A susceptibility gene signature for ERBB2-driven mammary tumour development and metastasis in collaborative cross mice.

BACKGROUND: Deeper insights into ERBB2-driven cancers are essential to develop new treatment approaches for ERBB2+ breast cancers (BCs). We employed the Collaborative Cross (CC) mouse model to unearth genetic factors underpinning Erbb2-driven mammary tumour development and metastasis. METHODS: 732 F1 hybrid female mice between FVB/N MMTV-Erbb2 and 30 CC strains were monitored for mammary tumour phenotypes. GWAS pinpointed SNPs that influence various tumour phenotypes. Multivariate analyses and models were used to construct the polygenic score and to develop a mouse tumour susceptibility gene signature (mTSGS), where the corresponding human ortholog was identified and designated as hTSGS. The importance and clinical value of hTSGS in human BC was evaluated using public datasets, encompassing TCGA, METABRIC, GSE96058, and I-SPY2 cohorts. The predictive power of mTSGS for response to chemotherapy was validated in vivo using genetically diverse MMTV-Erbb2 mice. FINDINGS: Distinct variances in tumour onset, multiplicity, and metastatic patterns were observed in F1-hybrid female mice between FVB/N MMTV-Erbb2 and 30 CC strains. Besides lung metastasis, liver and kidney metastases emerged in specific CC strains. GWAS identified specific SNPs significantly associated with tumour onset, multiplicity, lung metastasis, and liver metastasis. Multivariate analyses flagged SNPs in 20 genes (Stx6, Ramp1, Traf3ip1, Nckap5, Pfkfb2, Trmt1l, Rprd1b, Rer1, Sepsecs, Rhobtb1, Tsen15, Abcc3, Arid5b, Tnr, Dock2, Tti1, Fam81a, Oxr1, Plxna2, and Tbc1d31) independently tied to various tumour characteristics, designated as a mTSGS. hTSGS scores (hTSGSS) based on their transcriptional level showed prognostic values, superseding clinical factors and PAM50 subtype across multiple human BC cohorts, and predicted pathological complete response independent of and superior to MammaPrint score in I-SPY2 study. The power of mTSGS score for predicting chemotherapy response was further validated in an in vivo mouse MMTV-Erbb2 model, showing that, like findings in human patients, mouse tumours with low mTSGS scores were most likely to respond to treatment. INTERPRETATION: Our investigation has unveiled many new genes predisposing individuals to ERBB2-driven cancer. Translational findings indicate that hTSGS holds promise as a biomarker for refining treatment strategies for patients with BC. FUNDING: The U.S. Department of Defense (DoD) Breast Cancer Research Program (BCRP) (BC190820), United States; MCIN/AEI/10.13039/501100011039 (PID2020-118527RB-I00, PDC2021-121735-I00), the "European Union Next Generation EU/PRTR," the Regional Government of Castile and León (CSI144P20), European Union.

Genetic and microbial determinants of azoxymethane-induced colorectal tumor susceptibility in Collaborative Cross mice and their implication in human cancer.

The insights into interactions between host genetics and gut microbiome (GM) in colorectal tumor susceptibility (CTS) remains lacking. We used Collaborative Cross mouse population model to identify genetic and microbial determinants of Azoxymethane-induced CTS. We identified 4417 CTS-associated single nucleotide polymorphisms (SNPs) containing 334 genes that were transcriptionally altered in human colorectal cancers (CRCs) and consistently clustered independent human CRC cohorts into two subgroups with different prognosis. We discovered a set of genera in early-life associated with CTS and defined a 16-genus signature that accurately predicted CTS, the majority of which were correlated with human CRCs. We identified 547 SNPs associated with abundances of these genera. Mediation analysis revealed GM as mediators partially exerting the effect of SNP UNC3869242 within Duox2 on CTS. Intestine cell-specific depletion of Duox2 altered GM composition and contribution of Duox2 depletion to CTS was significantly influenced by GM. Our findings provide potential novel targets for personalized CRC prevention and treatment.

Nutrient-sensing growth hormone secretagogue receptor in macrophage programming and meta-inflammation.

OBJECTIVE: Obesity-associated chronic inflammation, aka meta-inflammation, is a key pathogenic driver for obesity-associated comorbidity. Growth hormone secretagogue receptor (GHSR) is known to mediate the effects of nutrient-sensing hormone ghrelin in food intake and fat deposition. We previously reported that global Ghsr ablation protects against diet-induced inflammation and insulin resistance, but the site(s) of action and mechanism are unknown. Macrophages are key drivers of meta-inflammation. To unravel the role of GHSR in macrophages, we generated myeloid-specific Ghsr knockout mice (LysM-Cre;Ghsrf/f). METHODS: LysM-Cre;Ghsrf/f and control Ghsrf/f mice were subjected to 5 months of high-fat diet (HFD) feeding to induce obesity. In vivo, metabolic profiling of food intake, physical activity, and energy expenditure, as well as glucose and insulin tolerance tests (GTT and ITT) were performed. At termination, peritoneal macrophages (PMs), epididymal white adipose tissue (eWAT), and liver were analyzed by flow cytometry and histology. For ex vivo studies, bone marrow-derived macrophages (BMDMs) were generated from the mice and treated with palmitic acid (PA) or lipopolysaccharide (LPS). For in vitro studies, macrophage RAW264.7 cells with Ghsr overexpression or Insulin receptor substrate 2 (Irs2) knockdown were studied. RESULTS: We found that Ghsr expression in PMs was increased under HFD feeding. In vivo, HFD-fed LysM-Cre;Ghsrf/f mice exhibited significantly attenuated systemic inflammation and insulin resistance without affecting food intake or body weight. Tissue analysis showed that HFD-fed LysM-Cre;Ghsrf/f mice have significantly decreased monocyte/macrophage infiltration, pro-inflammatory activation, and lipid accumulation, showing elevated lipid-associated macrophages (LAMs) in eWAT and liver. Ex vivo, Ghsr-deficient macrophages protected against PA- or LPS-induced pro-inflammatory polarization, showing reduced glycolysis, increased fatty acid oxidation, and decreased NF-κB nuclear translocation. At molecular level, GHSR metabolically programs macrophage polarization through PKA-CREB-IRS2-AKT2 signaling pathway. CONCLUSIONS: These novel results demonstrate that macrophage GHSR plays a key role in the pathogenesis of meta-inflammation, and macrophage GHSR promotes macrophage infiltration and induces pro-inflammatory polarization. These exciting findings suggest that GHSR may serve as a novel immunotherapeutic target for the treatment of obesity and its associated comorbidity.

Genetic architecture of the acute and persistent immune cell response after radiation exposure.

Hematologic toxicity is a common side effect of multimodal cancer therapy. Nearly all animal studies investigating the causes of radiotherapy-induced hematologic toxicity use inbred strains with limited genetic diversity and do not reflect the diverse responses observed in humans. We used the population-based Collaborative Cross (CC) mouse resource to investigate the genetic architecture of the acute and persistent immune response after radiation exposure by measuring 22 immune parameters in 1,720 CC mice representing 35 strains. We determined relative acute and persistent radiation resistance scores at the individual strain level considering contributions from all immune parameters. Genome-wide association analysis identified quantitative trait loci associated with baseline and radiation responses. A cross-species radiation resistance score predicted recurrence-free survival in medulloblastoma patients. We present a community resource of immune parameters and genome-wide association analyses before and after radiation exposure for future investigations of the contributions of host genetics on radiosensitivity.

An unanticipated discourse of HB-EGF with VANGL2 signaling during embryo implantation.

Implantation is the first direct encounter between the embryo and uterus during pregnancy, and Hbegf is the earliest known molecular signaling for embryo-uterine crosstalk during implantation. The downstream effectors of heparin-binding EGF (HB-EGF) in implantation remain elusive due to the complexity of EGF receptor family. This study shows that the formation of implantation chamber (crypt) triggered by HB-EGF is disrupted by uterine deletion of Vangl2, a key planar cell polarity component (PCP). We found that HB-EGF binds to ERBB2 and ERBB3 to recruit VANGL2 for tyrosine phosphorylation. Using in vivo models, we show that uterine VAGL2 tyrosine phosphorylation is suppressed in Erbb2/Erbb3 double conditional knockout mice. In this context, severe implantation defects in these mice lend support to the critical role of HB-EGF-ERBB2/3-VANGL2 in establishing a two-way dialogue between the blastocyst and uterus. In addition, the result addresses an outstanding question how VANGL2 is activated during implantation. Taken together, these observations reveal that HB-EGF regulates the implantation process by influencing uterine epithelial cell polarity comprising VANGL2.

Overexpression of IGF-1 During Early Development Expands the Number of Mammary Stem Cells and Primes them for Transformation.

Insulin-like growth factor I (IGF-1) has been implicated in breast cancer due to its mitogenic and anti-apoptotic effects. Despite substantial research on the role of IGF-1 in tumor progression, the relationship of IGF-1 to tissue stem cells, particularly in mammary tissue, and the resulting tumor susceptibility has not been elucidated. Previous studies with the BK5.IGF-1 transgenic (Tg) mouse model reveals that IGF-1 does not act as a classical, post-carcinogen tumor promoter in the mammary gland. Pre-pubertal Tg mammary glands display increased numbers and enlarged sizes of terminal end buds, a niche for mammary stem cells (MaSCs). Here we show that MaSCs from both wild-type (WT) and Tg mice expressed IGF-1R and that overexpression of Tg IGF-1 increased numbers of MaSCs by undergoing symmetric division, resulting in an expansion of the MaSC and luminal progenitor (LP) compartments in pre-pubertal female mice. This expansion was maintained post-pubertally and validated by mammosphere assays in vitro and transplantation assays in vivo. The addition of recombinant IGF-1 promoted, and IGF-1R downstream inhibitors decreased mammosphere formation. Single-cell transcriptomic profiles generated from 2 related platforms reveal that IGF-1 stimulated quiescent MaSCs to enter the cell cycle and increased their expression of genes involved in proliferation, plasticity, tumorigenesis, invasion, and metastasis. This study identifies a novel, pro-tumorigenic mechanism, where IGF-1 increases the number of transformation-susceptible carcinogen targets during the early stages of mammary tissue development, and "primes" their gene expression profiles for transformation.

Impact of E-cig aerosol vaping on fetal and neonatal respiratory development and function.

Electronic cigarette (e-cig) use has increased over the past decade, and exposure to e-cig aerosols during pregnancy raises concern for maternal and fetal health. The developing fetal lung is known to be sensitive to prenatal tobacco product exposure. Utilizing a 3-pronged approach, we examined the effects of prenatal e-cig aerosols with, and without nicotine on respiratory development in a murine model. RNAseq analysis of fetal lungs revealed extensive dysregulation in gene expression. Morphologic assessment of distal airspaces in neonatal lungs display an emphysematic phenotype. Respiratory mechanics of neonates display signs of increased respiratory workload, with increased resistance and decreased compliance. These data are novel and provide evidence that prenatal e-cig exposure may result in altered lung function or development of disease.

Epithelial-specific ERBB3 deletion results in a genetic background-dependent increase in intestinal and colon polyps that is mediated by EGFR.

ERBB3 has gained attention as a potential therapeutic target to treat colorectal and other types of cancers. To confirm a previous study showing intestinal polyps are dependent upon ERBB3, we generated an intestinal epithelia-specific ERBB3 deletion in C57BL/6-ApcMin/+ mice. Contrary to the previous report showing a significant reduction in intestinal polyps with ablation of ERBB3 on a B6;129 mixed genetic background, we observed a significant increase in polyp number with ablation of ERBB3 on C57BL/6J compared to control littermates. We confirmed the genetic background dependency of ERBB3 by also analyzing polyp development on B6129 hybrid and B6;129 advanced intercross mixed genetic backgrounds, which showed that ERBB3 deficiency only reduced polyp number on the mixed background as previously reported. Increased polyp number with ablation of ERBB3 was also observed in C57BL/6J mice treated with azoxymethane showing the effect is model independent. Polyps forming in absence of ERBB3 were generally smaller than those forming in control mice, albeit the effect was greatest in genetic backgrounds with reduced polyp numbers. The mechanism for differential polyp number in the absence of ERBB3 was through altered proliferation. Backgrounds with increased polyp number with loss of ERBB3 showed an increase in cell proliferation even in non-tumor epithelia, while backgrounds showing reduced polyp number with loss of ERBB3 showed reduced cellular proliferation. Increase polyp number caused by loss of ERBB3 was mediated by increased epidermal growth factor receptor (EGFR) expression, which was confirmed by deletion of Egfr. Taken together, this study raises substantial implications on the use of ERBB3 inhibitors against colorectal cancer. The prediction is that some patients may have increased progression with ERBB3 inhibitor therapy, which is consistent with observations reported for ERBB3 inhibitor clinical trials.

Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2.

The N-Myc Downstream-Regulated Gene 4 (NDRG4), a prominent biomarker for colorectal cancer (CRC), is specifically expressed by enteric neurons. Considering that nerves are important members of the tumor microenvironment, we here establish different Ndrg4 knockout (Ndrg4-/- ) CRC models and an indirect co-culture of primary enteric nervous system (ENS) cells and intestinal organoids to identify whether the ENS, via NDRG4, affects intestinal tumorigenesis. Linking immunostainings and gastrointestinal motility (GI) assays, we show that the absence of Ndrg4 does not trigger any functional or morphological GI abnormalities. However, combining in vivo, in vitro, and quantitative proteomics data, we uncover that Ndrg4 knockdown is associated with enlarged intestinal adenoma development and that organoid growth is boosted by the Ndrg4-/- ENS cell secretome, which is enriched for Nidogen-1 (Nid1) and Fibulin-2 (Fbln2). Moreover, NID1 and FBLN2 are expressed in enteric neurons, enhance migration capacities of CRC cells, and are enriched in human CRC secretomes. Hence, we provide evidence that the ENS, via loss of Ndrg4, is involved in colorectal pathogenesis and that ENS-derived Nidogen-1 and Fibulin-2 enhance colorectal carcinogenesis.

A molecular subtype of colorectal cancers initiates independently of epidermal growth factor receptor and has an accelerated growth rate mediated by IL10-dependent anergy.

Although epidermal growth factor receptor (EGFR)-targeted therapies are approved for colorectal cancer (CRC) treatment, only 15% of CRC patients respond to EGFR inhibition. Here, we show that colorectal cancers (CRC) can initiate and grow faster through an EGFR-independent mechanism, irrespective of the presence of EGFR, in two different mouse models using tissue-specific ablation of Egfr. The growth benefit in the absence of EGFR is also independent of Kras status. An EGFR-independent gene expression signature, also observed in human CRCs, revealed that anergy-inducing genes are overexpressed in EGFR-independent polyps, suggesting increased infiltration of anergic lymphocytes promotes an accelerated growth rate that is partially caused by escape from cell-mediated immune responses. Many genes in the EGFR-independent gene expression signature are downstream targets of interleukin 10 receptor alpha (IL10RA). We further show that IL10 is detectable in serum from mice with EGFR-independent colon polyps. Using organoids in vitro and Src ablation in vivo, we show that IL10 contributes to growth of EGFR-independent CRCs, potentially mediated by the well-documented role of SRC in IL10 signaling. Based on these data, we show that the combination of an EGFR inhibitor with an anti-IL10 neutralizing antibody results in decreased cell proliferation in organoids and in decreased polyp size in pre-clinical models harboring EGFR-independent CRCs, providing a new therapeutic intervention for CRCs resistant to EGFR inhibitor therapies.

Host genetics and gut microbiota cooperatively contribute to azoxymethane-induced acute toxicity in Collaborative Cross mice.

Azoxymethane (AOM) is a widely used carcinogen to study chemical-induced colorectal carcinogenesis and is an agent for studying fulminant hepatic failure. The inter-strain susceptibility to acute toxicity by AOM has been reported, but its association with host genetics or gut microbiota remains largely unexplored. Here a cohort of genetically diverse Collaborative Cross (CC) mice was used to assess the contribution of host genetics and the gut microbiome to AOM-induced acute toxicity. We observed variation in AOM-induced acute liver failure across CC strains. Quantitative trait loci (QTL) analysis revealed three chromosome regions significantly associated with AOM toxicity. Genes located within these QTL, including peroxisome proliferator-activated receptor alpha (Ppara), were enriched for enzyme activator and nucleoside-triphosphatase regulator activity. We further demonstrated that the protein level of PPARα in liver tissues from sensitive strains was remarkably lower compared to levels in resistant strains, consistent with protective role of PPAR family in liver injury. We discovered that the abundance levels of gut microbial families Anaeroplasmataceae, Ruminococcaceae, Lactobacillaceae, Akkermansiaceae and Clostridiaceae were significantly higher in the sensitive strains compared to the resistant strains. Using a random forest classifier method, we determined that the relative abundance levels of these microbial families predicted AOM toxicity with the area under the receiver-operating curve (AUC) of 0.75. Combining the three genetic loci and five microbial families increased the predictive accuracy of AOM toxicity (AUC of 0.99). Moreover, we found that Ruminococcaceae and Lactobacillaceae acted as mediators between host genetics and AOM toxicity. In conclusion, this study shows that host genetics and specific microbiome members play a critical role in AOM-induced acute toxicity, which provides a framework for analysis of the health effects from environmental toxicants.

Derivation of stable embryonic stem cell-like, but transcriptionally heterogenous, induced pluripotent stem cells from non-permissive mouse strains.

Genetic background is known to play a role in the ability to derive pluripotent, embryonic stem cells (ESC), a trait referred to as permissiveness. Previously we demonstrated that induced pluripotent stem cells (iPSC) can be readily derived from non-permissive mouse strains by addition of serum-based media supplemented with GSK3B and MEK inhibitors, termed 2iS media, 3 days into reprogramming. Here, we describe the derivation of second type of iPSC colony from non-permissive mouse strains that can be stably maintained independently of 2iS media. The resulting cells display transcriptional heterogeneity similar to that observed in ESC from permissive genetic backgrounds derived in conventional serum containing media supplemented with leukemia inhibitor factor. However, unlike previous studies that report exclusive subpopulations, we observe both exclusive and simultaneous expression of naive and primed cell surface markers. Herein, we explore shifts in pluripotency in the presence of 2iS and characterize heterogenous subpopulations to determine their pluripotent state and role in heterogenous iPSCs derived from the non-permissive NOD/ShiLtJ strain. We conclude that heterogeneity is a naturally occurring, necessary quality of stem cells that allows for the maintenance of pluripotency. This study further demonstrates the efficacy of the 2iS reprogramming technique. It is also the first study to derive stable ESC-like stem cells from the non-permissive NOD/ShiLtJ and WSB/EiJ strains, enabling easier and broader research possibilities into pluripotency for these and similar non-permissive mouse strains and species.

A New Polygenic Model for Nonfamilial Colorectal Cancer Inheritance Based on the Genetic Architecture of the Azoxymethane-Induced Mouse Model.

The azoxymethane model of colorectal cancer (CRC) was used to gain insights into the genetic heterogeneity of nonfamilial CRC. We observed significant differences in susceptibility parameters across 40 mouse inbred strains, with 6 new and 18 of 24 previously identified mouse CRC modifier alleles detected using genome-wide association analysis. Tumor incidence varied in F1 as well as intercrosses and backcrosses between resistant and susceptible strains. Analysis of inheritance patterns indicates that resistance to CRC development is inherited as a dominant characteristic genome-wide, and that susceptibility appears to occur in individuals lacking a large-effect, or sufficient numbers of small-effect, polygenic resistance alleles. Our results suggest a new polygenic model for inheritance of nonfamilial CRC, and that genetic studies in humans aimed at identifying individuals with elevated susceptibility should be pursued through the lens of absence of dominant resistance alleles rather than for the presence of susceptibility alleles.

Using Collaborative Cross Mouse Population to Fill Data Gaps in Risk Assessment: A Case Study of Population-Based Analysis of Toxicokinetics and Kidney Toxicodynamics of Tetrachloroethylene.

BACKGROUND: Interindividual variability in susceptibility remains poorly characterized for environmental chemicals such as tetrachloroethylene (PERC). Development of population-based experimental models provide a potential approach to fill this critical need in human health risk assessment. OBJECTIVES: In this study, we aimed to better characterize the contribution of glutathione (GSH) conjugation to kidney toxicity of PERC and the degree of associated interindividual toxicokinetic (TK) and toxicodynamic (TD) variability by using the Collaborative Cross (CC) mouse population. METHODS: Male mice from 45 strains were intragastrically dosed with PERC ([Formula: see text]) or vehicle (5% Alkamuls EL-620 in saline), and time-course samples were collected for up to 24 h. Population variability in TK of S-(1,2,2-trichlorovinyl)GSH (TCVG), S-(1,2,2-trichlorovinyl)-L-cysteine (TCVC), and N-acetyl-S-(1,2,2-trichlorovinyl)-L-cysteine (NAcTCVC) was quantified in serum, liver, and kidney, and analyzed using a toxicokinetic model. Effects of PERC on kidney weight, fatty acid metabolism-associated genes [ Acot1 (Acyl-CoA thioesterase 1), Fabp1 (fatty acid-binding protein 1), and Ehhadh (enoyl-coenzyme A, hydratase/3-hydroxyacyl coenzyme A dehydrogenase)], and a marker of proximal tubular injury [KIM-1 (kidney injury molecule-1)/Hepatitis A virus cellular receptor 1 ( Havcr1)] were evaluated. Finally, quantitative data on interstrain variability in both formation of GSH conjugation metabolites of PERC and its kidney effects was used to calculate adjustment factors for the interindividual variability in both TK and TD. RESULTS: Mice treated with PERC had significantly lower kidney weight, higher kidney-to-body weight (BW) ratio, and higher expression of fatty acid metabolism-associated genes ( Acot1, Fabp1, and Ehhadh) and a marker of proximal tubular injury (KIM-1/ Havcr1). Liver levels of TCVG were significantly correlated with KIM-1/ Havcr1 in kidney, consistent with kidney injury being associated with GSH conjugation. We found that the default uncertainty factor for human variability may be marginally adequate to protect 95%, but not more, of the population for kidney toxicity mediated by PERC. DISCUSSION: Overall, this study demonstrates the utility of the CC mouse population in characterizing metabolism-toxicity interactions and quantifying interindividual variability. Further refinement of the characterization of interindividual variability can be accomplished by incorporating these data into in silico population models both for TK (such as a physiologically based pharmacokinetic model), as well as for toxicodynamic responses. https://doi.org/10.1289/EHP5105.

Population-Based Analysis of DNA Damage and Epigenetic Effects of 1,3-Butadiene in the Mouse.

Metabolism of 1,3-butadiene, a known human and rodent carcinogen, results in formation of reactive epoxides, a key event in its carcinogenicity. Although mice exposed to 1,3-butadiene present DNA adducts in all tested tissues, carcinogenicity is limited to liver, lung, and lymphoid tissues. Previous studies demonstrated that strain- and tissue-specific epigenetic effects in response to 1,3-butadiene exposure may influence susceptibly to DNA damage and serve as a potential mechanism of tissue-specific carcinogenicity. This study aimed to investigate interindividual variability in the effects of 1,3-butadiene using a population-based mouse model. Male mice from 20 Collaborative Cross strains were exposed to 0 or 635 ppm 1,3-butadiene by inhalation (6 h/day, 5 days/week) for 2 weeks. We evaluated DNA damage and epigenetic effects in target (lung and liver) and nontarget (kidney) tissues of 1,3-butadiene-induced carcinogenesis. DNA damage was assessed by measuring N-7-(2,3,4-trihydroxybut-1-yl)-guanine (THB-Gua) adducts. To investigate global histone modification alterations, we evaluated the trimethylation and acetylation of histones H3 and H4 across tissues. Changes in global cytosine DNA methylation were evaluated from the levels of methylation of LINE-1 and SINE B1 retrotransposons. We quantified the degree of variation across strains, deriving a chemical-specific human variability factor to address population variability in carcinogenic risk, which is largely ignored in current cancer risk assessment practice. Quantitative trait locus mapping identified four candidate genes related to chromatin remodeling whose variation was associated with interstrain susceptibility. Overall, this study uses 1,3-butadiene to demonstrate how the Collaborative Cross mouse population can be used to identify the mechanisms for and quantify the degree of interindividual variability in tissue-specific effects that are relevant to chemically induced carcinogenesis.

Long-Term Combinatorial Exposure to Trichloroethylene and Inorganic Arsenic in Genetically Heterogeneous Mice Results in Renal Tubular Damage and Cancer-Associated Molecular Changes.

Trichloroethylene (TCE) and inorganic arsenic (iAs) are environmental contaminants that can target the kidney. Chronic exposure to TCE is associated with increased incidence of renal cell carcinoma, while co-exposure to TCE and iAs likely occurs in exposed human populations, such as those near Superfund sites. In order to better understand the kidney health consequences of TCE and/or iAs exposure, a genetically heterogeneous mouse population derived from FVB/NJ and CAST/EiJ mouse strains and deficient for multidrug resistance genes (Abcb1atm1Bor , Abcb1btm1Bor ) was chronically exposed for 52-weeks to varying concentrations of TCE and iAs. Although no exposure group resulted in primary renal cell tumors, kidneys from exposed mice did have significant increases in histologic and biochemical evidence of renal tubular disease with each toxicant alone and with combined exposure, with males having significantly higher levels of damage. Although no added increase in tubular disease was observed with combination exposure compared to single toxicants, molecular changes in kidneys from mice that had the combined exposure were similar to those previous observed in an embryonic stem cell assay for the P81S TCE-induced renal cell carcinoma mutation in the Von Hippel-Lindau syndrome (VHL) gene. While this model more accurately reflects human exposure conditions, development of primary renal tumors observed in humans following chronic TCE exposure was not reproduced even after inclusion of genetic heterogeneity and co-carcinogenic iAs.

Diverse tumour susceptibility in Collaborative Cross mice: identification of a new mouse model for human gastric tumourigenesis.

OBJECTIVE: The Collaborative Cross (CC) is a mouse population model with diverse and reproducible genetic backgrounds used to identify novel disease models and genes that contribute to human disease. Since spontaneous tumour susceptibility in CC mice remains unexplored, we assessed tumour incidence and spectrum. DESIGN: We monitored 293 mice from 18 CC strains for tumour development. Genetic association analysis and RNA sequencing were used to identify susceptibility loci and candidate genes. We analysed genomes of patients with gastric cancer to evaluate the relevance of genes identified in the CC mouse model and measured the expression levels of ISG15 by immunohistochemical staining using a gastric adenocarcinoma tissue microarray. Association of gene expression with overall survival (OS) was assessed by Kaplan-Meier analysis. RESULTS: CC mice displayed a wide range in the incidence and types of spontaneous tumours. More than 40% of CC036 mice developed gastric tumours within 1 year. Genetic association analysis identified Nfκb1 as a candidate susceptibility gene, while RNA sequencing analysis of non-tumour gastric tissues from CC036 mice showed significantly higher expression of inflammatory response genes. In human gastric cancers, the majority of human orthologues of the 166 mouse genes were preferentially altered by amplification or deletion and were significantly associated with OS. Higher expression of the CC036 inflammatory response gene signature is associated with poor OS. Finally, ISG15 protein is elevated in gastric adenocarcinomas and correlated with shortened patient OS. CONCLUSIONS: CC strains exhibit tremendous variation in tumour susceptibility, and we present CC036 as a spontaneous laboratory mouse model for studying human gastric tumourigenesis.

Chronic exposure to e-cig aerosols during early development causes vascular dysfunction and offspring growth deficits.

Increasing popularity of electronic cigarettes (e-cigs), including among women of reproductive age, is attributed to its perceived safety compared to conventional tobacco. However, there is a major knowledge gap surrounding the effects of e-cig aerosols on pregnancy and fetal development. We aimed to evaluate the effects of vaping e-cigs during gestation on offspring growth and to asses if growth deficits are accompanied by altered maternal and fetal vascular hemodynamics. Sprague-Dawley dams were assigned to Pair-Fed Control, Pair-Fed Juice, or Juice+Nicotine groups, and then underwent either a prenatal or prenatal+postnatal exposure paradigm in a custom-engineered vaping system. Mass spectrometry identified major aerosolized constituents from e-cig vaping. The Juice+Nicotine group exhibited significantly decreased fetal weight and crown-rump length (↓46.56%, and ↓23.83%, respectively). Pre- and postnatal exposure to Juice+Nicotine resulted in decreased pup weight at postnatal day (PND) 4-10. Crown-rump length was decreased by 24.71% on PND 10. Blood flow in the Juice+Nicotine group was decreased in the maternal uterine and fetal umbilical circuits by 49.50% and 65.33%, respectively. We conclude that chronic exposure to e-cig aerosols containing nicotine during early development can have deleterious health effects on the exposed offspring. Vaping e-cigs containing nicotine during pregnancy lead to a reduction in offspring weight and crown-rump length, associated with a marked decrease in blood flow in both the maternal uterine and fetal umbilical circulation (a strong indicator of growth restriction). Thus, chronic exposure to e-cig aerosols containing nicotine can lead to potentially harmful developmental effects in early life.

Modulation of Tetrachloroethylene-Associated Kidney Effects by Nonalcoholic Fatty Liver or Steatohepatitis in Male C57BL/6J Mice.

Accounting for genetic and other (eg, underlying disease states) factors that may lead to inter-individual variability in susceptibility to xenobiotic-induced injury is a challenge in human health assessments. A previous study demonstrated that nonalcoholic fatty liver disease (NAFLD), one of the common underlying disease states, enhances tetrachloroethylene (PERC)-associated hepatotoxicity in mice. Interestingly, NAFLD resulted in a decrease in metabolism of PERC to nephrotoxic glutathione conjugates; we therefore hypothesized that NAFLD would protect against PERC-associated nephrotoxicity. Male C57BL/6J mice were fed a low-fat (LFD), high-fat (31% fat, HFD), or high-fat methionine/choline/folate-deficient (31% fat, MCD) diets. After 8 weeks mice were administered either a single dose of PERC (300 mg/kg i.g.) and euthanized at 1-36 h post dose, or five daily doses of PERC (300 mg/kg/d i.g.) and euthanized 4 h after last dose. Relative to LFD-fed mice, HFD- or MCD-fed mice exhibited decreased PERC concentrations and increased trichloroacetate (TCA) in kidneys. S-(1,2,2-trichlorovinyl)glutathione (TCVG), S-(1,2,2-trichlorovinyl)-l-cysteine (TCVC), and N-acetyl-S-(1,2,2,-trichlorovinyl)-l-cysteine (NAcTCVC) were also significantly lower in kidney and urine of HFD- or MCD-fed mice compared with LFD-fed mice. Despite differences in levels of nephrotoxic PERC metabolites in kidney, LFD- and MCD-fed mice demonstrated similar degree of nephrotoxicity. However, HFD-fed mice were less sensitive to PERC-induced nephrotoxicity. Thus, whereas both MCD- and HFD-induced fatty liver reduced the delivered dose of nephrotoxic PERC metabolites to the kidney, only HFD was protective against PERC-induced nephrotoxicity, possibly due to greater toxicodynamic sensitivity induced by methyl and choline deficiency. These results therefore demonstrate that pre-existing disease conditions can lead to a complex interplay of toxicokinetic and toxicodynamic changes that modulate susceptibility to the toxicity of xenobiotics.

Permissiveness to form pluripotent stem cells may be an evolutionarily derived characteristic in Mus musculus.

Mus musculus is the only known species from which embryonic stem cells (ESC) can be isolated under conditions requiring only leukemia inhibitory factor (LIF). Other species are non-permissive in LIF media, and form developmentally primed epiblast stem cells (EpiSC) similar to cells derived from post-implantation, egg cylinders. To evaluate whether non-permissiveness extends to induced pluripotent stem cells (iPSC), we derived iPSC from the eight founder strains of the mouse Collaborative Cross. Two strains, NOD/ShiLtJ and the WSB/EiJ, were non-permissive, consistent with the previous classification of NOD/ShiLtJ as non-permissive to ESC derivation. We determined non-permissiveness is recessive, and that non-permissive genomes do not compliment. We overcame iPSC non-permissiveness by using GSK3B and MEK inhibitors with serum, a technique we termed 2iS reprogramming. Although used for ESC derivation, GSK3B and MEK inhibitors have not been used during iPSC reprogramming because they inhibit survival of progenitor differentiated cells. iPSC derived in 2iS are more transcriptionally similar to ESC than EpiSC, indicating that 2iS reprogramming acts to overcome genetic background constraints. Finally, of species tested for ESC or iPSC derivation, only some M. musculus strains are permissive under LIF culture conditions suggesting that this is an evolutionarily derived characteristic in the M. musculus lineage.