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Light-based therapies to enhance pancreatic cancer chemotherapy
Abstracts / Pancreatology 17 (2017) S1eS142
Abstract ID: 1870. A common deletion variant of chymotrypsin B2 (CTRB2) causes misfolding but no endoplasmic reticulum stress Eszter Hegyi 1, Jonas Rosendahl 2, Peter Hegyi 3, Miklos Sahin-Toth 1 1 Department of Molecular and Cell Biology, Boston University Medical Center, Boston, MA, United States 2 Department of Internal Medicine I, Martin Luther University, Halle, Germany 3 Institute for Translational Medicine, First Department of Internal cs, Pe cs, Hungary Medicine, University of Pe
Introduction: Misfolding variants of digestive enzymes have been shown to induce endoplasmic reticulum (ER) stress and increase risk for chronic pancreatitis. A deletion of 585 nucleotides in the CTRB2 gene present in >10% of the population has been described recently. The deletion affects the entire exon 6 of CTRB2 and portions of the flanking introns resulting in a C-terminally truncated CTRB2 protein. Aims: To investigate the impact of the deletion on CTRB2 expression, folding, ER stress and to examine association with chronic pancreatitis. Patients & methods: Expression of CTRB2 mRNA was measured in human pancreatic cDNA samples by RT-PCR. Secretion of CTRB2 was measured from the conditioned medium of transiently transfected HEK 293T cells by SDS-PAGE and western blot. ER stress responses were assessed in AR42J cells by RT-PCR. Genetic association was measured in a Hungarian cohort and replicated in a German cohort. Results: The mRNA for the deletion variant was expressed at normal levels in the human pancreas. In contrast to wild-type CTRB2, the deletion variant was not secreted to the growth medium of transfected cells but accumulated intracellularly. Increased sensitivity of the deleted protein to proteolytic degradation by trypsin was observed. However, ER stress markers were not elevated. No significant genetic association was detected with chronic pancreatitis (OR¼1.03, p¼0.84). Conclusion: A common deletion variant of human CTRB2 causes misfolding but no ER stress. The deletion variant does not alter risk for chronic pancreatitis. The results are consistent with the notion that ER stress mediates the effects of misfolding digestive enzyme variants associated with chronic pancreatitis.
Abstract ID: 1874. NFkB In Pancreatic Cancer Stroma Suppresses Anti-Tumor Immunity Bharti Garg, Bhuwan Giri, Shrey Modi, Vrisketan Seth, Sulagna Banerjee, Sundran ramakrishnan, Ashok Saluja, Vikas Dudeja University of Miami, United States Introduction: Cancer-stromal cell interaction contributes to aggressiveness of pancreatic cancer (PDAC). pancreatic-stellate-cells in PDAC promotes tumor growth. While NFkB signaling in cancer cells is known to promote tumor growth, impact of NFkB signaling in stroma on PDAC growth is not known. Aims: Role of NFkB signaling in tumor stroma on the growth of pancreatic cancer Materials & methods: Cancer cells from tumor developing in KrasG12D TP53 PDX-1 cre (KPC) were co-injected with PSC from (WT) or p50-/- (from mice lacking functional NFkB) in WT or rag1-/- mice and tumor growth were evaluated. To confirm our findings in other cancers, LLC and B16 melanoma cells were co-injected with lung and dermal fibroblasts. To further evaluate the role of cytotoxic T cells, CD8 antibody mediated depletion approach was used. Results: Injection of p50-/- PSC with KPC cells led to decreased pancreatic cancer growth, as compared to co-injection with WT PSC. Similarly, stromal NFkB inhibition decreased growth of lung cancer and melanoma. Interestingly, lack of NFkB in tumor stroma led to increased
S17
infiltration of cytotoxic CD8+ T-cells. When KPC with PSC was injected in rag1-/- mice, lack of NFkB in tumor stroma did not inhibit tumor growth. When CD8+ cells were depleted with antibody, inhibition of NFkB was not able to reduce tumor growth, suggesting the role of cytotoxic t cells in this phenomenon. Conclusion: NFkB signaling in stroma promotes pancreatic cancer growth by protecting cancer cells from immune mediated-cytotoxicity. NFkB inhibition in stroma could emerge as novel strategy against pancreatic cancer, either alone or in combination with immunotherapy.
Abstract ID: 1875. Light-based therapies to enhance pancreatic cancer chemotherapy Pilar Acedo 1, Alexander Ney 1, Stephen Pereira 2, Alexander MacRobert 1 1
Division of Surgery and Interventional Science, University College London, United Kingdom 2 Institute for Liver and Digestive Health, University College London, United Kingdom Introduction: Our strategy is to target pancreatic cancer using a novel minimally invasive technique called Photochemical Internalisation (PCI), which can significantly enhance the efficacy of cancer chemotherapy. PCI is a light-triggered drug delivery system, which combines low dose Photodynamic Therapy (PDT) with chemotherapy to induce efficient cytosolic delivery of therapeutic compounds to their specific subcellular targets. Aims: To determine the efficacy of PCI in overcoming pancreatic cancer drug resistance compared to conventional monotherapy, using experimental models (2D, 3D, in vivo) and to identify the cell death mechanisms induced by these protocols. Materials & methods: In this study, porphyrin and chlorin-based photosensitisers were co-administered with saporin (a type I ribosomeinactivating protein) or gemcitabine, in human pancreatic cancer cells (Panc1 and MiaPaca2). Cell viability was assessed by MTT assays and Trypan Blue & Neutral Red stainings, alongside internalisation and intracellular localisation of both photosensitisers (flow cytometry, confocal microscopy). Results: Despite minimal cell death induced when the drugs or photoactivatable compounds were used in monotherapy, PCI synergistically enhanced saporin and gemcitabine cytotoxicity (p<0.001) using very low concentrations, in both cell lines. Moreover, we determined the mechanism of cell death triggered by these protocols. PCI induced endosomal disruption following light excitation and caspases 3/7 activation, in a time dependent fashion. Conclusion: Our findings demonstrate the potential of PCI to enhance the efficacy of cancer chemotherapy for pancreatic cancer using sub-lethal doses of PDT and open a new window for future clinical trials.
Abstract ID: 1876. Morphological and laboratory predictors of acute necrotizing pancreatitis Aliaksandr Varabei 1, Nikolai Kamyshnikov 1, Egi Vizhinis 1, Tamara Yuraga 1, Natali Litvinka 2, Yury Arlouski 1 1 2
Belarusian Medical Academy of Postgraduate Education, Belarus Biochemical Institute, Belarus
Introduction: Carrying out laboratory tests on the basis of new enzymatic testing tools inflammatory and destructive changes in the pancreas in acute necrotizing pancreatitis (ANP) is an important task of surgery. Aims: We used a new, domestic laboratory test based on the formation of a supramolecular complex of a fatty acid with hemoglobin as an indicator of pancreatic phospholipolysis.
Abstract ID: 1870. A common deletion variant of chymotrypsin B2 (CTRB2) causes misfolding but no endoplasmic reticulum stress Eszter Hegyi 1, Jonas Rosendahl 2, Peter Hegyi 3, Miklos Sahin-Toth 1 1 Department of Molecular and Cell Biology, Boston University Medical Center, Boston, MA, United States 2 Department of Internal Medicine I, Martin Luther University, Halle, Germany 3 Institute for Translational Medicine, First Department of Internal cs, Pe cs, Hungary Medicine, University of Pe
Introduction: Misfolding variants of digestive enzymes have been shown to induce endoplasmic reticulum (ER) stress and increase risk for chronic pancreatitis. A deletion of 585 nucleotides in the CTRB2 gene present in >10% of the population has been described recently. The deletion affects the entire exon 6 of CTRB2 and portions of the flanking introns resulting in a C-terminally truncated CTRB2 protein. Aims: To investigate the impact of the deletion on CTRB2 expression, folding, ER stress and to examine association with chronic pancreatitis. Patients & methods: Expression of CTRB2 mRNA was measured in human pancreatic cDNA samples by RT-PCR. Secretion of CTRB2 was measured from the conditioned medium of transiently transfected HEK 293T cells by SDS-PAGE and western blot. ER stress responses were assessed in AR42J cells by RT-PCR. Genetic association was measured in a Hungarian cohort and replicated in a German cohort. Results: The mRNA for the deletion variant was expressed at normal levels in the human pancreas. In contrast to wild-type CTRB2, the deletion variant was not secreted to the growth medium of transfected cells but accumulated intracellularly. Increased sensitivity of the deleted protein to proteolytic degradation by trypsin was observed. However, ER stress markers were not elevated. No significant genetic association was detected with chronic pancreatitis (OR¼1.03, p¼0.84). Conclusion: A common deletion variant of human CTRB2 causes misfolding but no ER stress. The deletion variant does not alter risk for chronic pancreatitis. The results are consistent with the notion that ER stress mediates the effects of misfolding digestive enzyme variants associated with chronic pancreatitis.
Abstract ID: 1874. NFkB In Pancreatic Cancer Stroma Suppresses Anti-Tumor Immunity Bharti Garg, Bhuwan Giri, Shrey Modi, Vrisketan Seth, Sulagna Banerjee, Sundran ramakrishnan, Ashok Saluja, Vikas Dudeja University of Miami, United States Introduction: Cancer-stromal cell interaction contributes to aggressiveness of pancreatic cancer (PDAC). pancreatic-stellate-cells in PDAC promotes tumor growth. While NFkB signaling in cancer cells is known to promote tumor growth, impact of NFkB signaling in stroma on PDAC growth is not known. Aims: Role of NFkB signaling in tumor stroma on the growth of pancreatic cancer Materials & methods: Cancer cells from tumor developing in KrasG12D TP53 PDX-1 cre (KPC) were co-injected with PSC from (WT) or p50-/- (from mice lacking functional NFkB) in WT or rag1-/- mice and tumor growth were evaluated. To confirm our findings in other cancers, LLC and B16 melanoma cells were co-injected with lung and dermal fibroblasts. To further evaluate the role of cytotoxic T cells, CD8 antibody mediated depletion approach was used. Results: Injection of p50-/- PSC with KPC cells led to decreased pancreatic cancer growth, as compared to co-injection with WT PSC. Similarly, stromal NFkB inhibition decreased growth of lung cancer and melanoma. Interestingly, lack of NFkB in tumor stroma led to increased
S17
infiltration of cytotoxic CD8+ T-cells. When KPC with PSC was injected in rag1-/- mice, lack of NFkB in tumor stroma did not inhibit tumor growth. When CD8+ cells were depleted with antibody, inhibition of NFkB was not able to reduce tumor growth, suggesting the role of cytotoxic t cells in this phenomenon. Conclusion: NFkB signaling in stroma promotes pancreatic cancer growth by protecting cancer cells from immune mediated-cytotoxicity. NFkB inhibition in stroma could emerge as novel strategy against pancreatic cancer, either alone or in combination with immunotherapy.
Abstract ID: 1875. Light-based therapies to enhance pancreatic cancer chemotherapy Pilar Acedo 1, Alexander Ney 1, Stephen Pereira 2, Alexander MacRobert 1 1
Division of Surgery and Interventional Science, University College London, United Kingdom 2 Institute for Liver and Digestive Health, University College London, United Kingdom Introduction: Our strategy is to target pancreatic cancer using a novel minimally invasive technique called Photochemical Internalisation (PCI), which can significantly enhance the efficacy of cancer chemotherapy. PCI is a light-triggered drug delivery system, which combines low dose Photodynamic Therapy (PDT) with chemotherapy to induce efficient cytosolic delivery of therapeutic compounds to their specific subcellular targets. Aims: To determine the efficacy of PCI in overcoming pancreatic cancer drug resistance compared to conventional monotherapy, using experimental models (2D, 3D, in vivo) and to identify the cell death mechanisms induced by these protocols. Materials & methods: In this study, porphyrin and chlorin-based photosensitisers were co-administered with saporin (a type I ribosomeinactivating protein) or gemcitabine, in human pancreatic cancer cells (Panc1 and MiaPaca2). Cell viability was assessed by MTT assays and Trypan Blue & Neutral Red stainings, alongside internalisation and intracellular localisation of both photosensitisers (flow cytometry, confocal microscopy). Results: Despite minimal cell death induced when the drugs or photoactivatable compounds were used in monotherapy, PCI synergistically enhanced saporin and gemcitabine cytotoxicity (p<0.001) using very low concentrations, in both cell lines. Moreover, we determined the mechanism of cell death triggered by these protocols. PCI induced endosomal disruption following light excitation and caspases 3/7 activation, in a time dependent fashion. Conclusion: Our findings demonstrate the potential of PCI to enhance the efficacy of cancer chemotherapy for pancreatic cancer using sub-lethal doses of PDT and open a new window for future clinical trials.
Abstract ID: 1876. Morphological and laboratory predictors of acute necrotizing pancreatitis Aliaksandr Varabei 1, Nikolai Kamyshnikov 1, Egi Vizhinis 1, Tamara Yuraga 1, Natali Litvinka 2, Yury Arlouski 1 1 2
Belarusian Medical Academy of Postgraduate Education, Belarus Biochemical Institute, Belarus
Introduction: Carrying out laboratory tests on the basis of new enzymatic testing tools inflammatory and destructive changes in the pancreas in acute necrotizing pancreatitis (ANP) is an important task of surgery. Aims: We used a new, domestic laboratory test based on the formation of a supramolecular complex of a fatty acid with hemoglobin as an indicator of pancreatic phospholipolysis.