Superresolution microscopy localizes endogenous Dvl2 to Wnt signaling-responsive biomolecular condensates.

During organismal development, homeostasis, and disease, Dishevelled (Dvl) proteins act as key signaling factors in beta-catenin-dependent and beta-catenin-independent Wnt pathways. While their importance for signal transmission has been genetically demonstrated in many organisms, our mechanistic understanding is still limited. Previous studies using overexpressed proteins showed Dvl localization to large, punctate-like cytoplasmic structures that are dependent on its DIX domain. To study Dvl's role in Wnt signaling, we genome engineered an endogenously expressed Dvl2 protein tagged with an mEos3.2 fluorescent protein for superresolution imaging. First, we demonstrate the functionality and specificity of the fusion protein in beta-catenin-dependent and beta-catenin-independent signaling using multiple independent assays. We performed live-cell imaging of Dvl2 to analyze the dynamic formation of the supramolecular cytoplasmic Dvl2_mEos3.2 condensates. While overexpression of Dvl2_mEos3.2 mimics the previously reported formation of abundant large "puncta," supramolecular condensate formation at physiological protein levels is only observed in a subset of cells with approximately one per cell. We show that, in these condensates, Dvl2 colocalizes with Wnt pathway components at gamma-tubulin and CEP164-positive centrosomal structures and that the localization of Dvl2 to these condensates is Wnt dependent. Single-molecule localization microscopy using photoactivated localization microscopy (PALM) of mEos3.2 in combination with DNA-PAINT demonstrates the organization and repetitive patterns of these condensates in a cell cycle-dependent manner. Our results indicate that the localization of Dvl2 in supramolecular condensates is coordinated dynamically and dependent on cell state and Wnt signaling levels. Our study highlights the formation of endogenous and physiologically regulated biomolecular condensates in the Wnt pathways at single-molecule resolution.

Extracellular vesicle-associated tyrosine kinase-like orphan receptors ROR1 and ROR2 promote breast cancer progression.

BACKGROUND: Extracellular vesicles (EVs) harbor a plethora of different biomolecules, which they can transport across cells. In cancer, tumor-derived EVs thereby support the creation of a favorable tumor microenvironment. So far, EV uptake and cargo delivery into target cells have been regarded as the main mechanisms for the pro-tumoral function of EVs. To test this hypothesis, we investigated the fate of the oncogenic transmembrane Wnt tyrosine kinase-like orphan receptor 1 and 2 (ROR1, ROR2) delivered via distinct EV subpopulations to breast cancer cells and aimed to unravel their impact on tumor progression. METHODS: EVs were isolated by differential ultracentrifugation from cell culture supernatant as well as plasma samples from healthy individuals (n = 27) and breast cancer patients (n = 41). EVs were thoroughly characterized by electron microscopy, nanoparticle tracking analysis, immunoblot, and flow cytometry. ROR transfer to target cells was observed using microscopy-based assays and biodistribution experiments were conducted in syngeneic mice. EV impact on cancer cell migration and invasion was tested in functional assays. RESULTS: We observed that the supernatant of ROR-overexpressing cells was sufficient for transferring the receptors to ROR-negative cells. Analyzing the secretome of the ROR-overexpressing cells, we detected a high enrichment of ROR1/2 on large and small EVs, but not on large oncosomes. Interestingly, the majority of ROR-positive EVs remained attached to the target cell surface after 24 h of stimulation and was quickly removed by treatment with trypsin. Nonetheless, ROR-positive EVs increased migration and invasion of breast cancer cells, even after chemically inhibiting EV uptake, in dependence of RhoA downstream signaling. In vivo, ROR-depleted EVs tended to distribute less into organs prone for the formation of breast cancer metastases. ROR-positive EVs were also significantly elevated in the plasma of breast cancer patients and allowed to separate them from healthy controls. CONCLUSIONS: The oncogenic Wnt receptors ROR1/2 are transferred via EVs to the surface of ROR-negative cancer cells, in which they induce an aggressive phenotype supporting tumor progression. Video Abstract.

In utero exposures to perfluoroalkyl substances and the human fetal liver metabolome in Scotland: a cross-sectional study.

BACKGROUND: Perfluoroalkyl and polyfluoroalkyl substances are classed as endocrine disrupting compounds but continue to be used in many products such as firefighting foams, flame retardants, utensil coatings, and waterproofing of food packaging. Perfluoroalkyl exposure aberrantly modulates lipid, metabolite, and bile acid levels, increasing susceptibility to onset and severity of metabolic diseases, such as diabetes and metabolic dysfunction-associated steatotic liver disease. To date, most studies in humans have focused on perfluoroalkyl-exposure effects in adults. In this study we aimed to show if perfluoroalkyls are present in the human fetal liver and if they have metabolic consequences for the human fetus. METHODS: In this cross-sectional study, human fetal livers from elective termination of pregnancies at the Aberdeen Pregnancy Counselling Service, Aberdeen, UK, were analysed by both targeted (bile acids and perfluoroalkyl substances) and combined targeted and untargeted (lipids and polar metabolites) mass spectrometry based metabolomic analyses, as well as with RNA-Seq. Only fetuses from normally progressing pregnancies (determined at ultrasound scan before termination), terminated for non-medical reasons, from women older than 16 years, fluent in English, and between 11 and 21 weeks of gestation were collected. Women exhibiting considerable emotional distress or whose fetuses had anomalies identified at ultrasound scan were excluded. Stringent bioinformatic and statistical methods such as partial correlation network analysis, linear regression, and pathway analysis were applied to this data to investigate the association of perfluoroalkyl exposure with hepatic metabolic pathways. FINDINGS: Fetuses included in this study were collected between Dec 2, 2004, and Oct 27, 2014. 78 fetuses were included in the study: all 78 fetuses were included in the metabolomics analysis (40 female and 38 male) and 57 fetuses were included in the RNA-Seq analysis (28 female and 29 male). Metabolites associated with perfluoroalkyl were identified in the fetal liver and these varied with gestational age. Conjugated bile acids were markedly positively associated with fetal age. 23 amino acids, fatty acids, and sugar derivatives in fetal livers were inversely associated with perfluoroalkyl exposure, and the bile acid glycolithocholic acid was markedly positively associated with all quantified perfluoroalkyl. Furthermore, 7α-hydroxy-4-cholesten-3-one, a marker of bile acid synthesis rate, was strongly positively associated with perfluoroalkyl levels and was detectable as early as gestational week 12. INTERPRETATION: Our study shows direct evidence for the in utero effects of perfluoroalkyl exposure on specific key hepatic products. Our results provide evidence that perfluoroalkyl exposure, with potential future consequences, manifests in the human fetus as early as the first trimester of gestation. Furthermore, the profiles of metabolic changes resemble those observed in perinatal perfluoroalkyl exposures. Such exposures are already linked with susceptibility, initiation, progression, and exacerbation of a wide range of metabolic diseases. FUNDING: UK Medical Research Council, Horizon Europe Program of the European Union, Seventh Framework Programme of the European Union, NHS Grampian Endowments grants, European Partnership for the Assessment of Risks from Chemicals, Swedish Research Council, Formas, Novo Nordisk Foundation, and the Academy of Finland.

Outlook of women in science: an interview with our authors.

Diversity in research teams ties alternative perspectives into research projects, and this can fast-forward scientific progress. Concerted efforts have been aimed at encouraging and supporting women to pursue a career in science, yet a gender disparity can still be observed at senior positions, with fewer women in leadership roles. To get insight into how the current landscape for women in science is perceived by different career stages, we interviewed female authors of Molecular Oncology from diverse career stages and disciplines about their inspiration, challenges they have faced as scientists as well as their thoughts on how gender diversity can be further enhanced.

Cytokine release syndrome-like serum responses after COVID-19 vaccination are frequent and clinically inapparent under cancer immunotherapy.

Patients with cancer frequently receive immune-checkpoint inhibitors (ICIs), which may modulate immune responses to COVID-19 vaccines. Recently, cytokine release syndrome (CRS) was observed in a patient with cancer who received BTN162b2 vaccination under ICI treatment. Here, we analyzed adverse events and serum cytokines in patients with 23 different tumors undergoing (n = 64) or not undergoing (n = 26) COVID-19 vaccination under ICI therapy in a prospectively planned German single-center cohort study (n = 220). We did not observe clinically relevant CRS (≥grade 2) after vaccination (95% CI 0-5.6%; Common Terminology of Adverse Events v.5.0) in this small cohort. Within 4 weeks after vaccination, serious adverse events occurred in eight patients (12.5% 95% CI 5.6-23%): six patients were hospitalized due to events common under cancer therapy including immune related adverse events and two patients died due to conditions present before vaccination. Despite absence of CRS symptoms, a set of pairwise-correlated CRS-associated cytokines, including CXCL8 and interleukin-6 was >1.5-fold upregulated in 40% (95% CI 23.9-57.9%) of patients after vaccination. Hence, elevated cytokine levels are common and not sufficient to establish CRS diagnosis.

Extracellular vesicles and oncogenic signaling.

In recent years, extracellular vesicles (EVs) emerged as potential diagnostic and prognostic markers for cancer therapy. While the field of EV research is rapidly developing and their application as vehicles for therapeutic cargo is being tested, little is still known about the exact mechanisms of signaling specificity and cargo transfer by EVs, especially in vivo. Several signaling cascades have been found to use EVs for signaling in the tumor-stroma interaction. These include potentially oncogenic, verbatim transforming, signaling cascades such as Wnt and TGF-ß signaling, and other signaling cascades that have been tightly associated with tumor progression and metastasis, such as PD-L1 and VEGF signaling. Multiple mechanisms of how these signaling cascades and EVs interplay to mediate these complex processes have been described, such as direct signal activation through pathway components on or in EVs or indirectly by influencing vesicle biogenesis, cargo sorting, or uptake dynamics. In this review, we summarize the current knowledge of EVs, their biogenesis, and our understanding of EV interactions with recipient cells with a focus on selected oncogenic and cancer-associated signaling pathways. After an in-depth look at how EVs mediate and influence signaling, we discuss potentially translatable EV functions and existing knowledge gaps.

ß-catenin-independent WNT signaling and Ki67 in contrast to the estrogen receptor status are prognostic and associated with poor prognosis in breast cancer liver metastases.

Liver metastasis development in breast cancer patients is common and confers a poor prognosis. So far, the prognostic significance of surgical resection and clinical relevance of biomarker analysis in metastatic tissue have barely been investigated. We previously demonstrated an impact of WNT signaling in breast cancer brain metastasis. This study aimed to investigate the value of established prognostic markers and WNT signaling components in liver metastases. Overall N = 34 breast cancer liver metastases (with matched primaries in 19/34 cases) were included in this retrospective study. Primaries and metastatic samples were analyzed for their expression of the estrogen (ER) and progesterone receptor, HER-2, Ki67, and various WNT signaling-components by immunohistochemistry. Furthermore, ß-catenin-dependent and -independent WNT scores were generated and analyzed for their prognostic value. Additionally, the influence of the alternative WNT receptor ROR on signaling and invasiveness was analyzed in vitro. ER positivity (HR 0.09, 95 % CI 0.01-0.56) and high Ki67 (HR 3.68, 95 % CI 1.12-12.06) in the primaries had prognostic impact. However, only Ki67 remained prognostic in the metastatic tissue (HR 2.46, 95 % CI 1.11-5.44). Additionally, the ß-catenin-independent WNT score correlated with reduced overall survival only in the metastasized situation (HR 2.19, 95 % CI 1.02-4.69, p = 0.0391). This is in line with the in vitro results of the alternative WNT receptors ROR1 and ROR2, which foster invasion. In breast cancer, the value of prognostic markers established in primary tumors cannot directly be translated to metastases. Our results revealed ß-catenin-independent WNT signaling to be associated with poor prognosis in patients with breast cancer liver metastasis.