Concentrations of potentially endocrine disrupting chemicals in car cabin air and dust - Effect of temperature and ventilation.

Materials in car cabins contain performance-enhancing semi-volatile organic compounds (SVOCs). As these SVOCs are not chemically bound to the materials, they can emit from the materials at slow rates to the surrounding, causing human exposure. This study aimed at increasing the understanding on abundance of SVOCs in car cabins by studying 18 potential endocrine disrupting chemicals in car cabin air (gas phase and airborne particles) and dust. We also studied how levels of these chemicals varied by temperature inside the car cabin along with ventilation settings, relevant to human exposure. A positive correlation was observed between temperature and SVOC concentration in both the gas and the particle phase, where average gas phase levels at 80 °C were a factor of 18-16,000 higher than average levels at 25 °C, while average particle phase levels were a factor of 4.6-40,000 higher for the studied substances. This study also showed that levels were below the limit of detection for several SVOCs during realistic driving conditions, i.e., with the ventilation activated. To limit human exposure to SVOCs in car cabins, it is recommended to ventilate a warm car before entering and have the ventilation on during driving, as both temperature and ventilation have a significant impact on SVOC levels.

Weight of evidence evaluation of the metabolism disrupting effects of triphenyl phosphate using an expert knowledge elicitation approach.

Identification of Endocrine-Disrupting Chemicals (EDCs) in a regulatory context requires a high level of evidence. However, lines of evidence (e.g. human, in vivo, in vitro or in silico) are heterogeneous and incomplete for quantifying evidence of the adverse effects and mechanisms involved. To date, for the regulatory appraisal of metabolism-disrupting chemicals (MDCs), no harmonised guidance to assess the weight of evidence has been developed at the EU or international level. To explore how to develop this, we applied a formal Expert Knowledge Elicitation (EKE) approach within the European GOLIATH project. EKE captures expert judgment in a quantitative manner and provides an estimate of uncertainty of the final opinion. As a proof of principle, we selected one suspected MDC -triphenyl phosphate (TPP) - based on its related adverse endpoints (obesity/adipogenicity) relevant to metabolic disruption and a putative Molecular Initiating Event (MIE): activation of peroxisome proliferator activated receptor gamma (PPARγ). We conducted a systematic literature review and assessed the quality of the lines of evidence with two independent groups of experts within GOLIATH, with the objective of categorising the metabolic disruption properties of TPP, by applying an EKE approach. Having followed the entire process separately, both groups arrived at the same conclusion, designating TPP as a "suspected MDC" with an overall quantitative agreement exceeding 85%, indicating robust reproducibility. The EKE method provides to be an important way to bring together scientists with diverse expertise and is recommended for future work in this area.

Tissue pharmacokinetics of antisense oligonucleotides.

Pharmacokinetics (PK) of antisense oligonucleotides (ASOs) is characterized by rapid distribution from plasma to tissue and slow terminal plasma elimination driven by re-distribution from tissue. Quantitative understanding of tissue PK and RNA knockdown for various ASO chemistries, conjugations, and administration routes is critical for successful drug discovery. Here, we report concentration-time and RNA knockdown profiles for a gapmer ASO with locked nucleic acid ribose chemistry in mouse liver, kidney, heart, and lung after subcutaneous and intratracheal administration. Additionally, the same ASO with liver targeting conjugation (galactosamine-N-acetyl) is evaluated for subcutaneous administration. Data indicate that exposure and knockdown differ between tissues and strongly depend on administration route and conjugation. In a second study, we show that tissue PK is similar between the three different ribose chemistries locked nucleic acid, constrained ethyl and 2'-O-methoxyethyl, both after subcutaneous and intratracheal administration. Further, we show that the half-life in mouse liver may vary with ASO sequence. Finally, we report less than dose-proportional increase in liver concentration in the dose range of 3-30 µmol/kg. Overall, our studies contribute pivotal data to support design and interpretation of ASO in vivo studies, thereby increasing the probability of delivering novel ASO therapies to patients.

HSP70 binds to specific non-coding RNA and regulates human RNA polymerase III.

Molecular chaperones are critical for protein homeostasis and are implicated in several human pathologies such as neurodegeneration and cancer. While the binding of chaperones to nascent and misfolded proteins has been studied in great detail, the direct interaction between chaperones and RNA has not been systematically investigated. Here, we provide the evidence for widespread interaction between chaperones and RNA in human cells. We show that the major chaperone heat shock protein 70 (HSP70) binds to non-coding RNA transcribed by RNA polymerase III (RNA Pol III) such as tRNA and 5S rRNA. Global chromatin profiling revealed that HSP70 binds genomic sites of transcription by RNA Pol III. Detailed biochemical analyses showed that HSP70 alleviates the inhibitory effect of cognate tRNA transcript on tRNA gene transcription. Thus, our study uncovers an unexpected role of HSP70-RNA interaction in the biogenesis of a specific class of non-coding RNA with wider implications in cancer therapeutics.

Polychlorinated alkanes in paired blood serum and breast milk in a Swedish cohort study: Matrix dependent partitioning differences compared to legacy POPs.

BACKGROUND: Polychlorinated alkanes (PCAs) constitute a large group of individual congeners originating from commercial chlorinated paraffin (CP) products with carbon chain lengths of PCAs-C10-13, PCAs-C14-17, and PCAs-C18-32, occasionally containing PCAs-C6-9 impurities. The extensive use of CPs has led to global environmental pollution of PCAs. This study aimed to quantify PCAs in paired serum and breast milk of lactating Swedish mothers, exploring their concentration relationship. METHODS: Twenty-five paired samples of mothers' blood serum and breast milk were analysed and concentrations were determined for PCAs C6-32 and compared to 4,4'-DDE, the PCB congener 2,2',4,4',5,5'-hexachlorobiphenyl (CB-153), and hexachlorobenzene (HCB). RESULTS: The median concentrations of PCAs-C6-9, PCAs-C10-13, PCAs-C14-17, PCAs-C18-32 and ΣPCAs in serum were 14, 790, 520, 16 and 1350 ng/g lipid weight (lw), respectively, and in breast milk 0.84, 36, 63, 6.0 and 107 ng/g lw. Levels of 4,4'-DDE, CB-153 and HCB were comparable in the two matrices, serum and breast milk at 17, 12 and 4.9 ng/g lw. The results show significant differences of PCAs-C10-13 and PCAs-C14-17 in breast milk with 22- and 6.2-times lower lw-based concentrations than those measured in serum. On wet weight the differences serum/breast milk ratios of PCAs-C6-9, PCAs-C10-13, PCAs-C14-17, PCAs-C18-32 and ΣPCAs were 1.7, 3.2, 1.0, 0.4 and 1.6, respectively, while the ratio for 4,4'-DDE, CB-153 and HCB were each close to 0.1. CONCLUSION: Swedish lactating mothers had high serum concentrations of PCAs-C10-13 and PCAs-C14-17, with the ΣPCAs median serum concentration of 1350 ng/g lw. The breast milk concentration, although considerably lower at 107 ng/g lw, still surpassed those of 4,4'-DDE, CB-153 and HCB, suggesting an exposure risk of infants to PCAs. The variation in blood and breast milk accumulation between PCAs and studied legacy POPs, is rarely discussed but warrants further studies on partitioning properties as well as associated toxicological implications.

Studying mixture effects on uptake and tissue distribution of PFAS in zebrafish (Danio rerio) using physiologically based kinetic (PBK) modelling.

Per- and polyfluoroalkyl substances (PFAS) are ubiquitously distributed in the aquatic environment. They include persistent, mobile, bioaccumulative, and toxic chemicals and it is therefore critical to increase our understanding on their adsorption, distribution, metabolism, excretion (ADME). The current study focused on uptake of seven emerging PFAS in zebrafish (Danio rerio) and their potential maternal transfer. In addition, we aimed at increasing our understanding on mixture effects on ADME by developing a physiologically based kinetic (PBK) model capable of handling co-exposure scenarios of any number of chemicals. All studied chemicals were taken up in the fish to varying degrees, whereas only perfluorononanoate (PFNA) and perfluorooctanoate (PFOA) were quantified in all analysed tissues. Perfluorooctane sulfonamide (FOSA) was measured at concerningly high concentrations in the brain (Cmax over 15 µg/g) but also in the liver and ovaries. All studied PFAS were maternally transferred to the eggs, with FOSA and 6:2 perfluorooctane sulfonate (6,2 FTSA) showing significant (p < 0.02) signs of elimination from the embryos during the first 6 days of development, while perfluorobutane sulfonate (PFBS), PFNA, and perfluorohexane sulfonate (PFHxS) were not eliminated in embryos during this time-frame. The mixture PBK model resulted in >85 % of predictions within a 10-fold error and 60 % of predictions within a 3-fold error. At studied levels of PFAS exposure, competitive binding was not a critical factor for PFAS kinetics. Gill surface pH influenced uptake for some carboxylates but not the sulfonates. The developed PBK model provides an important tool in understanding kinetics under complex mixture scenarios and this use of New Approach Methodologies (NAMs) is critical in future risk assessment of chemicals and early warning systems.

Physiology-informed toxicokinetic model for the zebrafish embryo test developed for bisphenols.

Zebrafish embryos (ZFE) is a widely used model organism, employed in various research fields including toxicology to assess e.g., developmental toxicity and endocrine disruption. Variation in effects between chemicals are difficult to compare using nominal dose as toxicokinetic properties may vary. Toxicokinetic (TK) modeling is a means to estimate internal exposure concentration or dose at target and to enable extrapolation between experimental conditions and species, thereby improving hazard assessment of potential pollutants. In this study we advance currently existing TK models for ZFE with physiological ZFE parameters and novel experimental bisphenol data, a class of chemicals with suspected endocrine activity. We developed a five-compartment model consisting of water, plastic, chorion, yolk sack and embryo in which surface area and volume changes as well as the processes of biotransformation and blood circulation influence mass fluxes. For model training and validation, we measured internal concentrations in ZFE exposed individually to BPA, bisphenol AF (BPAF) and Z (BPZ). Bayesian inference was applied for parameter calibration based on the training data set of BPZ. The calibrated TK model predicted internal ZFE concentrations of the majority of external test data within a 5-fold error and half of the data within a 2-fold error for bisphenols A, AF, F, and tetrabromo bisphenol A (TBBPA). We used the developed model to rank the hazard of seven bisphenols based on predicted internal concentrations and measured in vitro estrogenicity. This ranking indicated a higher hazard for BPAF, BPZ, bisphenol B and C (BPB, BPC) than for BPA.

KRAS mutation-driven angiopoietin 2 bestows anti-VEGF resistance in epithelial carcinomas.

Defining reliable surrogate markers and overcoming drug resistance are the most challenging issues for improving therapeutic outcomes of antiangiogenic drugs (AADs) in cancer patients. At the time of this writing, no biomarkers are clinically available to predict AAD therapeutic benefits and drug resistance. Here, we uncovered a unique mechanism of AAD resistance in epithelial carcinomas with KRAS mutations that targeted angiopoietin 2 (ANG2) to circumvent antivascular endothelial growth factor (anti-VEGF) responses. Mechanistically, KRAS mutations up-regulated the FOXC2 transcription factor that directly elevated ANG2 expression at the transcriptional level. ANG2 bestowed anti-VEGF resistance as an alternative pathway to augment VEGF-independent tumor angiogenesis. Most colorectal and pancreatic cancers with KRAS mutations were intrinsically resistant to monotherapies of anti-VEGF or anti-ANG2 drugs. However, combination therapy with anti-VEGF and anti-ANG2 drugs produced synergistic and potent anticancer effects in KRAS-mutated cancers. Together, these data demonstrate that KRAS mutations in tumors serve as a predictive marker for anti-VEGF resistance and are susceptible to combination therapy with anti-VEGF and anti-ANG2 drugs.

Cancer cell plasticity and MHC-II-mediated immune tolerance promote breast cancer metastasis to lymph nodes.

Tumor-draining lymph nodes (TDLNs) are important for tumor antigen-specific T cell generation and effective anticancer immune responses. However, TDLNs are often the primary site of metastasis, causing immune suppression and worse outcomes. Through cross-species single-cell RNA-Seq analysis, we identified features defining cancer cell heterogeneity, plasticity, and immune evasion during breast cancer progression and lymph node metastasis (LNM). A subset of cancer cells in the lymph nodes exhibited elevated MHC class II (MHC-II) gene expression in both mice and humans. MHC-II+ cancer cells lacked costimulatory molecule expression, leading to regulatory T cell (Treg) expansion and fewer CD4+ effector T cells in TDLNs. Genetic knockout of MHC-II reduced LNM and Treg expansion, while overexpression of the MHC-II transactivator, Ciita, worsened LNM and caused excessive Treg expansion. These findings demonstrate that cancer cell MHC-II expression promotes metastasis and immune evasion in TDLNs.

From Cohort to Cohort: A Similar Mixture Approach (SMACH) to Evaluate Exposures to a Mixture Leading to Thyroid-Mediated Neurodevelopmental Effects Using NHANES Data.

Prenatal exposure to a mixture (MIX N) of eight endocrine-disrupting chemicals has been associated with language delay in children in a Swedish pregnancy cohort. A novel approach was proposed linking this epidemiological association with experimental evidence, where the effect of MIX N on thyroid hormone signaling was assessed using the Xenopus eleuthero-embryonic thyroid assay (XETA OECD TG248). From this experimental data, a point of departure (PoD) was derived based on OECD guidance. Our aim in the current study was to use updated toxicokinetic models to compare exposures of women of reproductive age in the US population to MIX N using a Similar Mixture Approach (SMACH). Based on our findings, 66% of women of reproductive age in the US (roughly 38 million women) had exposures sufficiently similar to MIX N. For this subset, a Similar Mixture Risk Index (SMRIHI) was calculated comparing their exposures to the PoD. Women with SMRIHI > 1 represent 1.1 million women of reproductive age. Older women, Mexican American and other/multi race women were less likely to have high SMRIHI values compared to Non-Hispanic White women. These findings indicate that a reference mixture of chemicals identified in a Swedish cohort-and tested in an experimental model for establishment of (PoDs)-is also of health relevance in a US population.

Considerations in the Preclinical Assessment of the Safety of Antisense Oligonucleotides.

The nucleic acid therapeutics field has made tremendous progress in the past decades. Continuous advances in chemistry and design have led to many successful clinical applications, eliciting even more interest from researchers including both academic groups and drug development companies. Many preclinical studies in the field focus on improving the delivery of antisense oligonucleotide drugs (ONDs) and/or assessing their efficacy in target tissues, often neglecting the evaluation of toxicity, at least in early phases of development. A series of consensus recommendations regarding regulatory considerations and expectations have been generated by the Oligonucleotide Safety Working Group and the Japanese Research Working Group for the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use S6 and Related Issues (WGS6) in several white papers. However, safety aspects should also be kept in sight in earlier phases while screening and designing OND to avoid subsequent failure in the development phase. Experts and members of the network "DARTER," a COST Action funded by the Cooperation in Science and Technology of the EU, have utilized their collective experience working with OND, as well as their insights into OND-mediated toxicities, to generate a series of consensus recommendations to assess OND toxicity in early stages of preclinical research. In the past few years, several publications have described predictive assays, which can be used to assess OND-mediated toxicity in vitro or ex vivo to filter out potential toxic candidates before moving to in vivo phases of preclinical development, that is, animal toxicity studies. These assays also have the potential to provide translational insight since they allow a safety evaluation in human in vitro systems. Yet, small preliminary in vivo studies should also be considered to complement this early assessment. In this study, we summarize the state of the art and provide guidelines and recommendations on the different tests available for these early stage preclinical assessments.

Predicting Endocrine Disruption Using Conformal Prediction - A Prioritization Strategy to Identify Hazardous Chemicals with Confidence.

Receptor-mediated molecular initiating events (MIEs) and their relevance in endocrine activity (EA) have been highlighted in literature. More than 15 receptors have been associated with neurodevelopmental adversity and metabolic disruption. MIEs describe chemical interactions with defined biological outcomes, a relationship that could be described with quantitative structure-activity relationship (QSAR) models. QSAR uncertainty can be assessed using the conformal prediction (CP) framework, which provides similarity (i.e., nonconformity) scores relative to the defined classes per prediction. CP calibration can indirectly mitigate data imbalance during model development, and the nonconformity scores serve as intrinsic measures of chemical applicability domain assessment during screening. The focus of this work was to propose an in silico predictive strategy for EA. First, 23 QSAR models for MIEs associated with EA were developed using high-throughput data for 14 receptors. To handle the data imbalance, five protocols were compared, and CP provided the most balanced class definition. Second, the developed QSAR models were applied to a large data set (∼55,000 chemicals), comprising chemicals representative of potential risk for human exposure. Using CP, it was possible to assess the uncertainty of the screening results and identify model strengths and out of domain chemicals. Last, two clustering methods, t-distributed stochastic neighbor embedding and Tanimoto similarity, were used to identify compounds with potential EA using known endocrine disruptors as reference. The cluster overlap between methods produced 23 chemicals with suspected or demonstrated EA potential. The presented models could be utilized for first-tier screening and identification of compounds with potential biological activity across the studied MIEs.

Wnt inhibition alleviates resistance to immune checkpoint blockade in glioblastoma.

Wnt signaling plays a critical role in the progression and treatment outcome of glioblastoma (GBM). Here, we identified WNT7b as a heretofore unknown mechanism of resistance to immune checkpoint inhibition (αPD1) in GBM patients and murine models. Acquired resistance to αPD1 was found to be associated with the upregulation of Wnt7b and ß-catenin protein levels in GBM in patients and in a clinically relevant, stem-rich GBM model. Combining the porcupine inhibitor WNT974 with αPD1 prolonged the survival of GBM-bearing mice. However, this combination had a dichotomous response, with a subset of tumors showing refractoriness. WNT974 and αPD1 expanded a subset of DC3-like dendritic cells (DCs) and decreased the granulocytic myeloid-derived suppressor cells (gMDSCs) in the tumor microenvironment (TME). By contrast, monocytic MDSCs (mMDSCs) increased, while T-cell infiltration remained unchanged, suggesting potential TME-mediated resistance. Our preclinical findings warrant the testing of Wnt7b/ß-catenin combined with αPD1 in GBM patients with elevated Wnt7b/ß-catenin signaling.

Physiologically Based Toxicokinetic Modeling of Bisphenols in Zebrafish (Danio rerio) Accounting for Variations in Metabolic Rates, Brain Distribution, and Liver Accumulation.

Bisphenol A (BPA) is an industrial chemical, which has raised human health and environmental concerns due to its endocrine-disrupting properties. BPA analogues are less well-studied despite their wide use in consumer products. These analogues have been detected in water and aquatic organisms around the world, with some analogues showing toxic effects in various species including fish. Here, we present novel organ-specific time-course distribution data of bisphenol Z (BPZ) in female zebrafish (Danio rerio), including concentrations in the ovaries, liver, and brain, a rarely sampled organ with high toxicological relevance. Furthermore, fish-specific in vitro biotransformation rates were determined for 11 selected bisphenols. A physiologically based toxicokinetic (PBTK) model was adapted for four of these bisphenols, which was able to predict levels in the gonads, liver, and brain as well as the whole body within a 2-5-fold error with respect to experimental data, covering several important target organs of toxicity. In particular, predicted liver concentrations improved compared to currently available PBTK models. Predicted data indicate that studied bisphenols mainly distribute to the carcass and gonads and less to the brain. Our model provides a tool to increase our understanding on the distribution and kinetics of a group of emerging pollutants.

In Silico Identification of Potential Thyroid Hormone System Disruptors among Chemicals in Human Serum and Chemicals with a High Exposure Index.

Data on toxic effects are at large missing the prevailing understanding of the risks of industrial chemicals. Thyroid hormone (TH) system disruption includes interferences of the life cycle of the thyroid hormones and may occur in various organs. In the current study, high-throughput screening data available for 14 putative molecular initiating events of adverse outcome pathways, related to disruption of the TH system, were used to develop 19 in silico models for identification of potential thyroid hormone system-disrupting chemicals. The conformal prediction framework with the underlying Random Forest was used as a wrapper for the models allowing for setting the desired confidence level and controlling the error rate of predictions. The trained models were then applied to two different databases: (i) an in-house database comprising xenobiotics identified in human blood and ii) currently used chemicals registered in the Swedish Product Register, which have been predicted to have a high exposure index to consumers. The application of these models showed that among currently used chemicals, fewer were overall predicted as active compared to chemicals identified in human blood. Chemicals of specific concern for TH disruption were identified from both databases based on their predicted activity.

Preclinical Safety Assessment of Therapeutic Oligonucleotides.

During the last decade, therapeutic oligonucleotide drugs (OND) have witnessed a tremendous development in chemistry and mechanistic understanding that have translated into successful clinical applications. Depending on the specific OND mechanism, chemistry, and design, the DMPK and toxicity properties can vary significantly between different OND classes and delivery approaches, the latter including lipid formulations or conjugation approaches to enhance productive OND uptake. At the same time, with the only difference between compounds being the nucleobase sequence, ONDs with same mechanism of action, chemistry, and design show relatively consistent behavior, allowing certain extrapolations between compounds within an OND class. This chapter provides a summary of the most common toxicities, the improved mechanistic understanding and the safety assessment activities performed for therapeutic oligonucleotides during the drug discovery and development process. Several of the considerations described for therapeutic applications should also be of value for the scientists mainly using oligonucleotides as research tools to explore various biological processes.

Inflammatory cell-derived CXCL3 promotes pancreatic cancer metastasis through a novel myofibroblast-hijacked cancer escape mechanism.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is the most lethal malignancy and lacks effective treatment. We aimed to understand molecular mechanisms of the intertwined interactions between tumour stromal components in metastasis and to provide a new paradigm for PDAC therapy. DESIGN: Two unselected cohorts of 154 and 20 patients with PDAC were subjected to correlation between interleukin (IL)-33 and CXCL3 levels and survivals. Unbiased expression profiling, and genetic and pharmacological gain-of-function and loss-of-function approaches were employed to identify molecular signalling in tumour-associated macrophages (TAMs) and myofibroblastic cancer-associated fibroblasts (myoCAFs). The role of the IL-33-ST2-CXCL3-CXCR2 axis in PDAC metastasis was evaluated in three clinically relevant mouse PDAC models. RESULTS: IL-33 was specifically elevated in human PDACs and positively correlated with tumour inflammation in human patients with PDAC. CXCL3 was highly upregulated in IL-33-stimulated macrophages that were the primary source of CXCL3. CXCL3 was correlated with poor survival in human patients with PDAC. Mechanistically, activation of the IL-33-ST2-MYC pathway attributed to high CXCL3 production. The highest level of CXCL3 was found in PDAC relative to other cancer types and its receptor CXCR2 was almost exclusively expressed in CAFs. Activation of CXCR2 by CXCL3 induced a CAF-to-myoCAF transition and α-smooth muscle actin (α-SMA) was uniquely upregulated by the CXCL3-CXCR2 signalling. Type III collagen was identified as the CXCL3-CXCR2-targeted adhesive molecule responsible for myoCAF-driven PDAC metastasis. CONCLUSIONS: Our work provides novel mechanistic insights into understanding PDAC metastasis by the TAM-CAF interaction and targeting each of these signalling components would provide an attractive and new paradigm for treating pancreatic cancer.

Sodium hypochlorite as an oxidizing agent for removal of soil organic matter before microplastics analyses.

The omnipresence of microplastics (MPs) across Earth's surface has raised concerns about their environmental impact and created an urgent need for methods to identify them in complex soil and sedimentary matrices. However, detecting MPs in the O horizons of soils is difficult because plastic polymers share many physical and chemical properties with natural soil organic matter (SOM). In this study, we assessed whether sodium hypochlorite (NaOCl), a reagent that can oxidize SOM and simultaneously preserve mineral constituents, can be used for MP analysis and characterization in soil environments. In addition, we scrutinized how factors such as MP size, polymer type, extraction methods, and soil matrix affect the recovery of microplastic particles. We used both hydrophobic and density-dependent separation methods to assess the effects of our oxidation treatment on the recovery of MP. We observed that NaOCl effectively removed SOM without greatly altering the surface properties of resistant MP polymers (polypropylene, polylactic acid, low-density polyethylene, and polyethylene terephthalate), which were characterized using scanning electron microscopy and Fourier-transform infrared spectroscopy after SOM removal. The NaOCl treatment caused some chlorination and formation of additional C-OH bonds on polymer surfaces, which likely contributed to the reduced efficiency of the hydrophobic-based (oil) extraction. We conclude that NaOCl treatment can improve detection of MPs in SOM-rich soil and that recovery of MPs from soils is influenced by MP size, polymer type, extraction method, and soil type, which makes it challenging to develop a universal analytical method.

Leveraging microphysiological systems to address challenges encountered during development of oligonucleotide therapeutics.

Oligonucleotide therapeutics (ONTs) encompass classes of medicines that selectively target and potentially ameliorate previously untreatable and often rare diseases. Several unique classes of ONTs provide versatility, enabling direct modu­lation of gene expression by virtue of Watson-Crick base pairing or modulation of cell signaling through structural mimicry or interference with protein-receptor interactions. Due to a lack of suitable in vitro models capable of recapitulating or predicting in vivo effects of ONTs, their discovery and optimization has relied heavily on animal studies for predicting efficacy and safety in humans. Since ONTs often lack cross-species activity, animal models with genetic humanization and/or species-specific surrogate ONTs are often required. Human microphysiological systems (MPS) offer an oppor­tunity to reduce the use of animals and may enable evaluation of drug mechanisms, optimization of cell and tissue targeting ligands or delivery vehicles, and characterization of pharmacokinetics (PK), pharmacodynamics (PD), and safety of candidate ONTs. The lack of published examples for MPS applications with ONT demonstrates the need for a focused effort to characterize and build confidence in their utility. The goals of this review are to summarize the current landscape of ONTs and highlight potential opportunities and challenges for application of MPS during ONT discovery and development. In addition, this review aims to raise awareness with ONT drug developers and regulatory authorities on the potential impact of MPS with respect to characterizing pharmacology, ADME, and toxicity and to educate MPS platform developers on unique design attributes needed to fully appreciate MPS advantages in ONT development.

Dendritic cell paucity in mismatch repair-proficient colorectal cancer liver metastases limits immune checkpoint blockade efficacy.

Liver metastasis is a major cause of mortality for patients with colorectal cancer (CRC). Mismatch repair-proficient (pMMR) CRCs make up about 95% of metastatic CRCs, and are unresponsive to immune checkpoint blockade (ICB) therapy. Here we show that mouse models of orthotopic pMMR CRC liver metastasis accurately recapitulate the inefficacy of ICB therapy in patients, whereas the same pMMR CRC tumors are sensitive to ICB therapy when grown subcutaneously. To reveal local, nonmalignant components that determine CRC sensitivity to treatment, we compared the microenvironments of pMMR CRC cells grown as liver metastases and subcutaneous tumors. We found a paucity of both activated T cells and dendritic cells in ICB-treated orthotopic liver metastases, when compared with their subcutaneous tumor counterparts. Furthermore, treatment with Feline McDonough sarcoma (FMS)-like tyrosine kinase 3 ligand (Flt3L) plus ICB therapy increased dendritic cell infiltration into pMMR CRC liver metastases and improved mouse survival. Lastly, we show that human CRC liver metastases and microsatellite stable (MSS) primary CRC have a similar paucity of T cells and dendritic cells. These studies indicate that orthotopic tumor models, but not subcutaneous models, should be used to guide human clinical trials. Our findings also posit dendritic cells as antitumor components that can increase the efficacy of immunotherapies against pMMR CRC.