Steffen Teller

Differential roles of inflammatory cells in pancreatitis

The incidence of acute pancreatitis per 100 000 of population ranges from 5 to 80. Patients suffering from hemorrhagic‐necrotizing pancreatitis die in 10–24% of cases. 80% of all cases of acute pancreatitis are etiologically linked to gallstone disease immoderate alcohol consumption. As of today no specific causal treatment for acute pancreatitis exists. Elevated C‐reactive protein levels above 130 mg/L can also predict a severe course of acute pancreatitis. The essential medical treatment for acute pancreatitis is the correction of hypovolemia. Prophylactic antibiotics should be restricted to patients with necrotizing pancreatitis, infected necrosis or other infectious complications. However, as premature intracellular protease activation is known to be the primary event in acute pancreatitis. Severe acute pancreatitis is characterized by an early inflammatory immune response syndrome (SIRS) and a subsequent compensatory anti‐inflammatory response syndrome (CARS) contributing to severity as much as protease activation does. CARS suppresses the immune system and facilitates nosocomial infections including infected pancreatic necrosis, one of the most feared complications of the disease. A number of attempts have been made to suppress the early systemic inflammatory response but even if these mechanisms have been found to be beneficial in animal models they failed in daily clinical practice.

Investigation of Schwann Cells at Neoplastic Cell Sites Before the Onset of Cancer Invasion

BACKGROUND In neural invasion (NI), cancer cells are classically assumed to actively invade nerves and to cause local recurrence and pain. However, the opposite possibility, that nerves may reach cancer cells even in their preinvasive stage and thereby promote cancer spread, has not yet been genuinely considered. The present study analyzes the reaction of Schwann cells of peripheral nerves to carcinogenesis in pancreatic cancer and colon cancer. METHODS Two novel 3D migration and Schwann cell outgrowth assays were developed to monitor the timing and the specificity of Schwann cell migration and cancer invasion toward peripheral neurons through digital-time-lapse microscopy and after blockade of nerve growth factor (NGF) signalling via siRNA or a small-molecule inhibitor of the p75(NTR) receptor. The frequency and emergence of the Schwann cell markers Sox10, S100, ALDH1L1, and glial-fibrillary-acidic-protein (GFAP) around cancer precursor lesions were studied in human and conditional murine pancreatic and colon cancer specimens using multiple immunolabeling. RESULTS Schwann cells migrated toward pancreatic and colon cancer cells, but not toward benign cells, before the onset of cancer migration toward peripheral neurons. This chemoattraction was inhibited after blockade of p75(NTR)-signaling on Schwann and pancreatic cancer cells. Schwann cells were specifically detected around murine and human pancreatic intraepithelial neoplasias (PanINs) (mean percent of murine PanINs surrounded by Schwann cells = 78.9%, 95% CI = 70.9 to 86.8%, and mean percent of human PanINs surrounded by Schwann cells = 52.5%, 95% CI = 14.7 to 90.4%; human: n = 44, murine: n = 14) and intestinal adenomas (mean percent of murine adenomas surrounded by Schwann cells = 64.2%, 95% CI = 28.6 to 99.8%, and mean percent of human adenomas surrounded by Schwann cells = 17.2%, 95% CI = -126.9 to 161.4; human: n = 36, murine: n = 12). The Schwann cell presence in this premalignant stage was associated with the frequency of NI in the malignant phase. CONCLUSIONS Schwann cells have particular and specific affinity to cancer cells. Emergence of Schwann cells in the premalignant phase of pancreatic and colon cancer implies that, in contrast with the traditional assumption, nerves-and not cancer cells-migrate first during NI.

Activated Schwann cells in pancreatic cancer are linked to analgesia via suppression of spinal astroglia and microglia

Objective The impact of glia cells during GI carcinogenesis and in cancer pain is unknown. Here, we demonstrate a novel mechanism how Schwann cells (SCs) become activated in the pancreatic cancer (PCa) microenvironment and influence spinal activity and pain sensation. Design Human SCs were exposed to hypoxia, to pancreatic cancer cells (PCCs) and/or to T-lymphocytes. Both SC and intrapancreatic nerves of patients with PCa with known pain severity were assessed for glial intermediate filament and hypoxia marker expression, proliferation and for transcriptional alterations of pain-related targets. In conditional PCa mouse models with selective in vivo blockade of interleukin (IL)-6 signalling (Ptf1a-Cre;LSL-KrasG12D/KC interbred with IL6−/− or sgp130tg mice), SC reactivity, abdominal mechanosensitivity and spinal glial/neuronal activity were quantified. Results Tumour hypoxia, PCC and/or T-lymphocytes activated SC via IL-6-signalling in vitro. Blockade of the IL-6-signalling suppressed SC activation around PCa precursor lesions (pancreatic intraepithelial neoplasia (PanIN)) in KC;IL6−/− (32.06%±5.25% of PanINs) and KC;sgp130tg (55.84%±5.51%) mouse models compared with KC mice (78.27%±3.91%). Activated SCs were associated with less pain in human PCa and with decreased abdominal mechanosensitivity in KC mice (von Frey score of KC: 3.9±0.5 vs KC;IL6−/− mice: 5.9±0.9; and KC;sgp130tg: 10.21±1.4) parallel to attenuation of spinal astroglial and/or microglial activity. Activated SC exhibited a transcriptomic profile with anti-inflammatory and anti-nociceptive features. Conclusions Activated SC in PCa recapitulate the hallmarks of ‘reactive gliosis’ and contribute to analgesia due to suppression of spinal glia. Our findings propose a mechanism for how cancer might remain pain-free via the SC–central glia interplay during cancer progression.

Tumour-specific delivery of siRNA-coupled superparamagnetic iron oxide nanoparticles, targeted against PLK1, stops progression of pancreatic cancer

Objective Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignancies and is projected to be the second leading cause of cancer-related death by 2030. Despite extensive knowledge and insights into biological properties and genetic aberrations of PDAC, therapeutic options remain temporary and ineffective. One plausible explanation for the futile response to therapy is an insufficient and non-specific delivery of anticancer drugs to the tumour site. Design Superparamagnetic iron oxide nanoparticles (SPIONs) coupled with siRNA directed against the cell cycle-specific serine-threonine-kinase, Polo-like kinase-1 (siPLK1-StAv-SPIONs), could serve a dual purpose for delivery of siPLK1 to the tumour and for non-invasive assessment of efficiency of delivery in vivo by imaging the tumour response. siPLK1-StAv-SPIONs were designed and synthesised as theranostics to function via a membrane translocation peptide with added advantage of driving endosomal escape for mediating transportation to the cytoplasm (myristoylated polyarginine peptides) as well as a tumour-selective peptide (EPPT1) to increase intracellular delivery and tumour specificity, respectively. Results A syngeneic orthotopic as well as an endogenous cancer model was treated biweekly with siPLK1-StAv-SPIONs and tumour growth was monitored by small animal MRI. In vitro and in vivo experiments using a syngeneic orthotopic PDAC model as well as the endogenous LSL-KrasG12D, LSL-Trp53R172H, Pdx-1-Cre model revealed significant accumulation of siPLK1-StAv-SPIONs in PDAC, resulting in efficient PLK1 silencing. Tumour-specific silencing of PLK1 halted tumour growth, marked by a decrease in tumour cell proliferation and an increase in apoptosis. Conclusions Our data suggest siPLK1-StAv-SPIONs with dual specificity residues for tumour targeting and membrane translocation to represent an exciting opportunity for targeted therapy in patients with PDAC.

Early pancreatic cancer lesions suppress pain through CXCL12-mediated chemoattraction of Schwann cells

Significance Pancreatic ductal adenocarcinoma cancer (PDAC) cells have an exceptional propensity to invade nerves via pronounced crosstalk between nerves and cancer cells, but the mechanisms of this early neural invasion are yet unknown. By using genetically engineered mouse models, we show that in the precursor stage PDAC induces the generation of ready-to-use nerves for dissemination by secreting the chemokine CXCL12 that attracts glia (Schwann) cells of nerves. This migration of glia cells to cancerous cells at this very early stage intriguingly attenuates cancer-associated pain via downregulation of pain-associated targets in Schwann cells and via suppression of central glia. Hence, malignant transformed cells seem to disguise cancer-associated symptoms (such as pain) actively and thereby delay the early diagnosis of cancer. Pancreatic ductal adenocarcinoma (PDAC) cells (PCC) have an exceptional propensity to metastasize early into intratumoral, chemokine-secreting nerves. However, we hypothesized the opposite process, that precancerous pancreatic cells secrete chemokines that chemoattract Schwann cells (SC) of nerves and thus induce ready-to-use routes of dissemination in early carcinogenesis. Here we show a peculiar role for the chemokine CXCL12 secreted in early PDAC and for its receptors CXCR4/CXCR7 on SC in the initiation of neural invasion in the cancer precursor stage and the resulting delay in the onset of PDAC-associated pain. SC exhibited cancer- or hypoxia-induced CXCR4/CXCR7 expression in vivo and in vitro and migrated toward CXCL12-expressing PCC. Glia-specific depletion of CXCR4/CXCR7 in mice abrogated the chemoattraction of SC to PCC. PDAC mice with pancreas-specific CXCL12 depletion exhibited diminished SC chemoattraction to pancreatic intraepithelial neoplasia and increased abdominal hypersensitivity caused by augmented spinal astroglial and microglial activity. In PDAC patients, reduced CXCR4/CXCR7 expression in nerves correlated with increased pain. Mechanistically, upon CXCL12 exposure, SC down-regulated the expression of several pain-associated targets. Therefore, PDAC-derived CXCL12 seems to induce tumor infiltration by SC during early carcinogenesis and to attenuate pain, possibly resulting in delayed diagnosis in PDAC.

Association Analysis of Genetic Variants in the Myosin IXB Gene in Acute Pancreatitis

Introduction Impairment of the mucosal barrier plays an important role in the pathophysiology of acute pancreatitis. The myosin IXB (MYO9B) gene and the two tight-junction adaptor genes, PARD3 and MAGI2, have been linked to gastrointestinal permeability. Common variants of these genes are associated with celiac disease and inflammatory bowel disease, two other conditions in which intestinal permeability plays a role. We investigated genetic variation in MYO9B, PARD3 and MAGI2 for association with acute pancreatitis. Methods Five single nucleotide polymorphisms (SNPs) in MYO9B, two SNPs in PARD3, and three SNPs in MAGI2 were studied in a Dutch cohort of 387 patients with acute pancreatitis and over 800 controls, and in a German cohort of 235 patients and 250 controls. Results Association to MYO9B and PARD3 was observed in the Dutch cohort, but only one SNP in MYO9B and one in MAGI2 showed association in the German cohort (p < 0.05). Joint analysis of the combined cohorts showed that, after correcting for multiple testing, only two SNPs in MYO9B remained associated (rs7259292, p = 0.0031, odds ratio (OR) 1.94, 95% confidence interval (95% CI) 1.35-2.78; rs1545620, p = 0.0006, OR 1.33, 95% CI 1.16-1.53). SNP rs1545620 is a non-synonymous SNP previously suspected to impact on ulcerative colitis. None of the SNPs showed association to disease severity or etiology. Conclusion Variants in MYO9B may be involved in acute pancreatitis, but we found no evidence for involvement of PARD3 or MAGI2.

3D Cancer Migration Assay with Schwann Cells

In pancreatic cancer, neural invasion is one of the most common paths of cancer dissemination. Classically, cancer cells actively invade nerves and cause local recurrence and pain. Three-dimensional (3D) neural migration assay has become a standard tool for scientists to study neural invasion by confronting the involved cell types. This protocol introduces Schwann cells, i.e., the most prevalent cell type in peripheral nerves, in a novel heterotypic, glia-cancer-neuron, 3D migration assay for assessing their relevance in the early pathogenesis of neural invasion. Particularly, this assay allows the monitoring of the early Schwann cell migratory activity.