Applying Tissue Slice Culture in Cancer Research-Insights from Preclinical Proton Radiotherapy.

A challenge in cancer research is the definition of reproducible, reliable, and practical models, which reflect the effects of complex treatment modalities and the heterogeneous response of patients. Proton beam radiotherapy (PBRT), relative to conventional photon-based radiotherapy, offers the potential for iso-effective tumor control, while protecting the normal tissue surrounding the tumor. However, the effects of PBRT on the tumor microenvironment and the interplay with newly developed chemo- and immunotherapeutic approaches are still open for investigation. This work evaluated thin-cut tumor slice cultures (TSC) of head and neck cancer and organotypic brain slice cultures (OBSC) of adult mice brain, regarding their relevance for translational radiooncology research. TSC and OBSC were treated with PBRT and investigated for cell survival with a lactate dehydrogenase (LDH) assay, DNA repair via the DNA double strand break marker γH2AX, as well as histology with regards to morphology. Adult OBSC failed to be an appropriate model for radiobiological research questions. However, histological analysis of TSC showed DNA damage and tumor morphological results, comparable to known in vivo and in vitro data, making them a promising model to study novel treatment approaches in patient-derived xenografts or primary tumor material.

Flow Cytometric Isolation and Differentiation of Adipogenic Progenitor Cells into Brown and Brite/Beige Adipocytes.

Aside from mature adipocytes, adipose tissue harbors several distinct cell populations including immune cells, endothelial cells, and adipogenic progenitor cells (AdPCs). AdPCs represent the reservoir of regenerative cells that replenishes adipocytes during normal cellular turnover and during times of increased demand for triglyceride-storage capacity. The worldwide increase in pathologies associated with the metabolic syndrome, such as obesity and type-2 diabetes, has heightened public and scientific interest in adipose tissues and the cell biological processes of adipose tissue formation and function. Two distinct types of fat cells are known: White and brown adipocytes. Especially brown adipose tissue (BAT) has received considerable attention due to its unique capacity for thermogenic energy expenditure and potential role in the treatment of adiposity. Accordingly, the cold-induced conversion of white into brown-like adipocytes has become a feasible approach in humans and a study-subject in rodents to better understand the underlying molecular processes. Fluorescence-activated cell sorting (FACS) provides a method to isolate AdPCs and other cell populations from adipose tissue by using antibodies detecting unique surface markers. We here describe an approach to isolate cells committed to the adipogenic lineage and summarize established protocols to differentiate FACS-purified primary AdPCs into UCP1-expressing brown adipocytes under in vitro conditions.

Adipocyte Accumulation in the Bone Marrow during Obesity and Aging Impairs Stem Cell-Based Hematopoietic and Bone Regeneration.

Aging and obesity induce ectopic adipocyte accumulation in bone marrow cavities. This process is thought to impair osteogenic and hematopoietic regeneration. Here we specify the cellular identities of the adipogenic and osteogenic lineages of the bone. While aging impairs the osteogenic lineage, high-fat diet feeding activates expansion of the adipogenic lineage, an effect that is significantly enhanced in aged animals. We further describe a mesenchymal sub-population with stem cell-like characteristics that gives rise to both lineages and, at the same time, acts as a principal component of the hematopoietic niche by promoting competitive repopulation following lethal irradiation. Conversely, bone-resident cells committed to the adipocytic lineage inhibit hematopoiesis and bone healing, potentially by producing excessive amounts of Dipeptidyl peptidase-4, a protease that is a target of diabetes therapies. These studies delineate the molecular identity of the bone-resident adipocytic lineage, and they establish its involvement in age-dependent dysfunction of bone and hematopoietic regeneration.

Loss of BMP receptor type 1A in murine adipose tissue attenuates age-related onset of insulin resistance.

AIMS/HYPOTHESIS: Adipose tissue dysfunction is a prime risk factor for the development of metabolic disease. Bone morphogenetic proteins (BMPs) have previously been implicated in adipocyte formation. Here, we investigate the role of BMP signalling in adipose tissue health and systemic glucose homeostasis. METHODS: We employed the Cre/loxP system to generate mouse models with conditional ablation of BMP receptor 1A in differentiating and mature adipocytes, as well as tissue-resident myeloid cells. Metabolic variables were assessed by glucose and insulin tolerance testing, insulin-stimulated glucose uptake and gene expression analysis. RESULTS: Conditional deletion of Bmpr1a using the aP2 (also known as Fabp4)-Cre strain resulted in a complex phenotype. Knockout mice were clearly resistant to age-related impairment of insulin sensitivity during normal and high-fat-diet feeding and showed significantly improved insulin-stimulated glucose uptake in brown adipose tissue and skeletal muscle. Moreover, knockouts displayed significant reduction of variables of adipose tissue inflammation. Deletion of Bmpr1a in myeloid cells had no impact on insulin sensitivity, while ablation of Bmpr1a in mature adipocytes partially recapitulated the initial phenotype from aP2-Cre driven deletion. Co-cultivation of macrophages with pre-adipocytes lacking Bmpr1a markedly reduced expression of proinflammatory genes. CONCLUSIONS/INTERPRETATION: Our findings show that altered BMP signalling in adipose tissue affects the tissue's metabolic properties and systemic insulin resistance by altering the pattern of immune cell infiltration. The phenotype is due to ablation of Bmpr1a specifically in pre-adipocytes and maturing adipocytes rather than an immune cell-autonomous effect. Mechanistically, we provide evidence for a BMP-mediated direct crosstalk between pre-adipocytes and macrophages.

Mechanisms of aging-related impairment of brown adipocyte development and function.

Aging is one of the primary risk factors for the development of obesity, a pathology that develops due to an imbalance of increased energy consumption over reduced expenditure. Brown adipocytes are responsible for thermogenesis and could therefore counter obesity by increasing energy expenditure. It is by now well established that humans possess thermogenesis-competent brown adipocytes throughout life, and recent findings indicate that brown fat is actively involved in metabolic control and body weight regulation in adults. Aging is accompanied by a loss of classical brown adipocytes as well as the brown-like adipocytes found in white adipose tissue, suggesting that loss of their energy-expending capacity might contribute to an obesity-prone phenotype with increased age. We here discuss the hypothesis that the age-related loss of brown adipocyte regenerative capacity is a result of dysfunctional stem/progenitor cells. The possible molecular mechanisms that lead to an age-related decline in brown adipogenic stem/progenitor cell function include cell-autonomous and external effects. General loss of mitochondrial biogenesis and function has repeatedly been linked to age-related perturbation of metabolic processes. We also discuss the possibility that alterations in neuronal control by the sympathetic nervous system may contribute to impaired regeneration and thermogenesis in aged brown adipocytes. Finally, age-related changes of endocrine signals have been proposed to exacerbate the loss of brown adipose tissue. In conclusion, age-induced impairment of brown adipogenic stem/progenitor cell function could contribute to the loss of brown adipocyte regeneration, thereby promoting the development of obesity and other metabolic disorders with age.