Development of a 3D Biomimetic Tissue-Engineered Model of Cancer

Introduction: Three dimensional (3D) models of cancer have been a major focus in advances to research discoveries toward cancer progression and the involvement of the cancer stroma. Next to extracellular matrix components such as hyaluronic acid (HA), cancer associated fibroblasts (CAFs) are highly differentiated and heterogenous cancer stromal cells that promote tumour growth, angiogenesis and matrix remodelling. Methods: We utilised a novel 3D in vitro model of colorectal (CRC) and pancreatic cancer, composed of an artificial cancer mass (ACM) with varying invasive propensities and a stromal compartment. We incorporated extracellular matrix components and six colorectal patient-derived CAF samples to study their differential effects. The chemotherapy drug paclitaxel was tested on 3D models of pancreatic cancer with increasing complexity. Results: The main finding was that the invasive nature of a cancer mass directly influenced the complexity of a developing vascular network. All network alignments were quantified and endothelial structures aligned along the chemotactic gradient formed by the CRC mass. CRC and pancreatic cancer invades as clusters and sheets in contrast to their non-cancerous counterparts. Using quantitative PCR, we demonstrated the key genes and active proteins responsible for this invasion. The addition of stromal fibroblasts and endothelial cells provided a biomimetic microenvironment which was reflected by the upregulation of invasive genes. CAFs enhanced the distance and surface area of the invasive cancer mass whilst inhibiting vascular-like network formation. Pancreatic cancer cells within a highly complex 3D model responded to paclitaxel and dose response curves could be established. Conclusion: These results support, within a biomimetic 3D, in vitro framework, the underlying gene pathways involved in cancer invasion and vascularisation. Furthermore, a complex model of pancreatic cancer was developed accounting for the large stromal proportion and used as a drug testing platform

Schuld und Sühne im Anthropozän: Aspekte eines Phänomens in Anrufung und Selbsttechnik

In historischen und aktuellen wissenschaftlichen Klassikern der Umweltliteratur finden sich epistemische Linien, die das Aufkommen einer neuen Vorstellung von Schuld an oder vor der Umwelt vorbereiten und einbetten. Ältere Diskursbeiträge enthalten ein Schuldkonzept, das dem klassischen soziologischen Verständnis von Schuld entspricht. Sie begreifen sie prozessual – als normalisierendes Korrektiv, das vor allem staatliches Handeln reguliert. Hingegen zeichnen sich jüngere Beiträge durch die Konzeption einer tendenziell abschließenden Schuld aus, die primär die Identität handelnder Subjekte trifft. Aufgrund ihrer diskursiven Charakteristika kann diese grüne Schuld kaum mehr teleologisch und prozessual – etwa als Dimension neoliberaler Selbstregierung – gefasst werden. Mit einem subjektivierungstheoretisch informierten Zugang betrachte ich die Materialisierung dieser arationalen Schuld im Kontext ihrer identitätsstiftenden und totalisierenden Effekte als diskursive Beharrungstendenz. Dieser Beitrag dient der Reflexion soziologischer Kritik im Rahmen der Transformationsforschung; außerdem regt die dargelegte Konzeptualisierung einer arationalen Schuld die Theoriebildung der Subjektivierungstheorie an. By analysing historical and contemporary scholarly classics of environmentalism, I follow epistemic threads that link a notion of guilt to the transgression of perceived natural boundaries. Earlier works refer to this “green guilt” primarily with regard to state action. They correspond with the common sociological understanding of guilt as a normalizing processual corrective. However, younger works prepare for a different notion of green guilt that constitutes the identity of individuals. Moreover, the normalizing procedure that was earlier attached to the ascription of guilt disappears. Instead, deviant individuals are subject to a discourse that renders them inherently guilty and powerless. Informed by the poststructuralist theory of subjectivation, I discuss these characteristics as a materialization of arational guilt, since it cannot be grasped teleologically – e.g. as a dimension of neoliberal governmentality. This article offers a way of conceptualising green guilt as a discursive peculiarity that manifests itself solely in the subject’s identity. Within the framework of transformation research, my findings shed a light on a discursive pattern of persistence that is yet to be understood. (peer reviewed

Beikost als komplizierte Angelegenheit: erwartete Risiken bei der Umstellung auf feste Kost und daran gebundene Verantwortlichkeiten beim Elternpaar

"Der Artikel behandelt die Gestaltung der Übergangsphase von Milchernährung zu fester Kost und in diesem Kontext aktualisierte Geschlechter- und Rollenbilder innerhalb der Familie. Ausgangsfrage ist, welche normativen Anforderungen an Eltern bei der Ablösung der Stillernährung durch feste Kost entstehen oder fortgeschrieben werden. Datengrundlage bilden teilnehmende Beobachtungen in unterschiedlichen Bildungsangeboten eines Familienzentrums. Der Fokus der Analyse liegt auf den bei der Beikosteinführung erwarteten Risiken, die dem Bild der als sicher angesehenen Stillernährung gegenübergestellt werden. Der Artikel zeigt, dass von Eltern und Mitarbeiter_innen des Familienzentrums bei der Beikosteinführung viele unterschiedliche Risiken für das Kind erwartet werden. Der Artikel fragt nach den Strategien, die angewandt werden, um diese Risiken zu minimieren. Er zeigt, wie Beikosteinführung dabei im Setting von Familienbildung als anspruchsvolle Aufgabe entworfen wird und untersucht, wem diese Aufgabe zugewiesen wird und mit welchen Mutter- und Vaterschaftskonstruktionen diese Verantwortungsübernahme begründet wird." (Autorenreferat

Variation in clutch size in relation to nest size in birds

© 2014 The Authors. Nests are structures built to support and protect eggs and/or offspring from predators, parasites, and adverse weather conditions. Nests are mainly constructed prior to egg laying, meaning that parent birds must make decisions about nest site choice and nest building behavior before the start of egg-laying. Parent birds should be selected to choose nest sites and to build optimally sized nests, yet our current understanding of clutch size-nest size relationships is limited to small-scale studies performed over short time periods. Here, we quantified the relationship between clutch size and nest size, using an exhaustive database of 116 slope estimates based on 17,472 nests of 21 species of hole and non-hole-nesting birds. There was a significant, positive relationship between clutch size and the base area of the nest box or the nest, and this relationship did not differ significantly between open nesting and hole-nesting species. The slope of the relationship showed significant intraspecific and interspecific heterogeneity among four species of secondary hole-nesting species, but also among all 116 slope estimates. The estimated relationship between clutch size and nest box base area in study sites with more than a single size of nest box was not significantly different from the relationship using studies with only a single size of nest box. The slope of the relationship between clutch size and nest base area in different species of birds was significantly negatively related to minimum base area, and less so to maximum base area in a given study. These findings are consistent with the hypothesis that bird species have a general reaction norm reflecting the relationship between nest size and clutch size. Further, they suggest that scientists may influence the clutch size decisions of hole-nesting birds through the provisioning of nest boxes of varying sizes.Peer Reviewe

Selection of reference genes for diurnal and developmental time-course real-time PCR expression analyses in lettuce

Background: Real-time quantitative polymerase chain reaction (RT-qPCR) analysis is a low cost and sensitive technique that is widely used to measure levels of gene expression. Selecting and validating appropriate reference genes for normalising target gene expression should be the first step in any expression study to avoid inaccurate results. Results: In this study, ten candidate genes were tested for their suitability for use as reference genes in diurnal and developmental timecourse experiments in lettuce. The candidate reference genes were then used to normalise the expression pattern of the FLOWERING LOCUS T (FT) gene, one of key genes involved in the flowering time pathway whose expression is known to vary throughout the day and at different stages of development. Three reference genes, LsPP2A-1 (PROTEIN PHOSPHATASE 2A-1), LsPP2AA3 (PROTEIN PHOSPHATASE 2A REGULATORY SUBUNIT A3) and LsTIP41 (TAP42-INTERACTING PROTEIN OF 41 kDa), were the most stably expressed candidate reference genes throughout both the diurnal and developmental timecourse experiments. In the developmental experiment using just LsPP2A-1 and LsTIP41 as reference genes would be sufficient for accurate normalisation, whilst in the diurnal experiment all three reference genes, LsPP2A-1, LsPP2AA3 and LsTIP41, would be necessary. The FT expression pattern obtained demonstrates that the use of multiple and robust reference genes for RT-qPCR expression analyses results in a more accurate and reliable expression profile. Conclusions: Reference genes suitable for use in diurnal and developmental timecourse experiments in lettuce were identified and used to produce a more accurate and reliable analysis of lsFT expression levels than previously obtained in such timecourse experiments

RANKL neutralisation prevents osteoclast activation in a human in vitro ameloblastoma-bone model

Ameloblastoma is a benign, locally invasive epithelial odontogenic neoplasm of the jaw. Treatment of choice is jaw resection, often resulting in significant morbidity. The aim of this study was to recapitulate ameloblastoma in a completely humanised 3D disease model containing ameloblastoma cells, osteoblasts and activated osteoclasts to investigate the RANKL pathway within the ameloblastoma stromal environment and its response to the RANKL antibody denosumab. In vitro bone was engineered by culturing human osteoblasts (hOB) in a biomimetic, dense collagen type I matrix, resulting in extensive mineral deposits by day 21 forming alizarin red positive bone like nodules throughout the 3D model. Activated TRAP + human osteoclasts were confirmed through the differentiation of human CD14+ monocytes after 10 days within the model. Lastly, the ameloblastoma cell lines AM-1 and AM-3 were incorporated into the 3D model. RANKL release was validated through TACE/ADAM17 activation chemically or through hOB co-culture. Denosumab treatment resulted in decreased osteoclast activation in the presence of hOB and ameloblastoma cells. These findings stress the importance of accurately modelling tumour and stromal populations as a preclinical testing platform

Modelling stromal compartments to recapitulate the ameloblastoma tumour microenvironment

Tumour development and progression is dependent upon tumour cell interaction with the tissue stroma. Bioengineering the tumour-stroma microenvironment (TME) into 3D biomimetic models is crucial to gain insight into tumour cell development and progression pathways and identify therapeutic targets. Ameloblastoma is a benign but locally aggressive epithelial odontogenic neoplasm that mainly occurs in the jawbone and can cause significant morbidity and sometimes death. The molecular mechanisms for ameloblastoma progression are poorly understood. A spatial model recapitulating the tumour and stroma was engineered to show that without a relevant stromal population, tumour invasion is quantitatively decreased. Where a relevant stroma was engineered in dense collagen populated by gingival fibroblasts, enhanced receptor activator of nuclear factor kappa-B ligand (RANKL) expression was observed and histopathological properties, including ameloblastoma tumour islands, developed and were quantified. Using human osteoblasts (bone stroma) further enhanced the biomimicry of ameloblastoma histopathological phenotypes. This work demonstrates the importance of the two key stromal populations, osteoblasts, and gingival fibroblasts, for accurate 3D biomimetic ameloblastoma modelling

Modelling stromal compartments to recapitulate the ameloblastoma tumour microenvironment

Tumour development and progression is dependent upon tumour cell interaction with the tissue stroma. Bioengineering the tumour-stroma microenvironment (TME) into 3D biomimetic models is crucial to gain insight into tumour cell development and progression pathways and identify therapeutic targets. Ameloblastoma is a benign but locally aggressive epithelial odontogenic neoplasm that mainly occurs in the jawbone and can cause significant morbidity and sometimes death. The molecular mechanisms for ameloblastoma progression are poorly understood. A spatial model recapitulating the tumour and stroma was engineered to show that without a relevant stromal population, tumour invasion is quantitatively decreased. Where a relevant stroma was engineered in dense collagen populated by gingival fibroblasts, enhanced receptor activator of nuclear factor kappa-B ligand (RANKL) expression was observed and histopathological properties, including ameloblastoma tumour islands, developed and were quantified. Using human osteoblasts (bone stroma) further enhanced the biomimicry of ameloblastoma histopathological phenotypes. This work demonstrates the importance of the two key stromal populations, osteoblasts, and gingival fibroblasts, for accurate 3D biomimetic ameloblastoma modelling

RAF1-MEK/ERK pathway-dependent ARL4C expression promotes ameloblastoma cell proliferation and osteoclast formation

Ameloblastoma is an odontogenic neoplasm characterized by slow intraosseous growth with progressive jaw resorption. Recent reports have revealed that ameloblastoma harbours an oncogenic BRAFV600E mutation with mitogen-activated protein kinase (MAPK) pathway activation and described cases of ameloblastoma harbouring a BRAFV600E mutation in which patients were successfully treated with a BRAF inhibitor. Therefore, the MAPK pathway may be involved in the development of ameloblastoma; however, the precise mechanism by which it induces ameloblastoma is unclear. The expression of ADP-ribosylation factor (ARF)-like 4c (ARL4C), induced by a combination of the EGF-MAPK pathway and Wnt/beta-catenin signalling, has been shown to induce epithelial morphogenesis. It was also reported that the overexpression of ARL4C, due to alterations in the EGF/RAS-MAPK pathway and Wnt/beta-catenin signalling, promotes tumourigenesis. However, the roles of ARL4C in ameloblastoma are unknown. We investigated the involvement of ARL4C in the development of ameloblastoma. In immunohistochemical analyses of tissue specimens obtained from 38 ameloblastoma patients, ARL4C was hardly detected in non-tumour regions but tumours frequently showed strong expression of ARL4C, along with the expression of both BRAFV600E and RAF1 (also known as C-RAF). Loss-of-function experiments using inhibitors or siRNAs revealed that ARL4C elevation depended on the RAF1-MEK/ERK pathway in ameloblastoma cells. It was also shown that the RAF1-ARL4C and BRAFV600E-MEK/ERK pathways promoted cell proliferation independently. ARL4C-depleted tumour cells (generated by knockdown or knockout) exhibited decreased proliferation and migration capabilities. Finally, when ameloblastoma cells were co-cultured with mouse bone marrow cells and primary osteoblasts, ameloblastoma cells induced osteoclast formation. ARL4C elevation in ameloblastoma further promoted its formation capabilities through the increased RANKL expression of mouse bone marrow cells and/or primary osteoblasts. These results suggest that the RAF1-MEK/ERK-ARL4C axis, which may function in cooperation with the BRAFV600E-MEK/ERK pathway, promotes ameloblastoma development. (c) 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd

Murine GRPR and Stathmin Control in Opposite Directions both Cued Fear Extinction and Neural Activities of the Amygdala and Prefrontal Cortex

Extinction is an integral part of normal healthy fear responses, while it is compromised in several fear-related mental conditions in humans, such as post-traumatic stress disorder (PTSD). Although much research has recently been focused on fear extinction, its molecular and cellular underpinnings are still unclear. The development of animal models for extinction will greatly enhance our approaches to studying its neural circuits and the mechanisms involved. Here, we describe two gene-knockout mouse lines, one with impaired and another with enhanced extinction of learned fear. These mutant mice are based on fear memory-related genes, stathmin and gastrin-releasing peptide receptor (GRPR). Remarkably, both mutant lines showed changes in fear extinction to the cue but not to the context. We performed indirect imaging of neuronal activity on the second day of cued extinction, using immediate-early gene c-Fos. GRPR knockout mice extinguished slower (impaired extinction) than wildtype mice, which was accompanied by an increase in c-Fos activity in the basolateral amygdala and a decrease in the prefrontal cortex. By contrast, stathmin knockout mice extinguished faster (enhanced extinction) and showed a decrease in c-Fos activity in the basolateral amygdala and an increase in the prefrontal cortex. At the same time, c-Fos activity in the dentate gyrus was increased in both mutant lines. These experiments provide genetic evidence that the balance between neuronal activities of the amygdala and prefrontal cortex defines an impairment or facilitation of extinction to the cue while the hippocampus is involved in the context-specificity of extinction