Monarch Butterfly Ecology, Behavior, and Vulnerabilities in North Central United States Agricultural Landscapes.

The North American monarch butterfly (Danaus plexippus) is a candidate species for listing under the Endangered Species Act. Multiple factors are associated with the decline in the eastern population, including the loss of breeding and foraging habitat and pesticide use. Establishing habitat in agricultural landscapes of the North Central region of the United States is critical to increasing reproduction during the summer. We integrated spatially explicit modeling with empirical movement ecology and pesticide toxicology studies to simulate population outcomes for different habitat establishment scenarios. Because of their mobility, we conclude that breeding monarchs in the North Central states should be resilient to pesticide use and habitat fragmentation. Consequently, we predict that adult monarch recruitment can be enhanced even if new habitat is established near pesticide-treated crop fields. Our research has improved the understanding of monarch population dynamics at the landscape scale by examining the interactions among monarch movement ecology, habitat fragmentation, and pesticide use.

Optimization of QuEChERS Method for Simultaneous Determination of Neonicotinoid Residues in Pollinator Forage.

Consistent with the large-scale use of pesticide seed treatments in U.S. field crop production, there has been an increased use of neonicotinoid-treated corn and soybean seed over the past decade. Neonicotinoids can move downwind to adjacent off-field pollinator habitats in dust from planting and/or move downslope to habitats in surface water. The extent of potential neonicotinoid exposure to pollinators from neonicotinoid movement into these adjacent pollinator habitats is unclear. Pollen and leaf tissue extractions were completed using a quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction procedure. Samples were subjected to a clean-up step using dispersive solid-phase extraction (dSPE) techniques prior to analysis. The compounds in the extracts were separated on a reversed-phase column with gradient elution and confirmed with tandem mass spectrometry. The extraction method showed acceptable recoveries of analytes ranging from 78.4 to 93.6% and 89.4 to 101% for leaf tissue and pollen, respectively. The method's detection limits ranged from 0.04 to 0.3 ng/g in milkweed leaf tissue and 0.04 to 1.0 ng/g in pollen. The method is currently being employed in ongoing studies surveying pollen from a diversity of forbs and milkweed leaves obtained from habitat patches established within fields with a history of using neonicotinoid-treated seeds.

Development of clinically relevant in vivo metastasis models using human bone discs and breast cancer patient-derived xenografts.

BACKGROUND: Late-stage breast cancer preferentially metastasises to bone; despite advances in targeted therapies, this condition remains incurable. The lack of clinically relevant models for studying breast cancer metastasis to a human bone microenvironment has stunted the development of effective treatments for this condition. To address this problem, we have developed humanised mouse models in which breast cancer patient-derived xenografts (PDXs) metastasise to human bone implants with low variability and high frequency. METHODS: To model the human bone environment, bone discs from femoral heads of patients undergoing hip replacement surgery were implanted subcutaneously into NOD/SCID mice. For metastasis studies, 7 patient-derived xenograft tumours (PDX: BB3RC32, ER+ PR+ HER2-; BB2RC08, ER+ PR+ ER2-; BB6RC37, ER- PR- HER2- and BB6RC39, ER+ PR+ HER2+), MDA-MB-231-luc2, T47D-luc2 or MCF7-Luc2 cells were injected into the 4th mammary ducts and metastases monitored by luciferase imaging and confirmed on histological sections. Bone integrity, viability and vascularisation were assessed by uCT, calcein uptake and histomorphometry. Expression profiling of genes/proteins during different stages of metastasis were assessed by whole genome Affymetrix array, real-time PCR and immunohistochemistry. Importance of IL-1 was confirmed following anakinra treatment. RESULTS: Implantation of femoral bone provided a metabolically active, human-specific site for tumour cells to metastasise to. After 4 weeks, bone implants were re-vascularised and demonstrated active bone remodelling (as evidenced by the presence of osteoclasts, osteoblasts and calcein uptake). Restricting bone implants to the use of subchondral bone and introduction of cancer cells via intraductal injection maximised metastasis to human bone implants. MDA-MB-231 cells specifically metastasised to human bone (70% metastases) whereas T47D, MCF7, BB3RC32, BB2RC08, and BB6RC37 cells metastasised to both human bone and mouse bones. Importantly, human bone was the preferred metastatic site especially from ER+ PDX (100% metastasis human bone compared with 20-75% to mouse bone), whereas ER-ve PDX developed metastases in 20% of human and 20% of mouse bone. Breast cancer cells underwent a series of molecular changes as they progressed from primary tumours to bone metastasis including altered expression of IL-1B, IL-1R1, S100A4, CTSK, SPP1 and RANK. Inhibiting IL-1B signalling significantly reduced bone metastasis. CONCLUSIONS: Our reliable and clinically relevant humanised mouse models provide significant advancements in modelling of breast cancer bone metastasis.

Bioengineering the microanatomy of human skin.

Recreating the structure of human tissues in the laboratory is valuable for fundamental research, testing interventions, and reducing the use of animals. Critical to the use of such technology is the ability to produce tissue models that accurately reproduce the microanatomy of the native tissue. Current artificial cell-based skin systems lack thorough characterisation, are not representative of human skin, and can show variation. In this study, we have developed a novel full thickness model of human skin comprised of epidermal and dermal compartments. Using an inert porous scaffold, we created a dermal construct using human fibroblasts that secrete their own extracellular matrix proteins, which avoids the use of animal-derived materials. The dermal construct acts as a foundation upon which epidermal keratinocytes were seeded and differentiated into a stratified keratinised epithelium. In-depth morphological analyses of the model demonstrated very close similarities with native human skin. Extensive immunostaining and electron microscopy analysis revealed ultrastructural details such as keratohyalin granules and lamellar bodies within the stratum granulosum, specialised junctional complexes, and the presence of a basal lamina. These features reflect the functional characteristics and barrier properties of the skin equivalent. Robustness and reproducibility of in vitro models are important attributes in experimental practice, and we demonstrate the consistency of the skin construct between different users. In summary, a new model of full thickness human skin has been developed that possesses microanatomical features reminiscent of native tissue. This skin model platform will be of significant interest to scientists researching the structure and function of human skin.

Monarch butterfly-breeding habitat restoration: how movement ecology research can inform best practices for site selection.

Population dynamics, persistence, and distribution are emergent properties of animal movement behavior and the spatial configuration of resources. Monarch butterflies are a vagile species with an open-population structure. Selecting locations for monarch butterfly- breeding habitat restoration that aligns with natural movement behavior will facilitate efficient habitat utilization across the landscape, increase realized fecundity, and ultimately support increases in the overwintering population size in Mexico. Obtaining and interpreting empirical movement and space-use data through field and laboratory studies are fundamental to this effort. To gain insights into population responses at larger, spatially explicit landscape scales, the results from empirical studies can be incorporated into simulation models. Together, empirical and simulation studies can inform options for creating functional connectivity of monarch butterfly-breeding habitats. Given currently available information, we synthesize studies for the eastern monarch butterfly to illustrate how an improved understanding of movement ecology can assist in planning conservation practices.

Conservation risks and benefits of establishing monarch butterfly (Danaus plexippus) breeding habitats close to maize and soybean fields in the north central United States: A landscape-scale analysis of the impact of foliar insecticide on nonmigratory monarch butterfly populations.

Establishing habitat in agricultural landscapes of the north central United States is critical to reversing the decline of North America's eastern monarch butterfly (Danaus plexippus) population. Insecticide use could create population sinks and threaten recovery. Discouraging habitat establishment within a 38-m zone around crop fields is a suggested risk mitigation measure. In Story County, Iowa, United States, this mitigation would discourage habitat establishment in 84% of roadsides and 38% of noncrop land. It is unclear if the conservation benefits from establishing habitat close to crop fields outweigh suppression of population growth owing to insecticide exposure. Consequently, monarch conservation plans require spatially and temporally explicit landscape-scale assessments. Using an agent-based model that incorporates female monarch movement and egg laying, the number and location of eggs laid in Story County were simulated for four habitat scenarios: current condition, maximum new establishment, moderate establishment, and moderate establishment only outside a 38-m no-plant zone around crop fields. A demographic model incorporated mortality from natural causes and insecticide exposure to simulate adult monarch production over 10 years. Assuming no insecticide exposure, simulated adult production increased 24.7% and 9.3%, respectively, with maximum and moderate habitat establishment and no planting restrictions. A 3.5% increase was simulated assuming moderate habitat establishment with a 38-m planting restriction. Impacts on adult production were simulated for six representative insecticides registered for soybean aphid (Aphis glycines) management. Depending on the frequency of insecticide applications over a 10-year period, simulated production increased 8.2%-9.3%, assuming moderate habitat establishment with no planting restrictions. Results suggest that the benefits of establishing habitat close to crop fields outweigh the adverse effects of insecticide spray drift; that is, metapopulation extirpation is not a concern for monarchs. These findings are only applicable to species that move at spatial scales greater than the scale of potential spray-drift impacts. Integr Environ Assess Manag 2021;17:989-1002. © 2021 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Estimating Perceptual Range of Female Monarch Butterflies (Danaus plexippus) to Potted Vegetative Common Milkweed (Asclepias syriaca) and Blooming Nectar Resources.

Habitat loss in the summer breeding range contributes to eastern North American monarch (Lepidoptera: Nymphalidae) population decline. Habitat restoration efforts include increasing native prairie plants for adult forage and milkweed (Asclepias spp.) for oviposition and larval development. As the monarch is a vagile species, habitat establishment at a grain that matches the monarch perceptual range will facilitate efficient movement, decrease fitness costs of dispersal, and increase oviposition. We released 188 experimental monarch females 5, 25, 50, and 75 m downwind from potted milkweed and blooming forbs in 4-32 ha sod fields. Perceptual range was estimated from monarchs that flew towards and landed on the milkweed and forbs. Flight patterns of 49 non-experimental monarchs that landed on the resources were also observed. In our experimental, resource-devoid setting, wind-facilitated movement occurred most frequently. Monarchs performed direct displacement as evidenced by shallow turn angles and similarity of Euclidian and total distances traveled. We hypothesize similar monarch flight behavior when traveling over other resource-devoid areas, such as crop fields. Although the majority of experimental monarchs flew downwind, eight experimental and 49 non-experimental monarchs were observed flying upwind toward, and landing on, the potted resources from distances ranging from 3 to 125 m (mean = 30.98 m, median = 25 m, mode = 25 m). A conservative estimate of the perceptual range is 125 m, as longer distances cannot be precluded; however, the majority of observations were ≤50 m. Our findings suggest establishing habitat patches ~ 50 m apart would create functional connectivity across fragmented agricultural landscapes.

Neonicotinoids can cause arrested pupal ecdysis in Lepidoptera.

Recently, we reported a novel mode of action in monarch butterfly (Danaus plexippus) larvae exposed to neonicotinoid insecticides: arrest in pupal ecdysis following successful larval ecdysis. In this paper, we explore arrested pupal ecdysis in greater detail and propose adverse outcome pathways to explain how neonicotinoids cause this effect. Using imidacloprid as a model compound, we determined that final-instar monarchs, corn earworms (Helicoverpa zea), and wax moths (Galleria mellonella) showed high susceptibility to arrested pupal ecdysis while painted ladies (Vanessa cardui) and red admirals (Vanessa atalanta) showed low susceptibility. Fall armyworms (Spodoptera frugiperda) and European corn borers (Ostrinia nubilalis) were recalcitrant. All larvae with arrested ecdysis developed pupal cuticle, but with incomplete shedding of larval cuticle and unexpanded pupal appendages; corn earworm larvae successfully developed into adults with unexpanded appendages. Delayed initiation of pupal ecdysis was also observed with treated larvae. Imidacloprid exposure was required at least 26 h prior to pupal ecdysis to disrupt the molt. These observations suggest neonicotinoids may disrupt the function of crustacean cardioactive peptide (CCAP) neurons, either by directly acting on their nicotinic acetylcholine receptors or by acting on receptors of inhibitory neurons that regulate CCAP activity.

Use of Porous Polystyrene Scaffolds to Bioengineer Human Epithelial Tissues In Vitro.

In vitro epithelial models are valuable tools for both academic and industrial laboratories to investigate tissue physiology and disease. Epithelial tissues comprise the surface epithelium, basement membrane, and underlying supporting stromal cells. There are various types of epithelial tissue and they have a diverse and intricate architecture in vivo, which cannot be successfully recapitulated using two-dimensional (2D) cell culture. Tissue engineering strategies can be applied to bioengineer the organized, multilayered, and multicellular structure of epithelial tissues in vitro. Alvetex® is a porous, polystyrene scaffold that enables fibroblasts to synthesize a complex network of endogenous, humanized extracellular matrix proteins. This creates a physiologically relevant three-dimensional (3D) subepithelial microenvironment, enriched with mechanical and chemical cues, which supports the organization and differentiation of epithelial cells. Such technology has been used to bioengineer different epithelial architectures in vitro, including the simple, columnar structure of the intestine and the stratified, squamous, and keratinized structure of skin. Epithelial tissue models provide a useful platform for fundamental and translational research, with multifaceted applications including disease modeling, drug discovery, and product development.

Evaluating toxicity of Varroa mite (Varroa destructor)-active dsRNA to monarch butterfly (Danaus plexippus) larvae.

Varroa mites (Varroa destructor) are parasitic mites that, combined with other factors, are contributing to high levels of honey bee (Apis mellifera) colony losses. A Varroa-active dsRNA was recently developed to control Varroa mites within honey bee brood cells. This dsRNA has 372 base pairs that are homologous to a sequence region within the Varroa mite calmodulin gene (cam). The Varroa-active dsRNA also shares a 21-base pair match with monarch butterfly (Danaus plexippus) calmodulin mRNA, raising the possibility of non-target effects if there is environmental exposure. We chronically exposed the entire monarch larval stage to common (Asclepias syriaca) and tropical (Asclepias curassavica) milkweed leaves treated with concentrations of Varroa-active dsRNA that are one- and ten-fold higher than those used to treat honey bee hives. This corresponded to concentrations of 0.025-0.041 and 0.211-0.282 mg/g leaf, respectively. Potassium arsenate and a previously designed monarch-active dsRNA with a 100% base pair match to the monarch v-ATPase A mRNA (leaf concentration was 0.020-0.034 mg/g) were used as positive controls. The Varroa mite and monarch-active dsRNA's did not cause significant differences in larval mortality, larval or pupal development, pupal weights, or adult eclosion rates when compared to negative controls. Irrespective of control or dsRNA treatment, larvae that consumed approximately 7500 to 10,500-mg milkweed leaf within 10 to 12 days had the highest pupal weights. The lack of mortality and sublethal effects following dietary exposure to dsRNA with 21-base pair and 100% base pair match to mRNAs that correspond to regulatory genes suggest monarch mRNA may be refractory to silencing by dsRNA or monarch dsRNase may degrade dsRNA to a concentration that is insufficient to silence mRNA signaling.

Oviposition Response of Monarch Butterfly (Lepidoptera: Nymphalidae) to Imidacloprid-Treated Milkweed.

Monarch butterfly (Danaus plexippus) populations have declined over the last two decades, attributable in part to declines in its larval host plant, milkweed (Asclepias spp.), across its breeding range. Conservation efforts in the United States call for restoration of 1.3 billion milkweed stems into the Midwestern landscape. Reaching this goal will require habitat establishment in marginal croplands, where there is a high potential for exposure to agrochemicals. Corn and soybean crops may be treated with neonicotinoid insecticides systemically or through foliar applications to provide protection against insect pests. Here, we investigate whether ovipositing monarchs discriminate against milkweed plants exposed to the neonicotinoid insecticide imidacloprid, either systemically or through foliar application. In our first experiment, we placed gravid females in enclosures containing a choice of two cut stems for oviposition: one in 15 ml of a 0.5 mg/ml aqueous solution of imidacloprid and one in 15 ml water. In a second experiment, females were given a choice of milkweed plants whose leaves were treated with 30 µl of a 0.825 mg/ml imidacloprid-surfactant solution or plants treated with surfactant alone. To evaluate oviposition preference, we counted and removed eggs from all plants daily for 3 d. We also collected video data on a subset of butterflies to evaluate landing behavior. Results indicate that neither systemic nor foliar treatment with imidacloprid influenced oviposition behavior in female monarchs. The implications of these findings for monarch conservation practices will be informed by the results of ongoing egg and larval toxicity studies.

Monarch Butterfly (Danaus plexippus) Life-Stage Risks from Foliar and Seed-Treatment Insecticides.

Conservation of North America's eastern monarch butterfly (Danaus plexippus) population would require establishment of milkweed (Asclepias spp.) and nectar plants in the agricultural landscapes of the north central United States. A variety of seed-treatment and foliar insecticides are used to manage early- and late-season pests in these landscapes. Thus, there is a need to assess risks of these insecticides to monarch butterfly life stages to inform habitat conservation practices. Chronic and acute dietary toxicity studies were undertaken with larvae and adults, and acute topical bioassays were conducted with eggs, pupae, and adults using 6 representative insecticides: beta-cyfluthrin (pyrethroid), chlorantraniliprole (anthranilic diamide), chlorpyrifos (organophosphate), imidacloprid, clothianidin, and thiamethoxam (neonicotinoids). Chronic dietary median lethal concentration values for monarch larvae ranged from 1.6 × 10-3 (chlorantraniliprole) to 5.3 (chlorpyrifos) µg/g milkweed leaf, with the neonicotinoids producing high rates of arrested pupal ecdysis. Chlorantraniliprole and beta-cyfluthrin were generally the most toxic insecticides to all life stages, and thiamethoxam and chlorpyrifos were generally the least toxic. The toxicity results were compared to insecticide exposure estimates derived from a spray drift model and/or milkweed residue data reported in the literature. Aerial applications of foliar insecticides are expected to cause high downwind mortality in larvae and eggs, with lower mortality predicted for adults and pupae. Neonicotinoid seed treatments are expected to cause little to no downslope mortality and/or sublethal effects in larvae and adults. Given the vagile behavior of nonmigratory monarchs, considering these results within a landscape-scale context suggests that adult recruitment will not be negatively impacted if new habitat is established in close proximity of maize and soybean fields in the agricultural landscapes of the north central United States. Environ Toxicol Chem 2021;40:1761-1777. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Meeting the common needs of a more effective and efficient testing and assessment paradigm for chemical risk management.

Significant advances have been made in human health and ecological risk assessment over the last decade. Substantial challenges, however, remain in providing credible scientific information in a timely and efficient manner to support chemical risk assessment and management decisions. A major challenge confronting risk managers is the need for critical information to address risk uncertainties in large chemical inventories such as high- and medium-production-volume industrial chemicals or pesticide inert ingredients. From a strategic and tactical viewpoint, an integrated approach that relies on all existing knowledge and uses a range of methods, including those from emerging and novel technologies, is needed to advance progressive and focused testing strategies, as well as to advance the utility and predictability of the risk assessment by providing more relevant information. A hypothesis-based approach that draws on all relevant information is consistent with the vision articulated in the 2007 report by the National Research Council, Toxicity Testing in the 21st Century: A Vision and a Strategy. This article describes the current practices in evaluating chemical risks and ongoing efforts to enhance the quality and efficiency of risk assessment and risk management decisions within the Office of Prevention, Pesticides, and Toxic Substances at the U.S. Environmental Protection Agency.

Prediction of mitochondrial genome-wide variation through sequencing of mitochondrion-enriched extracts.

Although mitochondrial DNA (mtDNA) haplotype variation is often applied for estimating population dynamics and phylogenetic relationships, economical and generalized methods for entire mtDNA genome enrichment prior to high-throughput sequencing are not readily available. This study demonstrates the utility of differential centrifugation to enrich for mitochondrion within cell extracts prior to DNA extraction, short-read sequencing, and assembly using exemplars from eight maternal lineages of the insect species, Ostrinia nubilalis. Compared to controls, enriched extracts showed a significant mean increase of 48.2- and 86.1-fold in mtDNA based on quantitative PCR, and proportion of subsequent short sequence reads that aligned to the O. nubilalis reference mitochondrial genome, respectively. Compared to the reference genome, our de novo assembled O. nubilalis mitochondrial genomes contained 82 intraspecific substitution and insertion/deletion mutations, and provided evidence for correction of mis-annotated 28 C-terminal residues within the NADH dehydrogenase subunit 4. Comparison to a more recent O. nubilalis mtDNA assembly from unenriched short-read data analogously showed 77 variant sites. Twenty-eight variant positions, and a triplet ATT codon (Ile) insertion within ATP synthase subunit 8, were unique within our assemblies. This study provides a generalizable pipeline for whole mitochondrial genome sequence acquisition adaptable to applications across a range of taxa.

Employing Very High Frequency (VHF) Radio Telemetry to Recreate Monarch Butterfly Flight Paths.

The overwintering population of eastern North American monarch butterflies (Danaus plexippus) has declined significantly. Loss of milkweed (Asclepias sp.), the monarch's obligate host plant in the Midwest United States, is considered to be a major cause of the decline. Restoring breeding habitat is an actionable step towards population recovery. Monarch butterflies are highly vagile; therefore, the spatial arrangement of milkweed in the landscape influences movement patterns, habitat utilization, and reproductive output. Empirical studies of female movement patterns within and between habitat patches in representative agricultural landscapes support recommendations for habitat restoration. To track monarch movement at distances beyond human visual range, we employed very high frequency radio telemetry with handheld antennae to collect movement bearings on a biologically relevant time scale. Attachment of 220-300 mg transmitters did not significantly affect behavior and flight capability. Thirteen radio-tagged monarchs were released in a restored prairie, and locations were estimated every minute for up to 39 min by simultaneous triangulation from four operators. Monarchs that left the prairie were tracked and relocated at distances up to 250 m. Assuming straight flights between locations, the majority of steps within the prairie were below 50 m. Steps associated with exiting the prairie exceeded 50 m with high directionality. Because butterflies do not fly in straight lines between stationary points, we also illustrate how occurrence models can use location data obtained through radio telemetry to estimate movement within a prairie and over multiple land cover types.

Assessing Field-Scale Risks of Foliar Insecticide Applications to Monarch Butterfly (Danaus plexippus) Larvae.

Establishment and maintenance of milkweed plants (Asclepias spp.) in agricultural landscapes of the north central United States are needed to reverse the decline of North America's eastern monarch butterfly (Danaus plexippus) population. Because of a lack of toxicity data, it is unclear how insecticide use may reduce monarch productivity when milkweed habitat is placed near maize and soybean fields. To assess the potential effects of foliar insecticides, acute cuticular and dietary toxicity of 5 representative active ingredients were determined: beta-cyfluthrin (pyrethroid), chlorantraniliprole (anthranilic diamide), chlorpyrifos (organophosphate), and imidacloprid and thiamethoxam (neonicotinoids). Cuticular median lethal dose values for first instars ranged from 9.2 × 10-3 to 79 µg/g larvae for beta-cyfluthrin and chlorpyrifos, respectively. Dietary median lethal concentration values for second instars ranged from 8.3 × 10-3 to 8.4 µg/g milkweed leaf for chlorantraniliprole and chlorpyrifos, respectively. To estimate larval mortality rates downwind from treated fields, modeled insecticide exposures to larvae and milkweed leaves were compared to dose-response curves obtained from bioassays with first-, second-, third-, and fifth-instar larvae. For aerial applications to manage soybean aphids, mortality rates at 60 m downwind were highest for beta-cyfluthrin and chlorantraniliprole following cuticular and dietary exposure, respectively, and lowest for thiamethoxam. To estimate landscape-scale risks, field-scale mortality rates must be considered in the context of spatial and temporal patterns of insecticide use. Environ Toxicol Chem 2020;39:923-941. © 2020 SETAC.

Engineering a Multilayered Skin Equivalent: The Importance of Endogenous Extracellular Matrix Maturation to Provide Robustness and Reproducibility.

Human skin equivalents (HSEs) are a valuable tool for both academic and industrial laboratories to further the understanding of skin physiology and associated diseases. Over the last few decades, there have been many advances in the development of HSEs that successfully recapitulate the structure of human skin in vitro; however a main limitation is variability due to the use of complex protocols and exogenous extracellular matrix (ECM) proteins. We have developed a robust and unique full-thickness skin equivalent that is highly reproducible due to the use of a consistent scaffold, commercially available cells, and defined low-serum media. The Alvetex® scaffold technology allows fibroblasts to produce their own endogenous ECM proteins within the scaffold, which alleviates the need for exogenous collagen, and supports the differentiation and stratification of the epidermis. Our full-thickness skin equivalent is generated using a detailed step-by-step protocol, which sequentially forms the multilayered structure of human skin in vitro. This model can be adapted for many downstream applications such as disease modeling and testing of active compounds for cosmetics.

Endogenous Production of IL1B by Breast Cancer Cells Drives Metastasis and Colonization of the Bone Microenvironment.

PURPOSE: Breast cancer bone metastases are incurable, highlighting the need for new therapeutic targets. After colonizing bone, breast cancer cells remain dormant, until signals from the microenvironment stimulate outgrowth into overt metastases. Here we show that endogenous production of IL1B by tumor cells drives metastasis and growth in bone. EXPERIMENTAL DESIGN: Tumor/stromal IL1B and IL1 receptor 1 (IL1R1) expression was assessed in patient samples and effects of the IL1R antagonist, Anakinra, or the IL1B antibody canakinumab on tumor growth and spontaneous metastasis were measured in a humanized mouse model of breast cancer bone metastasis. Effects of tumor cell-derived IL1B on bone colonization and parameters associated with metastasis were measured in MDA-MB-231, MCF7, and T47D cells transfected with IL1B/control. RESULTS: In tissue samples from >1,300 patients with stage II/III breast cancer, IL1B in tumor cells correlated with relapse in bone (HR = 1.85; 95% CI, 1.05-3.26; P = 0.02) and other sites (HR = 2.09; 95% CI, 1.26-3.48; P = 0.0016). In a humanized model of spontaneous breast cancer metastasis to bone, Anakinra or canakinumab reduced metastasis and reduced the number of tumor cells shed into the circulation. Production of IL1B by tumor cells promoted epithelial-to-mesenchymal transition (altered E-Cadherin, N-Cadherin, and G-Catenin), invasion, migration, and bone colonization. Contact between tumor and osteoblasts or bone marrow cells increased IL1B secretion from all three cell types. IL1B alone did not stimulate tumor cell proliferation. Instead, IL1B caused expansion of the bone metastatic niche leading to tumor proliferation. CONCLUSIONS: Pharmacologic inhibition of IL1B has potential as a novel treatment for breast cancer metastasis.

IL-1 drives breast cancer growth and bone metastasis in vivo.

BACKGROUND: We have recently identified interleukin 1B (IL-1B) as a potential biomarker for predicting breast cancer patients at increased risk for developing bone metastasis. In mouse models, IL-1B and its receptor (IL-1R1) are upregulated in breast cancer cells that metastasise to bone compared with cells that do not. We have now investigated the functional role of IL-1 by blocking IL-1R signalling with the clinically licensed antagonist, anakinra. METHODOLOGY: 6-week old female BALB/c mice received a subcutaneous or intra-venous injection of MDA-MB-231-IV or MCF7 cells. Anakinra (1mg/kg/day) or placebo was administered 3 days before (preventative) or 7 days later (treatment). Tumour volume, apoptosis (TUNEL, Caspase 3), proliferation (Ki67) and angiogenesis (CD34, VEGF and endothelin) were analysed. Effects on bone were measured by uCT, and TRAP, P1NP, IL-1B, TNF alpha and IL-6 ELISA. RESULTS: Anakinra significantly reduced growth of MDA-MB-231-IV tumours in bone from 6.50+/3.00mm2 (placebo) to 2.56+/-1.07mm2 (treatment) and 0.63+/-0.18mm2 (preventative). Anakinra also reduced the number of mice that developed bone metastasis from 90% (placebo) to 40% (treatment) and 10% (preventative). Anti-tumour effects were not confined to bone, subcutaneous tumour volumes reduced from 656.68mm3 (placebo) to 160.47mm3 (treatment) and 31.08mm3 (preventative). Anakinra did not increase tumour cell apoptosis but reduced proliferation and angiogenesis in addition to exerting significant effects on the tumour environment reducing bone turnover markers, IL-1B and TNF alpha. CONCLUSIONS: Our novel data demonstrate a functional role of IL-1 signalling in breast tumour progression and metastasis, supporting that anakinra could be repurposed for the treatment of breast cancer bone metastasis.

Influence of suspended solids on acute toxicity of carbofuran to Daphnia magna: I. Interactive effects.

This study explored the effects on Daphnia magna from exposure to the pesticide carbofuran in combination with stress from suspended solids exposure. Our objective was to assess whether suspended solids affects the toxicodynamic response of D. magna to carbofuran. A series of laboratory experiments was performed where animals were exposed to carbofuran concentrations ranging from 0 to 160 microg/l in combination with suspended solids concentrations ranging from 0 to 10000 mg/l. In the absence of suspended solids, effects of carbofuran were dose dependent and resulted in an EC(50) of 92 microg/l. Exposure to suspended solids, up to extreme levels that may be encountered in the environment and in the absence of carbofuran, showed no measurable toxicity. When D. magna were exposed to a constant carbofuran concentration, the numbers of affected organisms increased with increasing suspended solids concentrations. At a suspended solids concentration of 1000 mg/l, the EC(50) for carbofuran was reduced by half to 45 microg/l. The relationship between the toxicity of carbofuran (microg/l) and the concentration of suspended solids (mg/l) can be described with the following equation: carbofuran EC(50)=72 exp(-0.00014 [suspended solids]). An analysis of the data indicates that this relationship is consistent with a potentiated toxicity mechanism rather than an additive model.