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Vitamin D: Foe of the immune response in pancreatic cancer
Abstracts / Pancreatology 17 (2017) S1eS142
addition, fibrosis producing stellate cells became activated. All the effects were suppressed by treatment with a TGF-beta inhibitor. Slit1 and Robo2 deficient animals treated with caerulein to provoke acute pancreatitis also showed an increase in pancreatic stellate cell activation and enhanced collagen deposition compared to wild type controls, corroborating the in vitro findings. Where control mouse pancreas recovered from acute pancreatitis within one week, Slit1 or Robo2 deficient pancreas showed lasting collagen deposition. ROBO1 expression in patient samples correlated with typical pathways in ECM remodelling. Conclusion: This is the first report of a role of the Slit-Robo pathway in pancreatic cells with downstream activation of TGF-beta signaling and stromal remodelling. The findings warrant further investigation of the role of Slit-Robo signaling in pancreatic tumour development.
Abstract ID: 1769. Vitamin D: Foe of the immune response in pancreatic cancer Stefania Moz, Sara Furlanello, Elisa Gnatta, Alice Schiavo, Sara Teolato, Monica Facco, Gianpietro Semenzato, Mario Plebani, Daniela Basso Department of Medicine-DIMED, University of Padova, Italy Introduction: Vitamin D (VitD), involved in calcium homeostasis and relevant for the immune cell function, enhance and reduce PDAC risk. Immunosuppression, is induced by PDAC cells and parallels disease progression with accumulation of genetic aberrations. Aims: To verify whether VitD has effect on intracellular calcium and immunosuppression of PBMCs after they have been conditioned by media from PDAC cells expressing (BxPC3-SMAD4+) or not (BxPC3) SMAD4 gene. Materials & methods: PBMCs, were seeded in conditioned media (CM) from BxPC3 and BxPC3-SMAD4+ and in control medium (RPMI + 10 % FCS) in the presence or absence of the VitD (Calcipotriol, 100 nM) and cultured for 48, 72 and 96 hours. Intracellular calcium fluxes (Ca2+)i were analyzed by epifluorescence microscopy. TNF was measured in the supernatants (ELISA). T cell proliferation was evaluated by 3H-thymidine incorporation. Results: BxPC3 CM (X2¼7.021, p¼0.030), not BxPC3-SMAD4+ CM (X2¼0.430, p¼0.806), increased the number of PBMCs with increased (Ca2+)i with respect to control. Calcipotriol did not affect (Ca2+)i in control PBMCs (X2¼2.662, p¼0.264), while it reduced the number of (Ca2+)i of BxPC3-SMAD4+ (X2¼8.655, p¼0.013) and of BxPC3 (X2¼25.325, p<0.0001) conditioned PBMCs. TNF increased in control and conditioned PBMCs after 96 hours (Two way repeated measures anova: F¼7.664, p¼0.002) and Calcipotriol inhibited this increase (Tukey’s multiple comparison test: p<0.05). BxPC3 and BxPC3-SMAD4+ conditioned PBMCs induced T cell proliferation and calcipotriol reverted this effect (One way anova: F¼9.901, p¼0.0001). Conclusion: Calcipotriol appears to increase tumor-associated immunosuppression by decreasing lymphocyte proliferation and TNF release by PBMCs. Calcium fluxes seem to be related with this modification of the immune response.
Abstract ID: 1772. SMAD4 related transfer through exosomes of glycolytic enzymes and miR-1260a underlies the reverse Warburg effect in PDAC Sara Furlanello 1, Andrea Padoan 1, Thomas Brefort 2, Thomas Laufer 3, Carlo-Federico Zambon 4, Filippo Navaglia 1, Stefania Moz 1, Dania Bozzato 1, Giorgio Arrigoni 4, Daniela Basso 1 1
Department of Medicine DIMED, University of Padova, Italy Eurofins Medigenomix GmbH, Ebersberg, Germany 3 Comprehensive Biomarker Center GmbH, Heidelberg, Recently renamed to Hummingbird Diagnostics GmbH, Germany 2
S7
4 Department of Biomedical Sciences, University of Padova, Padova, Italy
Introduction: KRAS, TP53 and MYC regulate the Warburg effect in cancer cells. The reverse Warburg effect involves tumor stromal cells. Aims: To verify whether SMAD4+ loss and related exosomes are involved in Warburg and reverse Warburg effects in PDAC, together with potential pathogenetic proteins and/or microRNAs. Materials & methods: BxPC3 (SMAD4+ HD) and BxPC3-SMAD4+ (BxPC3 stably transfected with SMAD4+) exo-enriched conditioned media (CM) were obtained following ultracentrifugation (4 days in 1% FCS). miRNAs expression and protein profiling of BxPC3 and BxPC3-SMAD4+ CM exosomes were performed (miRNA microarrays and SILAC). Human hsamiR-1260a was transfected and silenced (miRIDIAN mimic and Hairpin inhibitor) in BxPC3-SMAD4+ cells. Glucose and lactate were measured in supernatants of pancreatic cancer cells and of PBMCs cultured in exoenriched CM and non conditioned media (4 days). Results: Analysis of SMAD4-associated exo-proteins identified glycolysis among the main de-regulated biological processes. One of the most SMAD4associated de-regulated exo miRNAs was hsa-miR-1260a. BxPC3 cells had higher glucose consumption (p<0.01) and lactate production (p¼0.01) than BxPC3-SMAD4+. PBMCs cultured in exo-enriched BxPC3 CM with respect to exo-enriched BxPC3-SMAD4+, had a higher glucose consumption and lactate production (p<0.05). hsa-mir-1260a mimic transfection of BxPC3-SMAD4+ lowered glucose consumption and lactate production with respect to hsamir-1260a inhibitor and non transfected cells. Exosomes from BxPC3SMAD4+ transfected with hsa-mir-1260a mimic lowered lactate production of PBMCs with respect to hsa-mir-1260a inhibitor (p<0.05). Conclusion: SMAD4 can stand among cancer-associated genetic alterations which regulate the Warburg effect. SMAD4-related enrichment of glycolytic enzymes and of hsa-mir-1260a in cancer derived exosomes might underlie the reverse Warburg effect.
Abstract ID: 1777. Effect of oxidized phospholipids on the inflammatory response in pancreatic acini Alberto Mateu, Isabel de Dios, Manuel A. Manso, Laura Ramudo University of Salamanca, Spain Introduction: Oxidized phospholipids (oxPLs) accumulated at sites of inflammation have been involved in the pathogenesis of a wide range of inflammatory diseases. During acute pancreatitis (AP), these lipid compounds could be generated as a result of the massive neutrophil infiltration within necrotic areas of peripancreatic adipose tissue and thus they may be responsible for developing more severe forms of AP. Due to the ability of oxPLs to aggravate or to reduce inflammation, they could modulate the inflammatory response triggered by pancreatic acinar cells, which are considered the initial source of inflammatory mediators in AP. Aims: We investigated the effect of 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) and oxidized POPC (oxPOPC) on the inflammatory response triggered in pancreatic acini. Materials & methods: Pancreatic acini were incubated in the absence or presence of either POPC or oxPOPC (< 0.1 mM). Cell damage was measured by lactate dehydrogenase activity. CCL2 and TLR4 mRNA expression was analyzed by RT-qPCR. By western blot, JNK-MAPK, JAK and IkBa in cytoplasm as well as p65-NF-kB and p-STAT3 in the nucleus were evaluated. Results: No effect was found in response to POPC. Conversely, in response to 0.01 mM of oxPOPC, JNK-MAPK and JAK acted as TLR4downstream signals, leading to CCL2 upregulation mainly through NF-kB activation. Moreover, TLR4 non-dependent mechanisms induced STAT3 activation in oxPOPC-treated acini. Conclusion: oxPOPC exerts TLR4-mediated pro-inflammatory effects in pancreatic acinar cells, suggesting that oxidized lipids play a pathophysiological role during AP.
addition, fibrosis producing stellate cells became activated. All the effects were suppressed by treatment with a TGF-beta inhibitor. Slit1 and Robo2 deficient animals treated with caerulein to provoke acute pancreatitis also showed an increase in pancreatic stellate cell activation and enhanced collagen deposition compared to wild type controls, corroborating the in vitro findings. Where control mouse pancreas recovered from acute pancreatitis within one week, Slit1 or Robo2 deficient pancreas showed lasting collagen deposition. ROBO1 expression in patient samples correlated with typical pathways in ECM remodelling. Conclusion: This is the first report of a role of the Slit-Robo pathway in pancreatic cells with downstream activation of TGF-beta signaling and stromal remodelling. The findings warrant further investigation of the role of Slit-Robo signaling in pancreatic tumour development.
Abstract ID: 1769. Vitamin D: Foe of the immune response in pancreatic cancer Stefania Moz, Sara Furlanello, Elisa Gnatta, Alice Schiavo, Sara Teolato, Monica Facco, Gianpietro Semenzato, Mario Plebani, Daniela Basso Department of Medicine-DIMED, University of Padova, Italy Introduction: Vitamin D (VitD), involved in calcium homeostasis and relevant for the immune cell function, enhance and reduce PDAC risk. Immunosuppression, is induced by PDAC cells and parallels disease progression with accumulation of genetic aberrations. Aims: To verify whether VitD has effect on intracellular calcium and immunosuppression of PBMCs after they have been conditioned by media from PDAC cells expressing (BxPC3-SMAD4+) or not (BxPC3) SMAD4 gene. Materials & methods: PBMCs, were seeded in conditioned media (CM) from BxPC3 and BxPC3-SMAD4+ and in control medium (RPMI + 10 % FCS) in the presence or absence of the VitD (Calcipotriol, 100 nM) and cultured for 48, 72 and 96 hours. Intracellular calcium fluxes (Ca2+)i were analyzed by epifluorescence microscopy. TNF was measured in the supernatants (ELISA). T cell proliferation was evaluated by 3H-thymidine incorporation. Results: BxPC3 CM (X2¼7.021, p¼0.030), not BxPC3-SMAD4+ CM (X2¼0.430, p¼0.806), increased the number of PBMCs with increased (Ca2+)i with respect to control. Calcipotriol did not affect (Ca2+)i in control PBMCs (X2¼2.662, p¼0.264), while it reduced the number of (Ca2+)i of BxPC3-SMAD4+ (X2¼8.655, p¼0.013) and of BxPC3 (X2¼25.325, p<0.0001) conditioned PBMCs. TNF increased in control and conditioned PBMCs after 96 hours (Two way repeated measures anova: F¼7.664, p¼0.002) and Calcipotriol inhibited this increase (Tukey’s multiple comparison test: p<0.05). BxPC3 and BxPC3-SMAD4+ conditioned PBMCs induced T cell proliferation and calcipotriol reverted this effect (One way anova: F¼9.901, p¼0.0001). Conclusion: Calcipotriol appears to increase tumor-associated immunosuppression by decreasing lymphocyte proliferation and TNF release by PBMCs. Calcium fluxes seem to be related with this modification of the immune response.
Abstract ID: 1772. SMAD4 related transfer through exosomes of glycolytic enzymes and miR-1260a underlies the reverse Warburg effect in PDAC Sara Furlanello 1, Andrea Padoan 1, Thomas Brefort 2, Thomas Laufer 3, Carlo-Federico Zambon 4, Filippo Navaglia 1, Stefania Moz 1, Dania Bozzato 1, Giorgio Arrigoni 4, Daniela Basso 1 1
Department of Medicine DIMED, University of Padova, Italy Eurofins Medigenomix GmbH, Ebersberg, Germany 3 Comprehensive Biomarker Center GmbH, Heidelberg, Recently renamed to Hummingbird Diagnostics GmbH, Germany 2
S7
4 Department of Biomedical Sciences, University of Padova, Padova, Italy
Introduction: KRAS, TP53 and MYC regulate the Warburg effect in cancer cells. The reverse Warburg effect involves tumor stromal cells. Aims: To verify whether SMAD4+ loss and related exosomes are involved in Warburg and reverse Warburg effects in PDAC, together with potential pathogenetic proteins and/or microRNAs. Materials & methods: BxPC3 (SMAD4+ HD) and BxPC3-SMAD4+ (BxPC3 stably transfected with SMAD4+) exo-enriched conditioned media (CM) were obtained following ultracentrifugation (4 days in 1% FCS). miRNAs expression and protein profiling of BxPC3 and BxPC3-SMAD4+ CM exosomes were performed (miRNA microarrays and SILAC). Human hsamiR-1260a was transfected and silenced (miRIDIAN mimic and Hairpin inhibitor) in BxPC3-SMAD4+ cells. Glucose and lactate were measured in supernatants of pancreatic cancer cells and of PBMCs cultured in exoenriched CM and non conditioned media (4 days). Results: Analysis of SMAD4-associated exo-proteins identified glycolysis among the main de-regulated biological processes. One of the most SMAD4associated de-regulated exo miRNAs was hsa-miR-1260a. BxPC3 cells had higher glucose consumption (p<0.01) and lactate production (p¼0.01) than BxPC3-SMAD4+. PBMCs cultured in exo-enriched BxPC3 CM with respect to exo-enriched BxPC3-SMAD4+, had a higher glucose consumption and lactate production (p<0.05). hsa-mir-1260a mimic transfection of BxPC3-SMAD4+ lowered glucose consumption and lactate production with respect to hsamir-1260a inhibitor and non transfected cells. Exosomes from BxPC3SMAD4+ transfected with hsa-mir-1260a mimic lowered lactate production of PBMCs with respect to hsa-mir-1260a inhibitor (p<0.05). Conclusion: SMAD4 can stand among cancer-associated genetic alterations which regulate the Warburg effect. SMAD4-related enrichment of glycolytic enzymes and of hsa-mir-1260a in cancer derived exosomes might underlie the reverse Warburg effect.
Abstract ID: 1777. Effect of oxidized phospholipids on the inflammatory response in pancreatic acini Alberto Mateu, Isabel de Dios, Manuel A. Manso, Laura Ramudo University of Salamanca, Spain Introduction: Oxidized phospholipids (oxPLs) accumulated at sites of inflammation have been involved in the pathogenesis of a wide range of inflammatory diseases. During acute pancreatitis (AP), these lipid compounds could be generated as a result of the massive neutrophil infiltration within necrotic areas of peripancreatic adipose tissue and thus they may be responsible for developing more severe forms of AP. Due to the ability of oxPLs to aggravate or to reduce inflammation, they could modulate the inflammatory response triggered by pancreatic acinar cells, which are considered the initial source of inflammatory mediators in AP. Aims: We investigated the effect of 1-palmitoyl-2-oleoyl-sn-glycero-3phosphocholine (POPC) and oxidized POPC (oxPOPC) on the inflammatory response triggered in pancreatic acini. Materials & methods: Pancreatic acini were incubated in the absence or presence of either POPC or oxPOPC (< 0.1 mM). Cell damage was measured by lactate dehydrogenase activity. CCL2 and TLR4 mRNA expression was analyzed by RT-qPCR. By western blot, JNK-MAPK, JAK and IkBa in cytoplasm as well as p65-NF-kB and p-STAT3 in the nucleus were evaluated. Results: No effect was found in response to POPC. Conversely, in response to 0.01 mM of oxPOPC, JNK-MAPK and JAK acted as TLR4downstream signals, leading to CCL2 upregulation mainly through NF-kB activation. Moreover, TLR4 non-dependent mechanisms induced STAT3 activation in oxPOPC-treated acini. Conclusion: oxPOPC exerts TLR4-mediated pro-inflammatory effects in pancreatic acinar cells, suggesting that oxidized lipids play a pathophysiological role during AP.