Combining resources to obtain a comprehensive survey of the bovine embryo transcriptome through deep sequencing and microarrays.

While most assisted reproductive technologies (ART) are considered routine for the reproduction of species of economical importance, such as the bovine, the impact of these manipulations on the developing embryo remains largely unknown. In an effort to obtain a comprehensive survey of the bovine embryo transcriptome and how it is modified by ART, resources were combined to design an embryo-specific microarray. Close to one million high-quality reads were produced from subtracted bovine embryo libraries using Roche 454 Titanium deep sequencing technology, which enabled the creation of an augmented bovine genome catalog. This catalog was enriched with bovine embryo transcripts, and included newly discovered indel type and 3'UTR variants. Using this augmented bovine genome catalog, the EmbryoGENE Bovine Microarray was designed and is composed of a total of 42,242 probes, including 21,139 known reference genes; 9,322 probes for novel transcribed regions (NTRs); 3,677 alternatively spliced exons; 3,353 3'-tiling probes; and 3,723 controls. A suite of bioinformatics tools was also developed to facilitate microrarray data analysis and database creation; it includes a quality control module, a Laboratory Information Management System (LIMS) and microarray analysis software. Results obtained during this study have already led to the identification of differentially expressed blastocyst targets, NTRs, splice variants of the indel type, and 3'UTR variants. We were able to confirm microarray results by real-time PCR, indicating that the EmbryoGENE bovine microarray has the power to detect physiologically relevant changes in gene expression.

Optimized protocol for immunophenotyping of melanoma and tumor-bearing skin from mouse.

While isolating immune cells from spleens and lungs is routinely achieved using flow cytometry, it is challenging to isolate viable immune cells from skin. Here, we describe a step-by-step protocol for skin digestion using a murine melanoma model, which is amenable for detection of low abundant immune cell populations including group 2 innate lymphoid cells.

Tungsten Increases Sex-Specific Osteoclast Differentiation in Murine Bone.

Tungsten is a naturally occurring metal that is increasingly used in industry and medical devices, and is labeled as an emerging environmental contaminant. Like many metals, tungsten accumulates in bone. Our previous data indicate that tungsten decreases differentiation of osteoblasts, bone-forming cells. Herein, we explored the impact of tungsten on osteoclast differentiation, which function in bone resorption. We observed significantly elevated osteoclast numbers in the trabecular bone of femurs following oral exposure to tungsten in male, but not female mice. In order to explore the mechanism(s) by which tungsten increases osteoclast number, we utilized in vitro murine primary and cell line pre-osteoclast models. Although tungsten did not alter the adhesion of osteoclasts to the extracellular matrix protein, vitronectin, we did observe that tungsten enhanced RANKL-induced differentiation into tartrate-resistant acid phosphatase (TRAP)-positive mononucleated osteoclasts. Importantly, tungsten alone had no effect on differentiation or on the number of multinucleated TRAP-positive osteoclasts. Enhanced RANKL-induced differentiation correlated with increased gene expression of differentiated osteoclast markers Nfatc1, Acp5, and Ctsk. Although tungsten did not alter the RANK surface receptor expression, it did modulate its downstream signaling. Co-exposure of tungsten and RANKL resulted in sustained positive p38 signaling. These findings demonstrate that tungsten enhances sex-specific osteoclast differentiation, and together with previous findings of decreased osteoblastogenesis, implicate tungsten as a modulator of bone homeostasis.

Sex-Specific Effects of Prenatal and Early Life Inorganic and Methylated Arsenic Exposure on Atherosclerotic Plaque Development and Composition in Adult ApoE-/- Mice.

BACKGROUND: Epidemiologic studies indicate that early life arsenic exposures are linked to an increased risk of cardiovascular diseases. Different oxidation and methylation states of arsenic exist in the environment and are formed in vivo via the action of arsenic (+3 oxidation state) methyltransferase (As3MT). Methylated arsenicals are pro-atherogenic postnatally, but pre- and perinatal effects are unclear. This is particularly important because methylated arsenicals are known to cross the placenta. OBJECTIVES: We tested the effects of early life exposure to inorganic and methylated arsenicals on atherosclerotic plaque formation and its composition in apolipoprotein E knock-out (apoE-/-) mice and evaluated whether apoE-/- mice lacking As3MT expression were susceptible to this effect. METHODS: We exposed apoE-/- or apoE-/-/As3MT-/- mice to 200 ppb inorganic or methylated arsenic in the drinking water from conception to weaning and assessed atherosclerotic plaques in the offspring at 18 wk of age. Mixed regression models were used to estimate the mean difference in each outcome relative to controls, adjusting for sex and including a random effects term to account for within-litter clustering. RESULTS: Early life exposure to inorganic arsenic, and more profoundly methylated arsenicals, resulted in significantly larger plaques in the aortic arch and sinus in both sexes. Lipid levels in these plaques were higher without a substantial difference in macrophage numbers. Smooth muscle cell content was not altered, but collagen content was lower. Importantly, there were sex-specific differences in these observations, where males had higher lipids and lower collagen in the plaque, but females did not. In mice lacking As3MT, arsenic did not alter the plaque size, although the size was highly variable. In addition, control apoE-/-/As3MT-/- mice had significantly larger plaque size compared with control apoE-/-. CONCLUSION: This study shows that early life exposure to inorganic and methylated arsenicals is pro-atherogenic with sex-specific differences in plaque composition and a potential role for As3MT in mice. https://doi.org/10.1289/EHP8171.

The MNK1/2-eIF4E Axis Supports Immune Suppression and Metastasis in Postpartum Breast Cancer.

Breast cancer diagnosed within 10 years following childbirth is defined as postpartum breast cancer (PPBC) and is highly metastatic. Interactions between immune cells and other stromal cells within the involuting mammary gland are fundamental in facilitating an aggressive tumor phenotype. The MNK1/2-eIF4E axis promotes translation of prometastatic mRNAs in tumor cells, but its role in modulating the function of nontumor cells in the PPBC microenvironment has not been explored. Here, we used a combination of in vivo PPBC models and in vitro assays to study the effects of inactivation of the MNK1/2-eIF4E axis on the protumor function of select cells of the tumor microenvironment. PPBC mice deficient for phospho-eIF4E (eIF4ES209A) were protected against lung metastasis and exhibited differences in the tumor and lung immune microenvironment compared with wild-type mice. Moreover, the expression of fibroblast-derived IL33, an alarmin known to induce invasion, was repressed upon MNK1/2-eIF4E axis inhibition. Imaging mass cytometry on PPBC and non-PPBC patient samples indicated that human PPBC contains phospho-eIF4E high-expressing tumor cells and CD8+ T cells displaying markers of an activated dysfunctional phenotype. Finally, inhibition of MNK1/2 combined with anti-PD-1 therapy blocked lung metastasis of PPBC. These findings implicate the involvement of the MNK1/2-eIF4E axis during PPBC metastasis and suggest a promising immunomodulatory route to enhance the efficacy of immunotherapy by blocking phospho-eIF4E. SIGNIFICANCE: This study investigates the MNK1/2-eIF4E signaling axis in tumor and stromal cells in metastatic breast cancer and reveals that MNK1/2 inhibition suppresses metastasis and sensitizes tumors to anti-PD-1 immunotherapy.

Inhibiting the MNK1/2-eIF4E axis impairs melanoma phenotype switching and potentiates antitumor immune responses.

Melanomas commonly undergo a phenotype switch, from a proliferative to an invasive state. Such tumor cell plasticity contributes to immunotherapy resistance; however, the mechanisms are not completely understood and thus are therapeutically unexploited. Using melanoma mouse models, we demonstrated that blocking the MNK1/2-eIF4E axis inhibited melanoma phenotype switching and sensitized melanoma to anti-PD-1 immunotherapy. We showed that phospho-eIF4E-deficient murine melanomas expressed high levels of melanocytic antigens, with similar results verified in patient melanomas. Mechanistically, we identified phospho-eIF4E-mediated translational control of NGFR, a critical effector of phenotype switching. Genetic ablation of phospho-eIF4E reprogrammed the immunosuppressive microenvironment, exemplified by lowered production of inflammatory factors, decreased PD-L1 expression on dendritic cells and myeloid-derived suppressor cells, and increased CD8+ T cell infiltrates. Finally, dual blockade of the MNK1/2-eIF4E axis and the PD-1/PD-L1 immune checkpoint demonstrated efficacy in multiple melanoma models regardless of their genomic classification. An increase in the presence of intratumoral stem-like TCF1+PD-1+CD8+ T cells, a characteristic essential for durable antitumor immunity, was detected in mice given a MNK1/2 inhibitor and anti-PD-1 therapy. Using MNK1/2 inhibitors to repress phospho-eIF4E thus offers a strategy to inhibit melanoma plasticity and improve response to anti-PD-1 immunotherapy.

MNK1 signaling induces an ANGPTL4-mediated gene signature to drive melanoma progression.

The BRAFV600E mutation occurs in more than 50% of cutaneous melanomas, and results in the constitutive activation of the mitogen-activated protein kinases (MAPK) pathway. MAP kinase-interacting serine/threonine-protein kinase 1 and 2 (MNK1/2) are downstream effectors of the activated MAPK pathway, and important molecular targets in invasive and metastatic cancer. Despite the well-known role of MNK1 in regulating mRNA translation, little is known concerning the impact of its aberrant activation on gene transcription. Here, we show that changes in the activity, or abundance, of MNK1 result in changes in the expression of pro-oncogenic and pro-invasive genes. Among the MNK1-upregulated genes, we identify Angiopoietin-like 4 (ANGPTL4), which in turn promotes an invasive phenotype via its ability to induce the expression of matrix metalloproteinases (MMPs). Using a pharmacologic inhibitor of MNK1/2, SEL201, we demonstrate that BRAFV600E-mutated cutaneous melanoma cells are reliant on MNK1/2 for invasion and lung metastasis.

Tungsten Blocks Murine B Lymphocyte Differentiation and Proliferation Through Downregulation of IL-7 Receptor/Pax5 Signaling.

Tungsten is an emerging environmental toxicant associated with several pediatric leukemia clusters, although a causal association has not been established. Our previous work demonstrated that tungsten exposure resulted in an accumulation of pre-B cells in the bone marrow, the same cell type that accumulates in pediatric acute lymphoblastic leukemia (ALL). To better understand the relevant molecular mechanisms, we performed RNA-sequencing on flow sorted pre-B cells from control and tungsten-exposed mice. Tungsten decreased the expression of multiple genes critical for B cell development, including members of the interleukin-7 receptor (IL-7R) and pre-B cell receptor signaling pathways, such as Jak1, Stat5a, Pax5, Syk, and Ikzf3. These results were confirmed in an in vitro model of B cell differentiation, where tungsten arrested differentiation at the pro-B cell stage and inhibited proliferation. These changes were associated with decreased expression of multiple genes in the IL-7R signaling pathway and decreased percentage of IL-7R, phosphorylated STAT5 double-positive cells. Supplementation with IL-7 or overexpression of Pax5, the transcription factor downstream of IL-7R, rescued the tungsten-induced differentiation block. Together, these data support the hypothesis that IL-7R/Pax5 signaling axis is critical to tungsten-mediated effects on pre-B cell development. Importantly, many of these molecules are modulated in ALL.

MNK1/NODAL Signaling Promotes Invasive Progression of Breast Ductal Carcinoma In Situ.

The mechanisms by which breast cancers progress from relatively indolent ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC) are not well understood. However, this process is critical to the acquisition of metastatic potential. MAPK-interacting serine/threonine-protein kinase 1 (MNK1) signaling can promote cell invasion. NODAL, a morphogen essential for embryogenic patterning, is often reexpressed in breast cancer. Here we describe a MNK1/NODAL signaling axis that promotes DCIS progression to IDC. We generated MNK1 knockout (KO) or constitutively active MNK1 (caMNK1)-expressing human MCF-10A-derived DCIS cell lines, which were orthotopically injected into the mammary glands of mice. Loss of MNK1 repressed NODAL expression, inhibited DCIS to IDC conversion, and decreased tumor relapse and metastasis. Conversely, caMNK1 induced NODAL expression and promoted IDC. The MNK1/NODAL axis promoted cancer stem cell properties and invasion in vitro. The MNK1/2 inhibitor SEL201 blocked DCIS progression to invasive disease in vivo. In clinical samples, IDC and DCIS with microinvasion expressed higher levels of phospho-MNK1 and NODAL versus low-grade (invasion-free) DCIS. Cumulatively, our data support further development of MNK1 inhibitors as therapeutics for preventing invasive disease. SIGNIFICANCE: These findings provide new mechanistic insight into progression of ductal carcinoma and support clinical application of MNK1 inhibitors to delay progression of indolent ductal carcinoma in situ to invasive ductal carcinoma.

Using the Apolipoprotein E Knock-Out Mouse Model to Define Atherosclerotic Plaque Changes Induced by Low Dose Arsenic.

Arsenic exposure increases the risk of atherosclerosis, the gradual occlusion of the large arteries with fibro-fatty plaque. While epidemiologic data provide convincing evidence this is true at higher exposures, it is unclear whether this may occur at low arsenic exposures, near the maximum contaminant level of 10 ppb. We have previously shown that 200 ppb arsenite in the drinking water increased the atherosclerosis in apolipoprotein E knock-out (apoE-/-) mice after 13 weeks, but the effects of lower concentrations were unknown. Therefore, here, we analyzed the effects of oral exposure to arsenite from 10 to 200 ppb after 13 weeks. Importantly, we found that even at the lowest concentration of arsenite, there was a significant increase in atherosclerotic plaque size. In our previous studies, we found that arsenite exposure resulted in decreased smooth muscle cells (SMCs) and collagen within the plaque. This change is indicative of a less stable phenotype that could increase the risk of rupture and subsequently, myocardial infarct or stroke in humans. In addition, we observed that lipid increased within the plaque without concomitant increase in macrophage content, suggesting that the macrophages were retaining more lipid intracellularly. We also assessed these plaque components in apoE-/- mice exposed to 10-200 ppb arsenite. Interestingly, we observed that macrophage lipid accumulation occurred at lower concentrations than the decreased SMC/collagen content. Together these data suggest that in the apoE-/- model, low arsenite concentrations are pro-atherogenic and that macrophage lipid homeostasis is more sensitive to arsenite-induced perturbation than the SMCs.

Effects of Inorganic Arsenic, Methylated Arsenicals, and Arsenobetaine on Atherosclerosis in the Mouse Model and the Role of As3mt-Mediated Methylation.

BACKGROUND: Arsenic is metabolized through a series of oxidative methylation reactions by arsenic (3) methyltransferase (As3MT) to yield methylated intermediates. Although arsenic exposure is known to increase the risk of atherosclerosis, the contribution of arsenic methylation and As3MT remains undefined. OBJECTIVES: Our objective was to define whether methylated arsenic intermediates were proatherogenic and whether arsenic biotransformation by As3MT was required for arsenic-enhanced atherosclerosis. METHODS: We utilized the apoE−/− mouse model to compare atherosclerotic plaque size and composition after inorganic arsenic, methylated arsenical, or arsenobetaine exposure in drinking water. We also generated apoE−/−/As3mt−/− double knockout mice to test whether As3MT-mediated biotransformation was required for the proatherogenic effects of inorganic arsenite. Furthermore, As3MT expression and function were assessed in in vitro cultures of plaque-resident cells. Finally, bone marrow transplantation studies were performed to define the contribution of As3MT-mediated methylation in different cell types to the development of atherosclerosis after inorganic arsenic exposure. RESULTS: We found that methylated arsenicals, but not arsenobetaine, are proatherogenic and that As3MT is required for arsenic to induce reactive oxygen species and promote atherosclerosis. Importantly, As3MT was expressed and functional in multiple plaque-resident cell types, and transplant studies indicated that As3MT is required in extrahepatic tissues to promote atherosclerosis. CONCLUSION: Taken together, our findings indicate that As3MT acts to promote cardiovascular toxicity of arsenic and suggest that human AS3MT SNPs that correlate with enzyme function could predict those most at risk to develop atherosclerosis among the millions that are exposed to arsenic. https://doi.org/10.1289/EHP806.

Tungsten Promotes Sex-Specific Adipogenesis in the Bone by Altering Differentiation of Bone Marrow-Resident Mesenchymal Stromal Cells.

Tungsten is a naturally occurring metal that increasingly is being incorporated into industrial goods and medical devices, and is recognized as an emerging contaminant. Tungsten preferentially and rapidly accumulates in murine bone in a concentration-dependent manner; however the effect of tungsten deposition on bone biology is unknown. Other metals alter bone homeostasis by targeting bone marrow-derived mesenchymal stromal cell (MSC) differentiation, thus, we investigated the effects of tungsten on MSCsin vitroandin vivoIn vitro, tungsten shifted the balance of MSC differentiation by enhancing rosiglitazone-induced adipogenesis, which correlated with an increase in adipocyte content in the bone of tungsten-exposed, young, male mice. Conversely, tungsten inhibited osteogenesis of MSCsin vitro; however, we found no evidence that tungsten inhibited osteogenesisin vivo Interestingly, two factors known to influence adipogenesis are sex and age of mice. Both female and older mice have enhanced adipogenesis. We extended our study and exposed young female and adult (9-month) male and female mice to tungsten for 4 weeks. Although tungsten accumulated to a similar extent in young female mice, it did not promote adipogenesis. Interestingly, tungsten did not accumulate in the bone of older mice; it was undetectable in adult male mice, and just above the limit of detect in adult female mice. Surprisingly, tungsten enhanced adipogenesis in adult female mice. In summary, we found that tungsten alters bone homeostasis by altering differentiation of MSCs, which could have significant implications for bone quality, but is highly dependent upon sex and age.

Tungsten targets the tumor microenvironment to enhance breast cancer metastasis.

The number of individuals exposed to high levels of tungsten is increasing, yet there is limited knowledge of the potential human health risks. Recently, a cohort of breast cancer patients was left with tungsten in their breasts following testing of a tungsten-based shield during intraoperative radiotherapy. While monitoring tungsten levels in the blood and urine of these patients, we utilized the 66Cl4 cell model, in vitro and in mice to study the effects of tungsten exposure on mammary tumor growth and metastasis. We still detect tungsten in the urine of patients' years after surgery (mean urinary tungsten concentration at least 20 months post-surgery = 1.76 ng/ml), even in those who have opted for mastectomy, indicating that tungsten does not remain in the breast. In addition, standard chelation therapy was ineffective at mobilizing tungsten. In the mouse model, tungsten slightly delayed primary tumor growth, but significantly enhanced lung metastasis. In vitro, tungsten did not enhance 66Cl4 proliferation or invasion, suggesting that tungsten was not directly acting on 66Cl4 primary tumor cells to enhance invasion. In contrast, tungsten changed the tumor microenvironment, enhancing parameters known to be important for cell invasion and metastasis including activated fibroblasts, matrix metalloproteinases, and myeloid-derived suppressor cells. We show, for the first time, that tungsten enhances metastasis in an animal model of breast cancer by targeting the microenvironment. Importantly, all these tumor microenvironmental changes are associated with a poor prognosis in humans.

Cellular and molecular characterization of the impact of laboratory setup on bovine in vitro embryo production.

One of the main objectives related to performing comparative analysis of embryonic transcriptomes is to share information with other reproductive biologists or commercial service providers. Biological extracts influence performance of in vitro production systems and affect the reproducibility of results between production sites; these sources of variation could impede the potential for knowledge transfer. The objective of the present study was to assess the impact of the production site when sharing a common in vitro embryo production protocol. Biological extracts and semen were shared between production sites and thus removed as potential sources of variation. To remove the impact of blastocyst staging, all comparisons used expanded blastocysts. Although blastocyst yields and the number of Tunel positive cells per embryo differed between production sites, blastocysts were morphologically very similar in regards to cell number, their allocation to either the trophoblast or inner cell mass, or their gender ratio. These observations were also confirmed at the gene expression level, as indicated by highly similar transcript abundances. Only 36 genes out of the 16,121 expressed during bovine prehatching development were statistically differentially expressed, of which a large proportion were associated with the apoptotic process. These results highlighted the impact of laboratory set up, including personnel experience, when replicating an in vitro production system. Although inherent differences may arise, given the similarity of results between production sites, we concluded that embryo production protocols have the potential to be transferred and shared.

Contribution of oocyte source and culture conditions to phenotypic and transcriptomic variation in commercially produced bovine blastocysts.

Bovine embryo production is practiced worldwide for commercial purposes. A major concern of embryo suppliers is the impact of in vitro production systems on embryo quality. In the present study, we compared Buffalo Rat Liver cell coculture with semidefined, medium-based culture, oocytes recovered postmortem with those obtained from live animals, and in vitro with in vivo embryo development. Gene expression levels in expanded blastocysts were measured using microarray and quantitative RT-PCR. The systems were similar in terms of blastocyst yield and rate of development, whereas embryo productivity was greater for immature oocytes collected in vivo. Although immature oocytes collected in vivo had greater developmental competence, they yielded blastocysts that were indistinguishable (in terms of level of gene expression) from embryos derived from immature oocytes recovered postmortem. Culture conditions had a significant impact on gene expression, particularly among genes involved in lipid metabolism. Numerous uncharacterized novel transcript regions were also influenced by in vitro treatments. In conclusion, ovum pick-up combined with in vitro culture in semidefined medium provided a high blastocyst yield, without the deleterious effects associated with coculture.