Analyzing Sex-Specific Dimorphism in Human Skeletal Stem Cells.

Sex-related differences are a current topic in contemporary science. In addition to hormonal regulation, cell-autonomous mechanisms are important in bone homeostasis and regeneration. In this study, human skeletal stem cells (SSCs) from female and male adults were cultured and analyzed with immunological assays and osteogenic differentiation assessments. Female SSCs exhibited a mean doubling time of 100.6 h, whereas male SSCs displayed a mean doubling time of 168.0 h. Immunophenotyping revealed the expression of the stem cell markers Nestin, CD133, and CD164, accompanied by the neural-crest marker SOX9. Furthermore, multiparameter flow cytometric analyses revealed a substantial population of multipotent SSCs, comprising up to 80% in both sexes. An analysis of the osteogenic differentiation potential demonstrated a strong mineralization in both male and female SSCs under physiological conditions. Recognizing the prevailing association of bone diseases with inflammatory processes, we also analyzed the osteogenic potential of SSCs from both sexes under pro-inflammatory conditions. Upon TNF-α and IL-1ß treatment, we observed no sexual dimorphism on osteogenesis. In summary, we demonstrated the successful isolation and characterization of SSCs capable of rapid osteogenic differentiation. Taken together, in vitro cultured SSCs might be a suitable model to study sexual dimorphisms and develop drugs for degenerative bone diseases.

Targeting Key Signaling Pathways in Glioblastoma Stem Cells for the Development of Efficient Chemo- and Immunotherapy.

Glioblastoma multiforme (GBM) is the most aggressive and most common malignant brain tumor with poor patient survival despite therapeutic intervention. On the cellular level, GBM comprises a rare population of glioblastoma stem cells (GSCs), driving therapeutic resistance, invasion, and recurrence. GSCs have thus come into the focus of therapeutic strategies, although their targeting remains challenging. In the present study, we took advantage of three GSCs-populations recently established in our lab to investigate key signaling pathways and subsequent therapeutic strategies targeting GSCs. We observed that NF-κB, a crucial transcription factor in GBM progression, was expressed in all CD44+/CD133+/Nestin+-GSC-populations. Exposure to TNFα led to activation of NF-κB-RELA and/or NF-κB-c-REL, depending on the GBM type. GSCs further expressed the proto-oncogene MYC family, with MYChigh GSCs being predominantly located in the tumor spheres ("GROW"-state) while NF-κB-RELAhigh GSCs were migrating out of the sphere ("GO"-state). We efficiently targeted GSCs by the pharmacologic inhibition of NF-κB using PTDC/Bortezomib or inhibition of MYC by KJ-Pyr-9, which significantly reduced GSC-viability, even in comparison to the standard chemotherapeutic drug temozolomide. As an additional cell-therapeutic strategy, we showed that NK cells could kill GSCs. Our findings offer new perspectives for developing efficient patient-specific chemo- and immunotherapy against GBM.

The Diminishment of Novel Endometrial Carcinoma-Derived Stem-like Cells by Targeting Mitochondrial Bioenergetics and MYC.

Cancer stem cells (CSCs) are a small subpopulation of tumor cells harboring properties that include self-renewal, multi-lineage differentiation, tumor reconstitution, drug resistance and invasiveness, making them key players in tumor relapse. In the present paper, we develop new CSC models and analyze the molecular pathways involved in survival to identify targets for the establishment of novel therapies. Endometrial carcinoma-derived stem-like cells (ECSCs) were isolated from carcinogenic gynecological tissue and analyzed regarding their expression of prominent CSC markers. Further, they were treated with the MYC-signaling inhibitor KJ-Pyr-9, chemotherapeutic agent carboplatin and type II diabetes medication metformin. ECSC populations express common CSC markers, such as Prominin-1 and CD44 antigen as well as epithelial-to-mesenchymal transition markers, Twist, Snail and Slug, and exhibit the ability to form free-floating spheres. The inhibition of MYC signaling and treatment with carboplatin as well as metformin significantly reduced the cell survival of ECSC-like cells. Further, treatment with metformin significantly decreased the mitochondrial membrane potential of ECSC-like cells, while the extracellular lactate concentration was increased. The established ECSC-like populations represent promising in vitro models to further study the contribution of ECSCs to endometrial carcinogenesis. Targeting MYC signaling as well as mitochondrial bioenergetics has shown promising results in the diminishment of ECSCs, although molecular signaling pathways need further investigations.

Analysis of Several Pathways for Efficient Killing of Prostate Cancer Stem Cells: A Central Role of NF-κB RELA.

Prostate cancer is a common cause of death worldwide. Here, we isolated cancer stem cells (CSCs) from four adenocarcinomas of the prostate (Gleason scores from 3 + 3 up to 4 + 5). CSCs were characterized by the expression of the stem cell markers TWIST, the epithelial cell adhesion molecule (EPCAM), the transcription factors SNAI1 (SNAIL) and SNAI2 (SLUG) and cancer markers such as CD44 and prominin-1 (CD133). All investigated CSC populations contained a fraction highly positive for aldehyde dehydrogenase (ALDH) function and displayed robust expressions of programmed cell death 1 (PD-1) ligands. Furthermore, we investigated immunotherapeutic approaches but had no success even with the clinically used PD-1 inhibitor pembrolizumab. In addition, we studied another death-inducing pathway via interferon gamma signaling and detected high-level upregulations of human leukocyte antigen A (HLA-A) and beta 2-microglobulin (B2M) with only moderate killing efficacy. To examine further killing mechanisms in prostate cancer stem cells (PCSCs), we analyzed NF-κB signaling. Surprisingly, two patient-specific populations of PCSCs were found: one with canonical NF-κB signaling and another one with blunted NF-κB activation, which can be efficiently killed by tumor necrosis factor (TNF). Thus, culturing of PCSCs and analysis of respective NF-κB induction potency after surgery might be a powerful tool for optimizing patient-specific treatment options, such as the use of TNF-inducing chemotherapeutics and/or NF-κB inhibitors.

Isolation and Characterization of Two Novel Colorectal Cancer Cell Lines, Containing a Subpopulation with Potential Stem-Like Properties: Treatment Options by MYC/NMYC Inhibition.

Cancer stem cells (CSC) are crucial mediators of cancer relapse. Here, we isolated two primary human colorectal cancer cell lines derived from a rectal neuroendocrine carcinoma (BKZ-2) and a colorectal adenocarcinoma (BKZ-3), both containing subpopulations with potential stem-like properties. Protein expression of CSC-markers prominin-1 and CD44 antigen was significantly higher for BKZ-2 and BKZ-3 in comparison to well-established colon carcinoma cell lines. High sphere-formation capacity further confirmed the existence of a subpopulation with potential stem-like phenotype. Epithelial-mesenchymal transition markers as well as immune checkpoint ligands were expressed more pronounced in BKZ-2. Both cell populations demonstrated N-myc proto-oncogene (NMYC) copy number gain. Myc proto-oncogene (MYC)/NMYC activity inhibitor all-trans retinoic acid (ATRA) significantly reduced the number of tumor spheres for both and the volume of BKZ-2 spheres. In contrast, the sphere volume of ATRA-treated BKZ-3 was increased, and only BKZ-2 cell proliferation was reduced in monolayer culture. Treatment with KJ-Pyr-9, a specific inhibitor of MYC/NMYC-myc-associated factor X interaction, decreased survival by the induction of apoptosis of both. In summary, here, we present the novel colorectal cancer cell lines BKZ-2 and BKZ-3 as promising cellular in vitro models for colorectal carcinomas and identify the MYC/NMYC molecular pathway involved in CSC-induced carcinogenesis with relevant therapeutic potential.

IKK1/2 protect human cells from TNF-mediated RIPK1-dependent apoptosis in an NF-κB-independent manner.

TNF signaling is directly linked to cancer development and progression. A broad range of tumor cells is able to evade cell death induced by TNF impairing the potential anti-cancer value of TNF in therapy. Although sensitizing cells to TNF-induced death therefore has great clinical implications, detailed mechanistic insights into TNF-mediated human cell death still remain unknown. Here, we analyzed human cells by applying CRISPR/Cas9n to generate cells deficient of IKK1, IKK2, IKK1/2 and RELA. Despite stimulation with TNF resulted in impaired NF-κB activation in all genotypes compared to wildtype cells, increased cell death was observable only in IKK1/2-double-deficient cells. Cell death could be detected by Caspase-3 activation and binding of Annexin V. TNF-induced programmed cell death in IKK1/2-/- cells was further shown to be mediated via RIPK1 in a predominantly apoptotic manner. Our findings demonstrate the IKK complex to protect from TNF-induced cell death in human cells independently to NF-κB RelA suggesting IKK1/2 to be highly promising targets for cancer therapy.

Inactivity Is Nycthemeral, Endogenously Generated, Homeostatically Regulated, and Melatonin Modulated in a Free-Living Platyhelminth Flatworm.

Introduction: Sleep either appeared once early in the evolution of animals, or at multiple instances over evolutionary time. Understanding whether sleep is a diagnostic trait for members of the kingdom Animalia has important implications for our understanding of the evolution of sleep and sleep functions. Unfortunately, knowledge on the phylogenetic breadth of sleep is restricted to vertebrates, a few arthropods and molluscs, and one species of nematode. There is a dearth of information on the other 30 or so animal phyla. Aims and Methods: Here, we provide original data on a previously unstudied group of animals with respect to sleep: platyhelminth flatworms. These free-living animals are relatively simple, with a rudimentary central nervous system and absence of many other specialized physiological systems. Results: Despite this simplicity, inactive flatworms appeared to be sleeping. Specifically, quiescence was organized in a circadian manner, occurring largely during the daytime. This basic rhythm persisted under constant darkness, suggesting that it was endogenously generated. Active flatworms responded more readily to stimulation, and flatworms recovered lost sleep by sleeping longer after a 3-hour period of inactivity deprivation. We were also able to increase inactivity in a dose-dependent manner with exposure to melatonin, a hormone that increases sleep in diurnal animals. Conclusions: Taken together, these data expand our understanding of the phylogenetic extent of sleep and reinforce the idea that sleep evolved early in the evolutionary history of animals. However, additional studies on other types of animals are required for a comprehensive understanding of the origin(s) and evolution of sleep.

Fall field crickets did not acclimate to simulated seasonal changes in temperature.

In nature, many organisms alter their developmental trajectory in response to environmental variation. However, studies of thermal acclimation have historically involved stable, unrealistic thermal treatments. In our study, we incorporated ecologically relevant treatments to examine the effects of environmental stochasticity on the thermal acclimation of the fall field cricket (Gryllus pennsylvanicus). We raised crickets for 5 weeks at either a constant temperature (25°C) or at one of three thermal regimes mimicking a seasonal decline in temperature (from 25 to 12°C). The latter three treatments differed in their level of thermal stochasticity: crickets experienced either no diel cycle, a predictable diel cycle, or an unpredictable diel cycle. Following these treatments, we measured several traits considered relevant to survival or reproduction, including growth rate, jumping velocity, feeding rate, metabolic rate, and cold tolerance. Contrary to our predictions, the acclimatory responses of crickets were unrelated to the magnitude or type of thermal variation. Furthermore, acclimation of performance was not ubiquitous among traits. We recommend additional studies of acclimation in fluctuating environments to assess the generality of these findings.

Isopods failed to acclimate their thermal sensitivity of locomotor performance during predictable or stochastic cooling.

Most organisms experience environments that vary continuously over time, yet researchers generally study phenotypic responses to abrupt and sustained changes in environmental conditions. Gradual environmental changes, whether predictable or stochastic, might affect organisms differently than do abrupt changes. To explore this possibility, we exposed terrestrial isopods (Porcellio scaber) collected from a highly seasonal environment to four thermal treatments: (1) a constant 20°C; (2) a constant 10°C; (3) a steady decline from 20° to 10°C; and (4) a stochastic decline from 20° to 10°C that mimicked natural conditions during autumn. After 45 days, we measured thermal sensitivities of running speed and thermal tolerances (critical thermal maximum and chill-coma recovery time). Contrary to our expectation, thermal treatments did not affect the thermal sensitivity of locomotion; isopods from all treatments ran fastest at 33° to 34°C and achieved more than 80% of their maximal speed over a range of 10° to 11°C. Isopods exposed to a stochastic decline in temperature tolerated cold the best, and isopods exposed to a constant temperature of 20°C tolerated cold the worst. No significant variation in heat tolerance was observed among groups. Therefore, thermal sensitivity and heat tolerance failed to acclimate to any type of thermal change, whereas cold tolerance acclimated more during stochastic change than it did during abrupt change.

Mothers forewarn offspring about predators: a transgenerational maternal effect on behavior.

Predation risk has long been known to exert a strong influence on behavior, but no study to date has determined whether predators influence offspring antipredator behavior via maternal effects. Here, we provide a unique example of a transgenerational maternal effect in antipredator behavior that takes the form of a "warning" about predators that female fall field crickets Gryllus pennsylvanicus transmit to their offspring. Specifically, the offspring of gravid crickets exposed to a wolf spider Hogna helluo exhibit greater antipredator immobility in response to Hogna chemical cues than do offspring of nonexposed females. These "forewarned" crickets exhibit greater survival in the presence of Hogna than do those not forewarned. Accordingly, gravid crickets from areas with significant Hogna populations produce offspring that are more responsive to Hogna cues than do those from nearby Hogna-free areas. Such transgenerational maternal effects may be more common than currently realized.

Energy availability influences microclimate selection of hibernating bats.

Many species hibernate to conserve energy during periods of low food and water availability. It has long been assumed that the optimal hibernation strategy involves long, deep bouts of torpor that minimize energy expenditure. However, hibernation has ecological (e.g. decreased predator avoidance) and physiological (e.g. sleep deprivation) costs that must be balanced with energy savings; therefore, individuals possessing sufficient energy reserves may reduce their use of deep torpor. We tested the hypothesis that energy (fat) availability influences temperature selection of two fat-storing bat species during hibernation. We predicted that individuals with small energy reserves would select colder temperatures for hibernation in order to minimize energy expenditure, while individuals with larger energy reserves would choose warmer temperatures to minimize the costs of hibernation. Results from our field experiment indicate that little brown myotis (Myotis lucifugus) hibernating in warm microclimates were significantly heavier than individuals hibernating in cooler microclimates. To determine if energy availability was mediating this relationship, we limited fatty acid availability with mercaptoacetate (MA) and quantified its effect on torpid metabolic rate (TMR) and thermal preference of big brown bats (Eptesicus fuscus). Administration of MA caused a 43% drop in TMR at 10 degrees C and caused bats to choose significantly colder temperatures for hibernation. Our results suggest that fat-storing bats minimize torpor expression using both physiological and behavioral mechanisms.

The perils of picky eating: dietary breadth is related to extinction risk in insectivorous bats.

Several recent papers evaluate the relationship between ecological characteristics and extinction risk in bats. These studies report that extinction risk is negatively related to geographic range size and positively related to habitat specialization. Here, we evaluate the hypothesis that extinction risk is also related to dietary specialization in insectivorous vespertilionid bats using both traditional and phylogenetically-controlled analysis of variance. We collected dietary data and The World Conservation Union (IUCN) rankings for 44 Australian, European, and North American bat species. Our results indicate that species of conservation concern (IUCN ranking near threatened or above) are more likely to have a specialized diet than are species of least concern. Additional analyses show that dietary breadth is not correlated to geographic range size or wing morphology, characteristics previously found to correlate with extinction risk. Therefore, there is likely a direct relationship between dietary specialization and extinction risk; however, the large variation in dietary breadth within species of least concern suggests that diet alone cannot explain extinction risk. Our results may have important implications for the development of predictive models of extinction risk and for the assignment of extinction risk to insectivorous bat species. Similar analyses should be conducted on additional bat families to assess the generality of this relationship between niche breadth and extinction risk.