One-step rapid tracking and isolation of senescent cells in cellular systems, tissues, or animal models via GLF16.

Identification and isolation of senescent cells is challenging, rendering their detailed analysis an unmet need. We describe a precise one-step protocol to fluorescently label senescent cells, for flow cytometry and fluorescence microscopy, implementing a fluorophore-conjugated Sudan Black-B analog, GLF16. Also, a micelle-based approach allows identification of senescent cells in vivo and in vitro, enabling live-cell sorting for downstream analyses and live in vivo tracking. Our protocols are applicable to cellular systems, tissues, or animal models where senescence is present. For complete details on the use and execution of this protocol, please refer to Magkouta et al.1.

Multi-level profiling of the Fmr1 KO rat unveils altered behavioral traits along with aberrant glutamatergic function.

Fragile X syndrome (FXS) is the most common cause of inherited intellectual disabilities and the most prevalent monogenic cause of autism. Although the knockout (KO) of the Fmr1 gene homolog in mice is primarily used for elucidating the neurobiological substrate of FXS, there is limited association of the experimental data with the pathophysiological condition in humans. The use of Fmr1 KO rats offers additional translational validity in this regard. Therefore, we employed a multi-level approach to study the behavioral profile and the glutamatergic and GABAergic neurotransmission status in pathophysiology-associated brain structures of Fmr1 KO rats, including the recordings of evoked and spontaneous field potentials from hippocampal slices, paralleled with next-generation RNA sequencing (RNA-seq). We found that these rats exhibit hyperactivity and cognitive deficits, along with characteristic bidirectional glutamatergic and GABAergic alterations in the prefrontal cortex and the hippocampus. These results are coupled to affected excitability and local inhibitory processes in the hippocampus, along with a specific transcriptional profile, highlighting dysregulated hippocampal network activity in KO rats. Overall, our data provide novel insights concerning the biobehavioral profile of FmR1 KO rats and translationally upscales our understanding on pathophysiology and symptomatology of FXS syndrome.

Senescent cells in giant cell arteritis display an inflammatory phenotype participating in tissue injury via IL-6-dependent pathways.

OBJECTIVES: Age is the strongest risk factor of giant cell arteritis (GCA), implying a possible pathogenetic role of cellular senescence. To address this question, we applied an established senescence specific multimarker algorithm in temporal artery biopsies (TABs) of GCA patients. METHODS: 75(+) TABs from GCA patients, 22(-) TABs from polymyalgia rheumatica (PMR) patients and 10(-) TABs from non-GCA/non-PMR patients were retrospectively retrieved and analysed. Synovial tissue specimens from patients with inflammatory arthritis and aorta tissue were used as disease control samples. Senescent cells and their histological origin were identified with specific cellular markers; IL-6 and MMP-9 were investigated as components of the senescent associated secretory phenotype by triple costaining. GCA or PMR artery culture supernatants were applied to fibroblasts, HUVECs and monocytes with or without IL-6R blocking agent to explore the induction of IL-6-associated cellular senescence. RESULTS: Senescent cells were present in GCA arteries at higher proportion compared with PMR (9.50% vs 2.66%, respectively, p<0.0001) and were mainly originated from fibroblasts, macrophages and endothelial cells. IL-6 was expressed by senescent fibroblasts, and macrophages while MMP-9 by senescent fibroblasts only. IL-6(+) senescent cells were associated with the extension of vascular inflammation (transmural inflammation vs adventitia limited disease: 10.02% vs 4.37%, respectively, p<0.0001). GCA but not PMR artery culture supernatant could induce IL-6-associated senescence that was partially inhibited by IL-6R blockade. CONCLUSIONS: Senescent cells with inflammatory phenotype are present in GCA arteries and are associated with the tissue inflammatory bulk, suggesting a potential implication in disease pathogenesis.

A fluorophore-conjugated reagent enabling rapid detection, isolation and live tracking of senescent cells.

Cellular senescence is a stress-response mechanism implicated in various physiological processes, diseases, and aging. Current detection approaches have partially addressed the issue of senescent cell identification in clinical specimens. Effective methodologies enabling precise isolation or live tracking of senescent cells are still lacking. In-depth analysis of truly senescent cells is, therefore, an extremely challenging task. We report (1) the synthesis and validation of a fluorophore-conjugated, Sudan Black-B analog (GLF16), suitable for in vivo and in vitro analysis of senescence by fluorescence microscopy and flow cytometry and (2) the development and application of a GLF16-carrying micelle vector facilitating GLF16 uptake by living senescent cells in vivo and in vitro. The compound and the applied methodology render isolation of senescent cells an easy, rapid, and precise process. Straightforward nanocarrier-mediated GLF16 delivery in live senescent cells comprises a unique tool for characterization of senescence at an unprecedented depth.

An optimized protocol for chromatin immunoprecipitation from murine inguinal white adipose tissue.

Chromatin immunoprecipitation (ChIP) protocols have been used to reveal protein-DNA interactions of various cell types and tissues; however, optimization is required for each specific type of sample. Here, we present a ChIP protocol from murine inguinal white adipose tissue. We describe steps for tissue harvesting, crosslinking, chromatin extraction, shearing, immunoprecipitation, and purification. We then detail procedures for analysis including library preparation, sequencing, and qRT-PCR validation. For complete details on the use and execution of this protocol, please refer to Antonia Katsouda et al. (2022).1.

Microtubule Dynamics Deregulation Induces Apoptosis in Human Urothelial Bladder Cancer Cells via a p53-Independent Pathway.

Bladder cancer (BLCA) is the sixth most common type of cancer and has a dismal prognosis if diagnosed late. To identify treatment options for BLCA, we systematically evaluated data from the Broad Institute DepMap project. We found that urothelial BLCA cell lines are among the most sensitive to microtubule assembly inhibition by paclitaxel treatment. Strikingly, we revealed that the top dependencies in BLCA cell lines include genes encoding proteins involved in microtubule assembly. This highlights the importance of microtubule network dynamics as a major vulnerability in human BLCA. In cancers such as ovarian and breast, where paclitaxel is the gold standard of care, resistance to paclitaxel treatment has been linked to p53-inactivating mutations. To study the response of BLCA to microtubule assembly inhibition and its mechanistic link with the mutational status of the p53 protein, we treated a collection of BLCA cell lines with a dose range of paclitaxel and performed a detailed characterization of the response. We discovered that BLCA cell lines are significantly sensitive to low concentrations of paclitaxel, independently of their p53 status. Paclitaxel induced a G2/M cell cycle arrest and growth inhibition, followed by robust activation of apoptosis. Most importantly, we revealed that paclitaxel triggered a robust DNA-damage response and apoptosis program without activating the p53 pathway. Integration of transcriptomics, epigenetic, and dependency data demonstrated that the response of BLCA to paclitaxel is independent of p53 mutational signatures but strongly depends on the expression of DNA repair genes. Our work highlights urothelial BLCA as an exceptional candidate for paclitaxel treatment. It paves the way for the rational use of a combination of paclitaxel and DNA repair inhibitors as an effective, novel therapeutic strategy.

Combinatorial targeting of a specific EMT/MET network by macroH2A variants safeguards mesenchymal identity.

Generation of induced pluripotent stem cells from specialized cell types provides an excellent model to study how cells maintain their stability, and how they can change identity, especially in the context of disease. Previous studies have shown that chromatin safeguards cell identity by acting as a barrier to reprogramming. We investigated mechanisms by which the histone macroH2A variants inhibit reprogramming and discovered that they work as gate keepers of the mesenchymal cell state by blocking epithelial transition, a step required for reprogramming of mouse fibroblasts. More specifically, we found that individual macroH2A variants regulate the expression of defined sets of genes, whose overall function is to stabilize the mesenchymal gene expression program, thus resisting reprogramming. We identified a novel gene network (MSCN, mesenchymal network) composed of 63 macroH2A-regulated genes related to extracellular matrix, cell membrane, signaling and the transcriptional regulators Id2 and Snai2, all of which function as guardians of the mesenchymal phenotype. ChIP-seq and KD experiments revealed a macroH2A variant-specific combinatorial targeting of the genes reconstructing the MSCN, thus generating robustness in gene expression programs to resist cellular reprogramming.

The Optimized γ-Globin Lentiviral Vector GGHI-mB-3D Leads to Nearly Therapeutic HbF Levels In Vitro in CD34+ Cells from Sickle Cell Disease Patients.

We have previously demonstrated that both the original γ-globin lentiviral vector (LV) GGHI and the optimized GGHI-mB-3D LV, carrying the novel regulatory elements of the 3D HPFH-1 enhancer and the 3' ß-globin UTR, can significantly increase HbF production in thalassemic CD34+ cells and ameliorate the disease phenotype in vitro. In the present study, we investigated whether the GGHI-mB-3D vector can also exhibit an equally therapeutic effect, following the transduction of sickle cell disease (SCD) CD34+ cells at MOI 100, leading to HbF increase coupled with HbS decrease, and thus, to phenotype improvement in vitro. We show that GGHI-mB-3D LV can lead to high and potentially therapeutic HbF levels, reaching a mean 2-fold increase to a mean value of VCN/cell of 1.0 and a mean transduction efficiency of 55%. Furthermore, this increase was accompanied by a significant 1.6-fold HbS decrease, a beneficial therapeutic feature for SCD. In summary, our data demonstrate the efficacy of the optimized γ-globin lentiviral vector to improve the SCD phenotype in vitro, and highlights its potential use in future clinical SCD trials.

MPST sulfurtransferase maintains mitochondrial protein import and cellular bioenergetics to attenuate obesity.

Given the clinical, economic, and societal impact of obesity, unraveling the mechanisms of adipose tissue expansion remains of fundamental significance. We previously showed that white adipose tissue (WAT) levels of 3-mercaptopyruvate sulfurtransferase (MPST), a mitochondrial cysteine-catabolizing enzyme that yields pyruvate and sulfide species, are downregulated in obesity. Here, we report that Mpst deletion results in fat accumulation in mice fed a high-fat diet (HFD) through transcriptional and metabolic maladaptation. Mpst-deficient mice on HFD exhibit increased body weight and inguinal WAT mass, reduced metabolic rate, and impaired glucose/insulin tolerance. At the molecular level, Mpst ablation activates HIF1α, downregulates subunits of the translocase of outer/inner membrane (TIM/TOM) complex, and impairs mitochondrial protein import. MPST deficiency suppresses the TCA cycle, oxidative phosphorylation, and fatty acid oxidation, enhancing lipid accumulation. Sulfide donor administration to obese mice reverses the HFD-induced changes. These findings reveal the significance of MPST for white adipose tissue biology and metabolic health and identify a potential new therapeutic target for obesity.

Activin-A impedes the establishment of CD4+ T cell exhaustion and enhances anti-tumor immunity in the lung.

BACKGROUND: Although tumor-infiltrating T cells represent a favorable prognostic marker for cancer patients, the majority of these cells are rendered with an exhausted phenotype. Hence, there is an unmet need to identify factors which can reverse this dysfunctional profile and restore their anti-tumorigenic potential. Activin-A is a pleiotropic cytokine, exerting a broad range of pro- or anti-inflammatory functions in different disease contexts, including allergic and autoimmune disorders and cancer. Given that activin-A exhibits a profound effect on CD4+ T cells in the airways and is elevated in lung cancer patients, we hypothesized that activin-A can effectively regulate anti-tumor immunity in lung cancer. METHODS: To evaluate the effects of activin-A in the context of lung cancer, we utilized the OVA-expressing Lewis Lung Carcinoma mouse model as well as the B16F10 melanoma model of pulmonary metastases. The therapeutic potential of activin-A-treated lung tumor-infiltrating CD4+ T cells was evaluated in adoptive transfer experiments, using CD4-/--tumor bearing mice as recipients. In a reverse approach, we disrupted activin-A signaling on CD4+ T cells using an inducible model of CD4+ T cell-specific knockout of activin-A type I receptor. RNA-Sequencing analysis was performed to assess the transcriptional signature of these cells and the molecular mechanisms which mediate activin-A's function. In a translational approach, we validated activin-A's anti-tumorigenic properties using primary human tumor-infiltrating CD4+ T cells from lung cancer patients. RESULTS: Administration of activin-A in lung tumor-bearing mice attenuated disease progression, an effect associated with heightened ratio of infiltrating effector to regulatory CD4+ T cells. Therapeutic transfer of lung tumor-infiltrating activin-A-treated CD4+ T cells, delayed tumor progression in CD4-/- recipients and enhanced T cell-mediated immunity. CD4+ T cells genetically unresponsive to activin-A, failed to elicit effective anti-tumor properties and displayed an exhausted molecular signature governed by the transcription factors Tox and Tox2. Of translational importance, treatment of activin-A on tumor-infiltrating CD4+ T cells from lung cancer patients augmented their immunostimulatory capacity towards autologous CD4+ and CD8+ T cells. CONCLUSIONS: In this study, we introduce activin-A as a novel immunomodulatory factor in the lung tumor microenvironment, which bestows exhausted CD4+ T cells with effector properties.

From Proteomic Mapping to Invasion-Metastasis-Cascade Systemic Biomarkering and Targeted Drugging of Mutant BRAF-Dependent Human Cutaneous Melanomagenesis.

Melanoma is classified among the most notoriously aggressive human cancers. Despite the recent progress, due to its propensity for metastasis and resistance to therapy, novel biomarkers and oncogenic molecular drivers need to be promptly identified for metastatic melanoma. Hence, by employing nano liquid chromatography-tandem mass spectrometry deep proteomics technology, advanced bioinformatics algorithms, immunofluorescence, western blotting, wound healing protocols, molecular modeling programs, and MTT assays, we comparatively examined the respective proteomic contents of WM115 primary (n = 3955 proteins) and WM266-4 metastatic (n = 6681 proteins) melanoma cells. It proved that WM115 and WM266-4 cells have engaged hybrid epithelial-to-mesenchymal transition/mesenchymal-to-epithelial transition states, with TGF-ß controlling their motility in vitro. They are characterized by different signatures of SOX-dependent neural crest-like stemness and distinct architectures of the cytoskeleton network. Multiple signaling pathways have already been activated from the primary melanoma stage, whereas HIF1α, the major hypoxia-inducible factor, can be exclusively observed in metastatic melanoma cells. Invasion-metastasis cascade-specific sub-routines of activated Caspase-3-triggered apoptosis and LC3B-II-dependent constitutive autophagy were also unveiled. Importantly, WM115 and WM266-4 cells exhibited diverse drug response profiles, with epirubicin holding considerable promise as a beneficial drug for metastatic melanoma clinical management. It is the proteome navigation that enables systemic biomarkering and targeted drugging to open new therapeutic windows for advanced disease.

Identification of a dynamic gene regulatory network required for pluripotency factor-induced reprogramming of mouse fibroblasts and hepatocytes.

The generation of induced pluripotent stem cells (iPSCs) from somatic cells provides an excellent model to study mechanisms of transcription factor-induced global alterations of the epigenome and genome function. Here, we have investigated the early transcriptional events of cellular reprogramming triggered by the co-expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) in mouse embryonic fibroblasts (MEFs) and mouse hepatocytes (mHeps). In this analysis, we identified a gene regulatory network composed of nine transcriptional regulators (9TR; Cbfa2t3, Gli2, Irf6, Nanog, Ovol1, Rcan1, Taf1c, Tead4, and Tfap4), which are directly targeted by OSKM, in vivo. Functional studies using single and double shRNA knockdowns of any of these factors caused disruption of the network and dramatic reductions in reprogramming efficiency, indicating that this network is essential for the induction and establishment of pluripotency. We demonstrate that the stochastic co-expression of 9TR network components occurs in a remarkably small number of cells, approximating the percentage of terminally reprogrammed cells as a result of dynamic molecular events. Thus, the early DNA-binding patterns of OSKM and the subsequent probabilistic co-expression of essential 9TR components in subpopulations of cells undergoing reprogramming steer the reconstruction of a gene regulatory network marking the transition to pluripotency.