TGF-signals from the membrane to the nucleus through serine/threonine kinase receptors and their downstream effectors, termed SMAD proteins 1 . The activated TGF-receptor induces phosphorylation of two such proteins, Smad2 and Smad3 (refs... more
TGF-signals from the membrane to the nucleus through serine/threonine kinase receptors and their downstream effectors, termed SMAD proteins 1 . The activated TGF-receptor induces phosphorylation of two such proteins, Smad2 and Smad3 (refs 2, 3, , 5, 6), which form hetero-oligomeric ...
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Chemistry, Confocal Microscopy, Cancer Research, Medicine, Immunocytochemistry, and 15 moreHuman, Drug Resistance, Coculture, Green Fluorescent Protein, Controlled Study, Luciferase Assay, Cancer Associated Fibroblasts, Cancers, Clinical Article, Gemcitabine, Enzyme Linked Immunosorbent Assay, Human Cell, Crizotinib, drug response, and Chemosensitivity
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Immunology, Biology, Cell Biology, Immunology of the Gut, Apoptosis, and 15 moreMedicine, Signal Transduction, Cell line, Humans, Mice, Animals, Alternative splicing, Phosphorylation, T cells, Lipopolysaccharides, Alternative Splicing, B Lymphocytes, Cell Proliferation, Protein Biosynthesis, and Immunoglobulin A
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Highlights • PDAC cells in the duodenal epithelium mimic intestinal cells and co-opt the basement membrane.• Intramucosal PDAC location is strongly coupled to the classical phenotype and to intestinal traits.• Intratumoral heterogeneity... more
Highlights • PDAC cells in the duodenal epithelium mimic intestinal cells and co-opt the basement membrane.• Intramucosal PDAC location is strongly coupled to the classical phenotype and to intestinal traits.• Intratumoral heterogeneity is linked to specific tissue compartments, which shape phenotype plasticity of PDAC cells.
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Aim of the study: The aim of this study was to validate a novel 3D spheroid co-culture model of pancreatic ductal adenocarcinoma (PDAC) cells and pancreatic stellate cells (PSCs), and to use said model as to investigate the effects of... more
Aim of the study: The aim of this study was to validate a novel 3D spheroid co-culture model of pancreatic ductal adenocarcinoma (PDAC) cells and pancreatic stellate cells (PSCs), and to use said model as to investigate the effects of PDAC cell and PSC cross-talk on cell proliferation, cell death, migration and chemo-resistance. Methods: An eGFP-positive PSC line and PDAC cell lines were cultured in 3D spheroid mono- and co-cultures. Activation of the PSCs within the co-culture model was investigated by real time PCR for whole spheroids and spheroid populations sorted by fluorescence activated cell sorting (FACS). To investigate cell proliferation the cell numbers within the cultures were followed over time and the proportions of the different cell types within the co-cultures were monitored by FACS. Results: The mRNA expression of the PSC activation marker ASMA as well as the ECM proteins collagen I, laminin and lumican, the PDGFbeta receptor (PDGFRb) and the transforming growth factor beta1 (TGFb-1) downstream effector connective tissue growth factor (CTGF), were increased in PSCs within co-cultures compared to mono-cultures. PSCs were initially expanding but then diminished within 3D spheroid co-cultures together with two separate PDAC cell lines (Panc-1 and HPAF-II). Panc-1 cell numbers were seemingly unaffected by the presence of PSCs, but with higher proportions of PSCs the Panc1 cell numbers within the co-cultures initially were lagging behind but then caught up to the numbers within the mono-cultures. This growth switch was correlated with the opposite switch seen for the PSCs. The proliferation of HPAF-II cells, was on the other hand induced within the co-cultures compared to mono-cultures. Conclusions: PSCs are activated within the 3D spheroid co-cultures. Both tumor cell and PSC proliferation is influenced by co-culturing the cells in the 3D spheroid model. Different PDAC cell – PSC combinations are affected differently. We currently are investigating the effects on cell death and apoptosis, migration and chemo-resistance. Citation Format: Jessica K. Norberg, Salvatore Nania, Rainer L. Heuchel, Matthias J. Löhr. Pancreatic cancer and stroma cell cross-talk affects cell proliferation in a 3D spheroid co-culture model. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C40.
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Sex-driven immune differences can affect tumor progression and the landscape of the tumor microenvironment. Deeper understanding of these differences in males and females can inform patient selection to improve sex-optimized immunotherapy... more
Sex-driven immune differences can affect tumor progression and the landscape of the tumor microenvironment. Deeper understanding of these differences in males and females can inform patient selection to improve sex-optimized immunotherapy treatments. In this study, single-cell RNA sequencing and protein analyses uncovered a subpopulation of myeloid cells in pancreatic lesions associated with an immune‐excluded tumor phenotype and effector T-cell exhaustion exclusively in females. This myeloid subpopulation was positively correlated with poor survival and genetic signatures of M2-like macrophages and T-cell exhaustion in females. The G-protein coupled receptor formyl peptide receptor 2 (FPR2) mediated these immunosuppressive effects. In vitro, treatment of myeloid cells with a specific FPR2 antagonist prevented exhaustion and enhanced cytotoxicity of effector cells. Proteomic analysis revealed high expression of immunosuppressive secretory proteins PGE2 and galectin-9, enriched integ...
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Biology and Immune system
Sex-driven immune differences can affect tumor progression and the landscape of the tumor microenvironment. Deeper understanding of these differences in males and females can inform patient selection to improve sex-optimized immunotherapy... more
Sex-driven immune differences can affect tumor progression and the landscape of the tumor microenvironment. Deeper understanding of these differences in males and females can inform patient selection to improve sex-optimized immunotherapy treatments. In this study, single-cell RNA sequencing and protein analyses uncovered a subpopulation of myeloid cells in pancreatic lesions associated with an immune-excluded tumor phenotype and effector T-cell exhaustion exclusively in females. This myeloid subpopulation was positively correlated with poor survival and genetic signatures of M2-like macrophages and T-cell exhaustion in females. The G-protein coupled receptor formyl peptide receptor 2 (FPR2) mediated these immunosuppressive effects. In vitro, treatment of myeloid cells with a specific FPR2 antagonist prevented exhaustion and enhanced cytotoxicity of effector cells. Proteomic analysis revealed high expression of immunosuppressive secretory proteins PGE2 and galectin-9, enriched integ...
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Objectives Transforming growth factor-β (TGF-β)–mediated accumulation of extracellular matrix proteins such as collagen I is a common feature of fibrosis. Pancreatic stellate cells play an integral role in the pathogenesis of... more
Objectives Transforming growth factor-β (TGF-β)–mediated accumulation of extracellular matrix proteins such as collagen I is a common feature of fibrosis. Pancreatic stellate cells play an integral role in the pathogenesis of pancreatitis, and their profibrotic ability is mainly mediated by TGF-β signaling. To specifically address the role of fibrogenic cells in experimental pancreatic fibrosis, we deleted Smad7, the main feedback inhibitor of TGF-β signaling in this cell type in mice. Methods A mouse strain harboring a conditional knockout allele of Smad7 (Smad7fl/fl ) with the tamoxifen-inducible inducible Col1a2-CreERT allele was generated and compared with wild-type mice challenged with the cerulein-based model of chronic pancreatitis. Results Pancreatic stellate cells lacking Smad7 had significantly increased collagen I and fibronectin production and showed a higher activation level in vitro. Surprisingly, the fibrotic index in the pancreata of treated conditional knockout mice...
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BackgroundImmunotherapy for pancreatic cancer (PC) is inefficient due to a highly immune-suppressive tumor microenvironment (TME) orchestrated by myeloid suppressor cells, which limit the infiltration and function of cytotoxic immune... more
BackgroundImmunotherapy for pancreatic cancer (PC) is inefficient due to a highly immune-suppressive tumor microenvironment (TME) orchestrated by myeloid suppressor cells, which limit the infiltration and function of cytotoxic immune cells. We have evidence that accumulation of a subpopulation of myeloid cells in human pancreatic lesions is associated with immune-exclusive tumor phenotype and effector T cell exhaustion by mechanisms involving the G-coupled protein receptor formyl peptide receptor 2 (FPR2), exclusively in women. We hypothesize that female FPR2+ myeloid cells in tumors induce immune exhaustion and contribute to immune-cold tumor phenotype.MethodsTo test our hypothesis, we first investigated the FPR2 RNA and protein expression in PC transcriptomic data and in murine and human PC tissues. Further, in vitro cytokine differentiated, alternatively tumor conditioned myeloid cells (TCM) were co-cultured with T cells to mimic their interaction in the TME. In vivo, PC cells we...
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Background Patients suffering from inflammatory bowel diseases (IBDs) express increased mucosal levels of transforming growth factor (TGF)-β compared with non-IBD controls. SMAD7 negatively regulates TGF-β signaling. An earlier study... more
Background Patients suffering from inflammatory bowel diseases (IBDs) express increased mucosal levels of transforming growth factor (TGF)-β compared with non-IBD controls. SMAD7 negatively regulates TGF-β signaling. An earlier study aiming to target Smad7 showed a lack of clinical benefit. It remains unknown whether inhibition of SMAD7 is beneficial in specific settings of IBD. We evaluated the effect of Smad7 deficiency on inflammation, fibrogenesis, and wound healing. Methods For the initiation of fibrosis in Smad7-/- (Smad7Δex-I) CD-1 mice, the dextran sodium sulfate–induced chronic colitis model and the heterotopic transplantation model of fibrosis were used. Wound closure of fibroblasts from Smad7-/- mice was determined using culture inserts and electric cell-substrate impedance sensing in vitro. Results In dextran sodium sulfate–induced chronic colitis, Smad7 deficiency was associated with ameliorated inflammation, as evidenced by decreased clinical score, histological score,...
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In sliding microfluidic platforms (“SlipChip” [1]), two plates with microfluidic wells slide in close contact to perform multiplexed reactions in a single operation. In-plane “sliding-flow” liquid transport, however, limits the potential... more
In sliding microfluidic platforms (“SlipChip” [1]), two plates with microfluidic wells slide in close contact to perform multiplexed reactions in a single operation. In-plane “sliding-flow” liquid transport, however, limits the potential assay operations to 1) sample compartmentalization/metering, 2) reagent addition, 3) mixing, and 4) aliquoting. Here, we introduce a three-plate sliding microfluidics platform, “Slip-X-Chip”, that additionally includes out-of-plane “cross-flow” liquid transport. Slip-X-Chip allows two additional assay operations: 5) sample concentration and 6) liquid exchange/washing, while retaining the simplicity of operation (one-step operation; no precise pipetting required; no external equipment). These additional assay operations extend the range and complexity of applications enabled by sliding microfluidics. We here demonstrate 1) splitting bead solutions in compartments with different concentrations and 2) compartmentalizing human cells from solution, followed by a viability assay. We foresee that Slip-X-Chip could be further adapted to, e.g., cell counting, cell staining, or ELISA.
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Highlights • PDAC cells in the duodenal epithelium mimic intestinal cells and co-opt the basement membrane.• Intramucosal PDAC location is strongly coupled to the classical phenotype and to intestinal traits.• Intratumoral heterogeneity... more
Highlights • PDAC cells in the duodenal epithelium mimic intestinal cells and co-opt the basement membrane.• Intramucosal PDAC location is strongly coupled to the classical phenotype and to intestinal traits.• Intratumoral heterogeneity is linked to specific tissue compartments, which shape phenotype plasticity of PDAC cells.
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Aim of the study: The aim of this study was to validate a novel 3D spheroid co-culture model of pancreatic ductal adenocarcinoma (PDAC) cells and pancreatic stellate cells (PSCs), and to use said model as to investigate the effects of... more
Aim of the study: The aim of this study was to validate a novel 3D spheroid co-culture model of pancreatic ductal adenocarcinoma (PDAC) cells and pancreatic stellate cells (PSCs), and to use said model as to investigate the effects of PDAC cell and PSC cross-talk on cell proliferation, cell death, migration and chemo-resistance. Methods: An eGFP-positive PSC line and PDAC cell lines were cultured in 3D spheroid mono- and co-cultures. Activation of the PSCs within the co-culture model was investigated by real time PCR for whole spheroids and spheroid populations sorted by fluorescence activated cell sorting (FACS). To investigate cell proliferation the cell numbers within the cultures were followed over time and the proportions of the different cell types within the co-cultures were monitored by FACS. Results: The mRNA expression of the PSC activation marker ASMA as well as the ECM proteins collagen I, laminin and lumican, the PDGFbeta receptor (PDGFRb) and the transforming growth fa...
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Bacille Calmette-Guérin (BCG) is the world's most widely distributed vaccine, used against tuberculosis (TB), in cancer immunotherapy, and in autoimmune diseases due to its immunomodulatory properties. To date, the effect of BCG... more
Bacille Calmette-Guérin (BCG) is the world's most widely distributed vaccine, used against tuberculosis (TB), in cancer immunotherapy, and in autoimmune diseases due to its immunomodulatory properties. To date, the effect of BCG vaccination on antibody responses to host proteins has not been reported. High-content peptide microarrays (HCPM) offer a unique opportunity to gauge specific humoral immune responses. The sera of BCG-vaccinated healthy adults were tested on a human HCPM platform (4953 randomly selected epitopes of human proteins) to detect specific immunoglobulin gamma (IgG) responses. Samples were obtained at 56, 112, and 252 days after vaccination. Immunohistology was performed on lymph node tissue from patients with TB lymphadenitis. Results were analysed with a combination of existing and novel statistical methods. IgG recognition of host peptides exhibited a peak at day 56 post BCG vaccination in all study subjects tested, which diminished over time. Primarily, IgG...
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The use of endoscopic papillectomy for resecting adenomas in the major duodenal papilla is increasing. This study focuses on the following three issues: Can endoscopic papillectomy be performed as a safe diagnostic and/or therapeutic... more
The use of endoscopic papillectomy for resecting adenomas in the major duodenal papilla is increasing. This study focuses on the following three issues: Can endoscopic papillectomy be performed as a safe diagnostic and/or therapeutic procedure in biopsy-verified or suspected ampullary adenoma? Does expression of mutated KRAS in resected adenomatous tissue predict long-term outcome? What other factors may affect long-term outcome and should, therefore, be considered in decision making prior to endoscopic papillectomy? Thirty-six prospectively collected patients who underwent endoscopic papillectomy at Karolinska University Hospital between 2005 and 2014 were analyzed. The rate of exact agreement between the histomorphological grading of the endoscopic biopsies and the papillectomy specimens was low (48%). Obstructive jaundice at presentation increased the risk of undetected adenocarcinoma (RR = 3.98; 95% CI = 1.46-10.85, p = 0.007). Lesions with malignancies were significantly larger...
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Treatment Outcome, Medicine, Biopsy, Humans, Mutation, and 8 moreFemale, Male, Clinical Sciences, Aged, Middle Aged, Adult, Adenoma, and Adenocarcinoma
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e16214Background: Pancreatic cancer (PDAC) is almost resistant to therapy, yet some patients profit from targeted therapy. We performed NGS on PDAC tissue and evidence-based, AI-supported evaluatio...
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The repression of the maternally inheritedIgf2allele has been proposed to depend on a methylation-sensitive chromatin insulator organized by the 11 zinc finger protein CTCF at theH19imprinting control region (ICR). Here we document that... more
The repression of the maternally inheritedIgf2allele has been proposed to depend on a methylation-sensitive chromatin insulator organized by the 11 zinc finger protein CTCF at theH19imprinting control region (ICR). Here we document that point mutations of the nucleotides in physical contact with CTCF within the endogenousH19ICR lead to loss of CTCF binding andIgf2imprinting only when passaged through the female germline. This effect is accompanied by a significant loss of methylation protection of the maternally derivedH19ICR. Because CTCF interacts with other imprinting control regions, it emerges as a central factor responsible for interpreting and propagating gamete-derived epigenetic marks and for organizing epigenetically controlled expression domains.
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Immunology, Biology, Immunology of the Gut, Apoptosis, Medicine, and 15 moreCell line, Humans, Mice, Animals, Alternative splicing, The, Phosphorylation, T cells, Lipopolysaccharides, Transforming Growth Factor Beta, Alternative Splicing, B Lymphocytes, Cell Proliferation, Protein Biosynthesis, and Immunoglobulin A
Background Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer related death. It is lethal in nearly all patients, due to an almost complete chemoresistance. Most if not all drugs that pass preclinical tests... more
Background Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer related death. It is lethal in nearly all patients, due to an almost complete chemoresistance. Most if not all drugs that pass preclinical tests successfully, fail miserably in the patient. This raises the question whether traditional 2D cell culture is the correct tool for drug screening. The objective of this study is to develop a simple, high-throughput 3D model of human PDAC cell lines, and to explore mechanisms underlying the transition from 2D to 3D that might be responsible for chemoresistance. Methods Several established human PDAC and a KPC mouse cell lines were tested, whereby Panc-1 was studied in more detail. 3D spheroid formation was facilitated with methylcellulose. Spheroids were studied morphologically, electron microscopically and by qRT-PCR for selected matrix genes, related factors and miRNA. Metabolic studies were performed, and a panel of novel drugs was tested against g...
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Pancreatic ductal adenocarcinoma (PDAC) is one of the most overlooked cancers despite its dismal median survival time of 6 months. The biggest challenges in improving patient survival are late diagnosis due to lack of diagnostic markers,... more
Pancreatic ductal adenocarcinoma (PDAC) is one of the most overlooked cancers despite its dismal median survival time of 6 months. The biggest challenges in improving patient survival are late diagnosis due to lack of diagnostic markers, and limited treatment options due to almost complete therapy resistance. The past decades of research identified the dense stroma and the complex interplay/crosstalk between the cancer- and the different stromal cells as the main culprits for the slow progress in improving patient outcome. For better ex vivo simulation of this complex tumor microenvironment the models used in PDAC research likewise need to become more diverse. Depending on the focus of the investigation, several in vitro and in vivo models for PDAC have been established in the past years. Particularly, 3D cell culture such as spheroids and organoids have become more frequently used. This review aims to examine current PDAC in vitro models, their inherent limitations, and their succe...