMA was cut into 192 sections to enable quantification at different depths of the tumour specimens. Five depths were selected for analysis of up to 15 cores per patient. Immunohistochemical staining was performed for tumour cells, activated fibroblasts, collagen, macrophages, T cells, cytotoxic T cells, endothelial cells and lymphatic vessels. Stained TMA sections were scored by two independent investigators. Quantified stromal components were correlated with overall survival and clinicopathological parameters, using each TMA depth individually as well as all available cores per patient. Results: Kaplan-Meier analysis of individual TMA sections stained for a-SMA, a marker of activated fibroblasts, revealed an association with overall survival in one section of the TMA (log-rank, p¼0.0024) that was not replicated across the other four. Analysis of cores across all TMA sections showed no significant association (log-rank, p¼0.21). Analyses of other individual and combined stromal elements will be presented, including comparisons with matched serum cytokine profiles. Conclusion: The heterogeneity of stromal components suggests TMA assessment at varying depths may be required when quantifying PDAC stroma.
Abstracts / Pancreatology 15 (2015) S1eS141
839. HuR mediated post-transcriptional regulation as a new potential therapeutic target in adjuvant treatment of pancreatic cancer
Light and fluorescent microscopy was used to visualise the monolayer and G-SPIONs, respectively. Results: Marked cell lysis occurred over areas of maximal magnetic field strength (MAG).
Zilvinas Dambrauskas, Aldona Jakstaite, Aurelija Maziukiene, Kristina Kmieliute, Antanas Gulbinas Institute for Digestive System Research, Lithuanian University of Health Sciences, Lithuania Introduction: HuR has been reported to regulate the expression of many proteins by different post-transcriptional mechanisms, including mRNA trafficking, mRNA stabilization, and protein translation. HuR is also involved in the post-transcriptional modification of cytoprotective molecules, such as cyclooxygenase-2 (COX-2) and heme oxygenase-1 (HO-1), which may be related to the increased resistance of pancreatic cancer to the chemotherapy. Aims: The aim of our study was to investigate the expression of HuR in pancreatic ductal adenocarcinoma (PDA). Assess the effects of HuR silencing and its role in chemoresistance of pancreatic cancer. Materials & methods: We compared the expression of HuR, COX-2 and HO-1 mRNA in PDA with normal pancreatic tissue. In addition, the HuR, COX-2 and HO-1 mRNA expression was analysed in four types of cancer cell lines that were treated with Gemcitabine (GEM). Cell viability and response to GEM were also analysed after HuR silencing. Results: HuR mRNA expression was 3.2 times lower in PDA tissue, while COX-2 and HO-1 mRNA expression were, accordingly, 2.3-fold and 7.2-fold higher, when compared to normal pancreatic tissue. Western blot analysis showed that COX-2 and HO-1 levels significantly decreased in cancer cells after HuR silencing. Furthermore, HuR silencing resulted in increased response to GEM treatment and 11.6 e 53.7 % decrease in cell viability, compared to control cell lines. Conclusion: HuR is associated with the upregulation of COX-2 and HO-1 expression in pancreatic cancer after treatment with GEM. HuR silencing significantly increases the effectiveness of GEM treatment in vitro and is a new important molecular target in treatment of human pancreatic cancer.
1095. Use of gemcitabine-loaded superparamagnetic iron oxide nanoparticles against pancreatic cancer cells in an artificial circulatory model Sumit Nandi 1, Paul Sykes 1, Erol Hasan 2, Michael Barrow 2, John Neoptolemos 1, Eithne Costello 1, Matthew Rosseinsky 2, John Hunt 3, Christopher Halloran 1 1
NIHR Pancreas Biomedical Research Unit, Department of Molecular and Clinical Cancer Medicine, University of Liverpool, United Kingdom 2 Department of Chemistry, University of Liverpool, United Kingdom 3 Institute of Ageing and Chronic Disease, University of Liverpool, United Kingdom
Introduction: Nanotechnology can provide novel and targeted therapy for pancreatic cancer. Gemcitabine-loaded superparamagnetic iron oxide nanoparticles (G-SPIONs) have proven cytotoxic effect against static pancreatic cancer cell lines in vitro. This model, however does not replicate how these particles could behave when administered intravenously. Aims: To determine: i) whether G-SPIONs can be removed from a dynamic artificial circulation by magnetic targeting and ii) whether they have a cytotoxic effect upon pancreatic cancer cell lines. Materials & methods: Culture media (RPMI) was continuously circulated through two specifically manufactured 25cm2 flasks containing monolayers of 2x106 MiaPaCa-2 cells within a 37 C/5%CO2 incubator. Monolayers were subjected to control media or 2% rhodamine-labelled GSPIONs, with/without a magnet under the flasks for 16/24/48/72 hours. LDH assay was performed as a measure of cell lysis following treatment.
Time (hr)
No MAG (% lysis)
MAG (%)
16 24 48 72
4.79 8.77 69.47 67.12
32.05 34.07 73.21 70.18
(p<0.001) (p<0.001) (p¼0.02) (p¼0.004)
Light microscopy confirmed destruction of monolayers, especially over MAG. Co-localisation fluorescent microscopy demonstrated uptake of GSPIONs by cells with increased signal seen over areas of MAG. Conclusion: G-SPIONs can successfully be drawn from circulation and destroy monolayers. The magnetic targeting mechanism has most effect at early time points and although has reducing effect, is still significant at 72 hours. Bio-magnetic targeting in custom-made nano-vehicles provides a potential novel theranostic solution to chemotherapy for pancreatic cancer.
1099. Evaluation of stromal quantification in pancreatic ductal adenocarcinoma using a tissue microarray Anthony Evans 1, Frances Oldfield 1, Katharine Hand 1, Christopher Halloran 1, Fiona Campbell 2, John Neoptolemos 1, William Greenhalf 1, Eithne Costello 1 1
University of Liverpool, Molecular and Clinical Cancer Medicine, United Kingdom 2 Royal Liverpool University Hospital, Department of Pathology, United Kingdom Introduction: Pancreatic ductal adenocarcinoma (PDAC) is characterised by dense desmoplastic stroma, which comprises cancer-associated fibroblasts, extracellular matrix proteins, immune cells, nerve fibres, lymphatic and blood vessels. Tissue microarrays (TMAs) are commonly used to quantify protein expression in PDAC, although their accuracy for quantifying stromal elements is not tested. Aims: To determine whether TMAs can accurately enable quantification of specific stromal elements in PDAC. Materials & methods: A TMA was constructed using tumour samples from 48 patients with PDAC, sampling from three regions per patient. The TMA was cut into 192 sections to enable quantification at different depths of the tumour specimens. Five depths were selected for analysis of up to 15 cores per patient. Immunohistochemical staining was performed for tumour cells, activated fibroblasts, collagen, macrophages, T cells, cytotoxic T cells, endothelial cells and lymphatic vessels. Stained TMA sections were scored by two independent investigators. Quantified stromal components were correlated with overall survival and clinicopathological parameters, using each TMA depth individually as well as all available cores per patient. Results: Kaplan-Meier analysis of individual TMA sections stained for a-SMA, a marker of activated fibroblasts, revealed an association with overall survival in one section of the TMA (log-rank, p¼0.0024) that was not replicated across the other four. Analysis of cores across all TMA sections showed no significant association (log-rank, p¼0.21). Analyses of other individual and combined stromal elements will be presented, including comparisons with matched serum cytokine profiles. Conclusion: The heterogeneity of stromal components suggests TMA assessment at varying depths may be required when quantifying PDAC stroma.