eine experimentelle Arbeit in vitro und in vivo

Hintergrund: Das maligne Melanom ist eines der häufigsten Malignome weltweit. Obwohl sich die Therapie in den vergangenen Jahren durch neue Medikamente deutlich verbessert hat, ist die Lebenserwartung im fortgeschrittenen Stadium weiter schlecht. Taurolidin, ein Derivat der Aminosäure Taurin, hat in verschiedenen präklinischen aber auch klinischen Studien seine Wirksamkeit bei gleichzeitig überschaubaren Nebenwirkungen unter Beweis gestellt. In dieser Studie wurde der Einfluss von Taurolidin auf das maligne Melanom in vitro und in vivo untersucht. Methodik: Die kommerziell erwerblichen Melanomzelllinien, MEWO und SKMEL28, sowie 8 Patientenzelllinien wurde zunächst in vitro mit steigenden Konzentrationen, 50 bis 1000 µM, Taurolidin behandelt. Danach wurde der Einfluss Taurolidins in den Konzentrationen 100 und 500 µM mit 100 U/ml Interferon-alpha oder Tumornekrose-Faktor-alpha oder der Kombination auf MEWO und SKMEL28 untersucht. Abschließend wurde mittels eines Annexin-V-Assays die prozentuale Rate induzierter Apoptose auf MEWO und SKMEL28 sowie auf die Patientenzelllinien evaluiert. Im zweiten Schritt erhielten C57/BL6 Mäuse (n=80) 1x106 Zellen der Linie B16 B78 D14 subkutan in die Nackenfalte sowie 1.5x106 in die Milz. Nach einer Woche Inkubation wurden die Tiere in zwei Therapieäste, intravenös versus intraperitoneal, a vier Therapiegruppen randomisiert. Die vier Therapiearme waren: Ringerlaktat, 1%, 2% und 3% Taurolidin. Die Behandlung erfolgte intraperitoneal zweimal täglich für sieben Tage und intravenös mittels vier Bolusinjektionen in die Schwanzvene in sieben Tagen. Zwei Wochen nach Therapieende wurden die Versuche beendet und die Tiere obduziert. Ergebnisse: In vitro: Steigende Dosierungen und längere Inkubationszeiten von Taurolidin als Monotherapie induzierten steigende Zytotoxizität in allen Zelllinien. Die Apoptoserate stieg simultan bis zu einer Konzentration von 500 µM. Bei weiterer Steigerung stieg der Anteil der nekrotischen Zellen. Die Kombination mit den Zytokinen änderte die Apoptoserate nicht, jedoch verbesserte die Kombinationstherapie mit Interferon-alpha die Zytotoxizität signifikant. In vivo: Die intraperitoneale Therapie verursachte eine dosisabhängige Reduktion des intraperitonealen (p=0,001) und Gesamttumorgewichts (p=0,003). Das subkutane Tumorgewicht wurde nicht signifikant reduziert (p=0,132). Die intravenöse Therapie reduzierte das subkutane Tumorgewicht (p=0,016) und das Gesamttumorgewicht (p=0,013) signifikant. Das intraperitoneale Gewicht wurde nicht signifikant reduziert (p=0,122). Die Anzahl der Metastasen konnte in beiden Applikationsformen nur in der höchsten Konzentration 3% signifikant reduziert werden. Zusammenfassung: Taurolidin hat eine dosisabhänge zytotoxische Wirkung auf Melanomzellen in vitro und in vivo. Die Apoptoserate ist ebenfalls dosisabhängig und steigt bis zu einer Konzentration von 500 µM in vitro. Eine Kombination mit Interferon-alpha steigert den zytotoxischen, aber nicht den apoptotischen Effekt. Weitere Studien sind notwendig um den genauen Vorteil einer Therapie mit Taurolidin zu evaluieren.Background: Malignant melanoma is one of the most common malignancies world-wide. Despite therapeutic advancement due to new drug developments, the overall survival in advanced stages remains poor. Taurolidine, a derivate oft he amino acid taurine, has proven its usefulness including mild adverse effects in several preclinical and clinicial trials. This study is investigating the effect of Taurolidine on malignant melanoma in vitro and in vivo. Materials and Methods: Well established melanoma cell lines MEWO and SKMEL28 as well as eight patient derived cell lines have been treated with increasing concentrations of Taurolidine, 50 to 1000 µM. Subsequently, Taurolidine in concentrations 100 and 500 µM has been combined with 100 U/ml Interferone-alfa or Tumornecrose-factor-alfa or the combination of such. Lastly, an Annexin-V-Assay has been used to determine apoptosis rate on all cell lines. In an additional in vivo experiment, C57/BL6 mice (n=80) received 1x106 B16 B78 D14 subcutaneously in the neck as well as 1.5x106 cells intrasplenically. Following one week of incubation, mice were randomized in 2 treatment arms, intraperitoneally versus intravenously, containing four treatment groups: Ringer’s solution, 1%, 2% und 3% Taurolidine. Animals were treated twice daily for 7 days intraperitoenally or four bolus injections in seven days into the tail vein. Two weeks after therapy end the animals were euthanized and undwerwent an autopsy. Results: In vitro: Increasing doses and incubation times of Taurolidine increased cell death in all cell lines tested. Apoptosis rate increased up to a concentration up of 500 µM Taurolidine with subsequent increase of necrosis rate. The combiantion with both cytokines did not change the apoptotic pattern but a combination with interferone-alfa increased the cytotoxic effect significantly. In vivo: Intreaperitoneal (p=0,001) and total tumor growth (p=0,003) have been reduced significantly subsequent to an intraperitoenal treatment whereas subcutaneous tumor growth has not been reduced significantly (p=0,132). Intravenous treatment caused significant reduction oft the subcutaneous (p=0,016) and total tumor growth (p=0,013). Intraperitoneal tumor weight has not been reduced significantly (p=0,122). The highest concentration of Taurolidine reduced the number of tumorous lesions significantly. Conclusion: Taurolidine prompts a cytoxic effect on melanoma cells both in vitro and in vivo. Apoptosis rate is dose dependent and increases up to a concentration of 500 µM in vitro. A combination with Interferone-alfa increases cell death but does not affect apoptosis. Follow up studies are needed to further evaluate the effect of Taurolidine on malignant melanoma

The Tumor Immune Microenvironment in Pancreatic Ductal Adenocarcinoma: Neither Hot nor Cold

Pancreatic ductal adenocarcinoma (PDAC) is the most common pancreatic tumor and is associated with poor prognosis and treatment response. The tumor microenvironment (TME) is recognized as an important factor in metastatic progression across cancers. Despite extensive study of the TME in PDAC, the cellular and molecular signaling networks remain poorly understood, largely due to the tremendous heterogeneity across tumors. While earlier work characterized PDAC as an immunologically privileged tumor poorly recognized by the immune system, recent studies revealed the important and nuanced roles of immune cells in the pathogenesis of PDAC. Distinct lymphoid, myeloid, and stromal cell types in the TME exert opposing influences on PDAC tumor trajectory, suggesting a more complex organization than the classical “hot” versus “cold” tumor distinction. We review the pro- and antitumor immune processes found in PDAC and briefly discuss their leverage for the development of novel therapeutic approaches in the field

SPECT/CT Imaging, Biodistribution and Radiation Dosimetry of a 177Lu-DOTA-Integrin αvβ6 Cystine Knot Peptide in a Pancreatic Cancer Xenograft Model

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive malignant neoplasms, as many cases go undetected until they reach an advanced stage. Integrin avß6 is a cell surface receptor overexpressed in PDAC. Consequently, it may serve as a target for the development of probes for imaging diagnosis and radioligand therapy. Engineered cystine knottin peptides specific for integrin avß6 have recently been developed showing high affinity and stability. This study aimed to evaluate an integrin avß6-specific knottin molecular probe containing the therapeutic radionuclide Lu-177 for targeting of PDAC. Methods: The expression of integrin avß6 in PDAC cell lines BxPC-3 and Capan-2 was analyzed using RT-qPCR and immunofluorescence. In vitro competition and saturation radioligand binding assays were performed to calculate the binding affinity of the DOTA-coupled tracer loaded with and without lutetium to BxPC-3 and Capan-2 cell lines as well as the maximum number of binding sites in these cell lines. To evaluate tracer accumulation in the tumor and organs, SPECT/CT, biodistribution and dosimetry projections were carried out using a Capan-2 xenograft tumor mouse model. Results: RT-qPCR and immunofluorescence results showed high expression of integrin avß6a 6 in BxPC-3 and Capan-2 cells. A competition binding assay revealed high affinity of the tracer with IC50 values of 1.69 nM and 9.46 nM for BxPC-3 and Capan-2, respectively. SPECT/CT and biodistribution analysis of the conjugate Lu-177-DOTA-integrin avß6 knottin demonstrated accumulation in Capan-2 xenograft tumors (3.13 +/- 0.63%IA/g at day 1 post injection) with kidney uptake at 19.2 +/- 2.5 %IA/g, declining much more rapidly than in tumors. Conclusion: Lu-177-DOTA-integrin alpha v beta 6 knottin was found to be a high-affinity tracer for PDAC tumors with considerable tumor accumulation and moderate, rapidly declining kidney uptake. These promising results warrant a preclinical treatment study to establish therapeutic efficacy

Gut Microbiome in Inflammatory Bowel Disease: Role in Pathogenesis, Dietary Modulation, and Colitis-Associated Colon Cancer

The gut microbiome has increasingly been recognized as a critical and central factor in inflammatory bowel disease (IBD). Here, we review specific microorganisms that have been suggested to play a role in the pathogenesis of IBD and the current state of fecal microbial transplants as a therapeutic strategy in IBD. We discuss specific nutritional and dietary interventions in IBD and their effects on gut microbiota composition. Finally, we examine the role and mechanisms of the gut microbiome in mediating colitis-associated colon cancer

Highlighting the Undetectable: Fluorescence Molecular Imaging in Gastrointestinal Endoscopy

Flexible high-definition white-light endoscopy is the current gold standard in screening for cancer and its precursor lesions in the gastrointestinal tract. However, miss rates are high, especially in populations at high risk for developing gastrointestinal cancer (e.g., inflammatory bowel disease, Lynch syndrome, or Barrett's esophagus) where lesions tend to be flat and subtle. Fluorescence molecular endoscopy (FME) enables intraluminal visualization of (pre)malignant lesions based on specific biomolecular features rather than morphology by using fluorescently labeled molecular probes that bind to specific molecular targets. This strategy has the potential to serve as a valuable tool for the clinician to improve endoscopic lesion detection and real-time clinical decision-making. This narrative review presents an overview of recent advances in FME, focusing on probe development, techniques, and clinical evidence. Future perspectives will also be addressed, such as the use of FME in patient stratification for targeted therapies and potential alliances with artificial intelligence. KEY MESSAGES: • Fluorescence molecular endoscopy is a relatively new technology that enables safe and real-time endoscopic lesion visualization based on specific molecular features rather than on morphology, thereby adding a layer of information to endoscopy, like in PET-CT imaging. • Recently the transition from preclinical to clinical studies has been made, with promising results regarding enhancing detection of flat and subtle lesions in the colon and esophagus. However, clinical evidence needs to be strengthened by larger patient studies with stratified study designs. • In the future fluorescence molecular endoscopy could serve as a valuable tool in clinical workflows to improve detection in high-risk populations like patients with Barrett's esophagus, Lynch syndrome, and inflammatory bowel syndrome, where flat and subtle lesions tend to be malignant up to five times more often. • Fluorescence molecular endoscopy has the potential to assess therapy responsiveness in vivo for targeted therapies, thereby playing a role in personalizing medicine. • To further reduce high miss rates due to human and technical factors, joint application of artificial intelligence and fluorescence molecular endoscopy are likely to generate added value

The tyrosine kinase inhibitor imatinib mesylate suppresses uric acid crystal-induced acute gouty arthritis in mice

International audienceGouty arthritis is caused by the deposition of monosodium urate (MSU) crystals in joints. Despite many treatment options for gout, there is a substantial need for alternative treatments for patients unresponsive to current therapies. Tyrosine kinase inhibitors have demonstrated therapeutic benefit in experimental models of antibody-dependent arthritis and in rheumatoid arthritis in humans, but to date, the potential effects of such inhibitors on gouty arthritis has not been evaluated. Here we demonstrate that treatment with the tyrosine kinase inhibitor imatinib mesylate (imatinib) can suppress inflammation induced by injection of MSU crystals into subcutaneous air pouches or into the ankle joint of wild type mice. Moreover, imatinib treatment also largely abolished the lower levels of inflammation which developed in IL-1R1-/- or KitW-sh/W-sh mice, indicating that this drug can inhibit IL-1-independent pathways, as well as mast cell-independent pathways, contributing to pathology in this model. Imatinib treatment not only prevented ankle swelling and synovial inflammation when administered before MSU crystals but also diminished these features when administrated after the injection of MSU crystals, a therapeutic protocol more closely mimicking the clinical situation in which treatment occurs after the development of an acute gout flare. Finally, we also assessed the efficiency of local intra-articular injections of imatinib-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles in this model of acute gout. Treatment with low doses of this long-acting imatinib:PLGA formulation was able to reduce ankle swelling in a therapeutic protocol. Altogether, these results raise the possibility that tyrosine kinase inhibitors might have utility in the treatment of acute gout in humans

Image_10_GPR15 in colon cancer development and anti-tumor immune responses.jpeg

IntroductionThe chemoattractant receptor, G protein-coupled receptor 15 (GPR15), promotes colon homing of T cells in health and colitis. GPR15 function in colon cancer is largely unexplored, motivating our current studies.MethodsIn human study, immune cells were isolated from tumor tissues and healthy surgical tumor margins (STM), and their proportions as well as expression of GPR15 was analyzed by flow cytometry. In mouse studies, colon cancer was induced in GPR15-deficient (KO) and GPR15-suficient (Het) mice using azoxymethane (AOM) and dextran sulfate sodium (DSS) solution in drinking water. Serial endoscopy was performed in mice to monitor and visualize the distal region of colon. Mice were euthanized 10 weeks after the initial DSS administration, and the colon length and the number of polyps were recorded. Next, we identified the effects of GPR15L on established tumors in the MC38-colorectal cancer (CRC) mouse model. Immune cells were isolated from the mice colons or tumors and assessed by flow cytometry.ResultsOur analysis of human CRC tissue revealed a significant reduction in GPR15+ immune cell frequencies in tumors compared to ‘tumor-free’ surgical margins. Similarly, our data analysis using The Cancer Genome Atlas (TCGA) indicated that lower GPR15 expression is associated with poor survival in human colon cancer. In the AOM/DSS colitis-associated colon cancer model, we observed increased colonic polyps and lower survival in Gpr15+-KO compared to Gpr15-Het mice. Analysis of immune cell infiltrates in the colonic polyps showed significantly decreased CD8+ T cells and increased IL-17+ CD4+ and IL-17+ CD8+ T cells in Gpr15-KO than in Het mice. Consistent with a protective role of GPR15, administration of GPR15L to established tumors in the MC38-CRC model increased CD45+ cell infiltration, enhanced TNFa expression on CD4+ and CD8+ T cells at the tumor site and dramatically reduced tumor burden.DiscussionOur findings highlight an important, unidentified role of the GPR15-GPR15L axis in promoting a tumor-suppressive immune microenvironment and unveils a novel, colon-specific therapeutic target for CRC.</p

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IntroductionThe chemoattractant receptor, G protein-coupled receptor 15 (GPR15), promotes colon homing of T cells in health and colitis. GPR15 function in colon cancer is largely unexplored, motivating our current studies.MethodsIn human study, immune cells were isolated from tumor tissues and healthy surgical tumor margins (STM), and their proportions as well as expression of GPR15 was analyzed by flow cytometry. In mouse studies, colon cancer was induced in GPR15-deficient (KO) and GPR15-suficient (Het) mice using azoxymethane (AOM) and dextran sulfate sodium (DSS) solution in drinking water. Serial endoscopy was performed in mice to monitor and visualize the distal region of colon. Mice were euthanized 10 weeks after the initial DSS administration, and the colon length and the number of polyps were recorded. Next, we identified the effects of GPR15L on established tumors in the MC38-colorectal cancer (CRC) mouse model. Immune cells were isolated from the mice colons or tumors and assessed by flow cytometry.ResultsOur analysis of human CRC tissue revealed a significant reduction in GPR15+ immune cell frequencies in tumors compared to ‘tumor-free’ surgical margins. Similarly, our data analysis using The Cancer Genome Atlas (TCGA) indicated that lower GPR15 expression is associated with poor survival in human colon cancer. In the AOM/DSS colitis-associated colon cancer model, we observed increased colonic polyps and lower survival in Gpr15+-KO compared to Gpr15-Het mice. Analysis of immune cell infiltrates in the colonic polyps showed significantly decreased CD8+ T cells and increased IL-17+ CD4+ and IL-17+ CD8+ T cells in Gpr15-KO than in Het mice. Consistent with a protective role of GPR15, administration of GPR15L to established tumors in the MC38-CRC model increased CD45+ cell infiltration, enhanced TNFa expression on CD4+ and CD8+ T cells at the tumor site and dramatically reduced tumor burden.DiscussionOur findings highlight an important, unidentified role of the GPR15-GPR15L axis in promoting a tumor-suppressive immune microenvironment and unveils a novel, colon-specific therapeutic target for CRC.</p