Invasion, proliferation and influence of cancer cell line MDA-MB-231 on organotypic tissue using Precision Cut Lung Slices (PCLS)

Cancer is a major public health problem with breast cancer being among the leading causes of cancer mortality worldwide. The actual cause of death is the formation of metastasis which often occurs in the lung. Among the cell types present in the tumor microenvironment, macrophages have proven to be the dominant leukocyte population. An increasing amount of publications points to evidence that a macrophage sub-population actively supports and promotes the initiation, growth and development of tumor tissue. Indeed, Bingle et al. [2002] have shown in their meta-analysis that over 80% of studies show a correlation between macrophage density and poor patient prognosis. As a result, the local immune response to tumor initiation is a valuable research topic and the development of models for testing of cancer therapeutics is highly sought after. To this end, we utilize an innovative organotypic tumor invasion model, using living human precision-cut-lung-slices (PCLS) and cancer cells in an effort to focus on the local immunological reactions of the tissue during the first stages of metastasis formation. An AdGFP-transduced human breast cancer cell line MDA-MB-231 was added to human PCLS over a period of one week. Viability assays such as LIVE/DEAD staining and LDH measurements were performed to assure intact human tissue. Tissue immune staining against CD68 was used to visualize locations of macrophage-MDA-MB-231 interaction whereas a staining against Ki67 as a proliferation marker was performed to visualize proliferating cells. Immune response and neoangiogenesis were determined by cytokines IL-10 and IL-1beta, angiogenesis marker VEGF and tumor marker GM-CSF, respectively. Human Precision Cut Lung Slices (PCLS) remain viable during infection and invasion with cancer cells. Cancer cells expand and proliferate in human lung tissue, which could be shown through quantification of the Ki67- positive MDA- MB- 231 cells as well as videos of the dynamic changes within the tissue. CD78- positive cells were found to be associated with MDA-MB-231 cells as colo-calization of CD68- positive macrophages and MDA-MB-231 was found during the entire invasion period. The VEGF/GM-CSF release correlated with the MDA-MB-231 growth curves whereas IL- 1beta showed a strong pro-inflammatory signal 5 hours after seeding and a continuous decline thereafter. First experiments to adapt the model to different species such as mice were performed that show that the cancer cells also attach themselves to the tissue matrix of other species. Here we mimic cancer cell proliferation in the microenvironment of human lung tissue without and thus provide insights into functional local immune responses with a human physiology background

Human organotypic cancer model

Multiple xenocraft mouse models have been generated to understand cancer, with the disadvantages of less predictive, expensive or technically complicated procedures. Here we present an innovative ex vivo organotypic tumor invasion model using living human precision-cut lung slices (PCLS) and cancer cells. An AdGFP transduced human breast cancer cell line MDA MB 231 was added to human PCLS over a period of one week. Viability assays show intact human tissue during the infection with the cancer cells. Growth curves and Ki67 staining reflect proliferation of cancer cells over the observation period time in human PCLS. Immune response and neoangiogenesis were determined by the cytokine markers VEGF, IL 10, IL 1beta and GM CSF. The decrease of the proinflammatory cytokine IL 1beta was linked to the number of MDA MB 231 associated macrophages in human PCLS. The model mimics cancer cell proliferation in the microenvironment of human tissue without using artificial substances. It provides the possibility to gain insights into functional local immune responses with human physiology background. The model can be adjusted to other cancer targeted organs. In terms of the 3R concept, this alternative model does not require any animal experiments and takes advantage of human tissue

Effect of 25-hydroxyvitamin D levels on the internalising dimension as a transdiagnostic risk factor: Mendelian randomisation study

Background Observational studies indicate a relationship between vitamin D (25-hydroxyvitamin D; 25OHD) deficiency and the development of internalising disorders, especially depression. However, causal inference approaches (e.g. Mendelian randomisation) did not confirm this relationship. Findings from biobehavioural research suggests that new insights are revealed when focusing on psychopathological dimensions rather than on clinical diagnoses. This study provides further evidence on the relationship between 25OHD and the internalising dimension. Aims This investigation aimed at examining the causality between 25OHD and internalising disorders including a common internalising factor. Method We performed a two-sample Mendelian randomisation using genome-wide association study (GWAS) summary data for 25OHD (417 580 participants), major depressive disorder (45 591 cases; 97 674 controls), anxiety (5580 cases; 11 730 controls), post-traumatic stress disorder (12 080 cases; 33 446 controls), panic disorder (2248 cases; 7992 controls), obsessive–compulsive disorder (2688 cases; 7037 controls) and anorexia nervosa (16 992 cases; 55 525 controls). GWAS results of the internalising phenotypes were combined to a common factor representing the internalising dimension. We performed several complementary analyses to reduce the risk of pleiotropy and used a second 25OHD GWAS for replication. Results We found no causal relationship between 25OHD and any of the internalising phenotypes studied, nor with the common internalising factor. Several pleiotropy-robust methods corroborated the null association. Conclusions Following current transdiagnostic approaches to investigate mental disorders, our results focused on the shared genetic basis between different internalising phenotypes and provide no evidence for an effect of 25OHD on the internalising dimension

Clinically validated markers of the extracellular matrix remodelling are altered by potential anti-fibrotic compounds in a human lung fibrosis ex vivo model

Background: Pulmonary fibrosis (PF) is characterized by excessive extracellular matrix (ECM) remodeling. Clinically validated ECM neoepitopes markers, related to progressive PF, may be useful for the evaluation of potential anti-fibrotic effects. Aim: The aim was to evaluate ECM remodelling (ECMR) in a human ex vivo precision-cut lung slice (PCLS) model. Methods: Human PF tissue was collected from two donors during lung transplantation. Within 24hours, the lungs were processed into PCLSs. The slices were cultured 2 pr/well and in triplicates for 48hours in serum free medium with 1nM-10μM nintedanib or 100pM-1µM mTOR/PI3K inhibitor omipalisib (GSK2126458). Responsiveness was tested using Lipopolysaccaride (LPS) and cytotoxicity using lactate dehydrogenase (LDH). Markers of collagen type I, III and VI formation (P1NP, PRO-C3, PRO-C6) and type III collagen degradation (C3M) were assessed in the supernatants. The subpleural and central lung regions were used and evaluated by hematoxylin and eosin staining. Results: The tissue from both donors was responsive to LPS and no toxicity was seen with the selected compound doses using LDH. P1NP, PRO-C3, and C3M were significantly reduced by 1nM-1µM omipalisib (p<0.05-0.001) including the IC50 value around 40-50nM. Non-significant reductions of PRO-C6 were seen. Data varied depending on region and donor. Similar trends were observed for nintedanib, however no significant changes were seen in this model. Conclusion: We found that an mTOR/PI3K inhibitor decreased markers of ECMR in a human PF ex vivo model, potential as a tool for evaluating anti-fibrotic compounds in a 3D PF structure

Non-human primate orthologues of TMPRSS2 cleave and activate the influenza virus hemagglutinin

The cellular serine protease TMPRSS2, a member of the type II transmembrane serine protease (TTSP) family, cleaves and activates the hemagglutinin of influenza A viruses (FLUAV) in cell culture and is essential for spread of diverse FLUAV in mice. Non-human primates (NHP), in particular rhesus and cynomolgus macaques, serve as animal models for influenza and experimental FLUAV infection of common marmosets has recently also been reported. However, it is currently unknown whether the NHP orthologues of human TMPRSS2 cleave and activate FLUAV hemagglutinin and contribute to viral spread in respiratory tissue. Here, we cloned and functionally analyzed the macaque and marmoset orthologues of human TMPRSS2. In addition, we analyzed the macaque orthologues of human TMPRSS4 and HAT, which also belong to the TTSP family. We found that all NHP orthologues of human TMPRSS2, TMPRSS4 and HAT cleave and activate HA upon directed expression and provide evidence that endogenous TMPRSS2 is expressed in the respiratory epithelium of rhesus macaques. Finally, we demonstrate that a serine protease inhibitor active against TMPRSS2 suppresses FLUAV spread in precision-cut lung slices of human, macaque and marmoset origin. These results indicate that FLUAV depends on serine protease activity for spread in diverse NHP and in humans. Moreover, our findings suggest that macaques and marmosets may serve as models to study FLUAV activation by TMPRSS2 in human patients

Alpha-1 Antitrypsin inhibits ATP-mediated release of Interleukin-1β via CD36 and Nicotinic Acetylcholine receptors

While interleukin (IL)-1β is a potent pro-inflammatory cytokine involved in host defense, high levels can cause life-threatening sterile inflammation including systemic inflammatory response syndrome. Hence, the control of IL-1β secretion is of outstanding biomedical importance. In response to a first inflammatory stimulus such as lipopolysaccharide, pro-IL-1β is synthesized as a cytoplasmic inactive pro-form. Extracellular ATP originating from injured cells is a prototypical second signal for inflammasome-dependent maturation and release of IL-1β. The human anti-protease alpha-1 antitrypsin (AAT) and IL-1β regulate each other via mechanisms that are only partially understood. Here, we demonstrate that physiological concentrations of AAT efficiently inhibit ATP-induced release of IL-1β from primary human blood mononuclear cells, monocytic U937 cells, and rat lung tissue, whereas ATP-independent IL-1β release is not impaired. Both, native and oxidized AAT are active, suggesting that the inhibition of IL-1β release is independent of the anti-elastase activity of AAT. Signaling of AAT in monocytic cells involves the lipid scavenger receptor CD36, calcium-independent phospholipase A2β, and the release of a small soluble mediator. This mediator leads to the activation of nicotinic acetylcholine receptors, which efficiently inhibit ATP-induced P2X7 receptor activation and inflammasome assembly. We suggest that AAT controls ATP-induced IL-1β release from human mononuclear blood cells by a novel triple-membrane-passing signaling pathway. This pathway may have clinical implications for the prevention of sterile pulmonary and systemic inflammation