Super-resolution GSDIM microscopy unveils distinct nanoscale characteristics of DNA repair foci under diverse genotoxic stress.

DNA double-strand breaks initiate the DNA damage response (DDR), leading to the accumulation of repair proteins at break sites and the formation of the-so-called foci. Various microscopy methods, such as wide-field, confocal, electron, and super-resolution microscopy, have been used to study these structures. However, the impact of different DNA-damaging agents on their (nano)structure remains unclear. Utilising GSDIM super-resolution microscopy, here we investigated the distribution of fluorescently tagged DDR proteins (53BP1, RNF168, MDC1) and γH2AX in U2OS cells treated with γ-irradiation, etoposide, cisplatin, or hydroxyurea. Our results revealed that both foci structure and their nanoscale ultrastructure, including foci size, nanocluster characteristics, fluorophore density and localisation, can be significantly altered by different inducing agents, even ones with similar mechanisms. Furthermore, distinct behaviours of DDR proteins were observed under the same treatment. These findings have implications for cancer treatment strategies involving these agents and provide insights into the nanoscale organisation of the DDR.

Using picoliter droplet deposition to track clonal competition in adherent and organoid cancer cell cultures.

Clonal growth and competition underlie processes of key relevance in etiology, progression and therapy response across all cancers. Here, we demonstrate a novel experimental approach, based on multi-color, fluorescent tagging of cell nuclei, in combination with picoliter droplet deposition, to study the clonal dynamics in two- and three-dimensional cell cultures. The method allows for the simultaneous visualization and analysis of multiple clones in individual multi-clonal colonies, providing a powerful tool for studying clonal dynamics and identifying clonal populations with distinct characteristics. Results of our experiments validate the utility of the method in studying clonal dynamics in vitro, and reveal differences in key aspects of clonal behavior of different cancer cell lines in monoculture conditions, as well as in co-cultures with stromal fibroblasts.

High-Content and High-Throughput Clonogenic Survival Assay Using Fluorescence Barcoding.

The Clonogenic Survival Assay (CSA) is a fundamental tool employed to assess cell survival and proliferative potential in cancer research. Despite its importance, CSA faces limitations, primarily its time- and labor-intensive nature and its binary output. To overcome these limitations and enhance CSA's utility, several approaches have been developed, focusing on increasing the throughput. However, achieving both high-content and high-throughput analyses simultaneously has remained a challenge. In this paper, we introduce LeGO-CSA, an extension of the classical CSA that employs the imaging of cell nuclei barcoded with fluorescent lentiviral gene ontology markers, enabling both high-content and high-throughput analysis. To validate our approach, we contrasted it with results from a classical assay and conducted a proof-of-concept screen of small-molecule inhibitors targeting various pathways relevant to cancer treatment. Notably, our results indicate that the classical CSA may underestimate clonogenicity and unveil intriguing aspects of clonal cell growth. We demonstrate the potential of LeGO-CSA to offer a robust approach for assessing cell survival and proliferation with enhanced precision and throughput, with promising implications for accelerating drug discovery and contributing to a more comprehensive understanding of cellular behavior in cancer.

Levamisole suppresses activation and proliferation of human T cells by the induction of a p53-dependent DNA damage response.

Levamisole (LMS) is a small molecule used in the treatment of idiopathic nephrotic syndrome (INS). The pathogenesis of INS remains unknown, but evidence points toward an immunological basis of the disease. Recently, LMS has been shown to increase the relapse-free survival in INS patients. While LMS has been hypothesized to exert an immunomodulatory effect, its mechanism of action remains unknown. Here, we show that LMS decreased activation and proliferation of human T cells. T-cell activation-associated cytokines such as IL-2, TNF-α, and IFN-γ were reduced upon LMS treatment, whereas IL-4 and IL-13 were increased. Gene expression profiling confirmed that the suppressive effects of LMS as genes involved in cell cycle progression were downregulated. Furthermore, genes associated with p53 activation were upregulated by LMS. In agreement, LMS treatment resulted in p53 phosphorylation and increased expression of the p53 target gene FAS. Accordingly, LMS sensitized activated T cells for Fas-mediated apoptosis. LMS treatment resulted in a mid-S phase cell cycle arrest accompanied by γH2AX-foci formation and phosphorylation of CHK1. Our findings indicate that LMS acts as an immunosuppressive drug that directly affects the activation and proliferation of human T cells by induction of DNA damage and the activation of a p53-dependent DNA damage response.

Perturbation of Copper Homeostasis Sensitizes Cancer Cells to Elevated Temperature.

Temporary elevation of tumor temperature, also known as hyperthermia, is a safe and well-tolerated treatment modality. The efficacy of hyperthermia can be improved by efficient thermosensitizers, and various candidate drugs, including inhibitors of the heat stress response, have been explored in vitro and in animal models, but clinically relevant thermosensitizers are lacking. Here, we employ unbiased in silico approaches to uncover new mechanisms and compounds that could be leveraged to increase the thermosensitivity of cancer cells. We then focus on elesclomol, a well-performing compound, which amplifies cell killing by hyperthermia by 5- to 20-fold in cell lines and outperforms clinically applied chemotherapy when combined with hyperthermia in vitro. Surprisingly, our findings suggest that the thermosensitizing effects of elesclomol are independent of its previously reported modes of action but depend on copper shuttling. Importantly, we show that, like elesclomol, multiple other copper shuttlers can thermosensitize, suggesting that disturbing copper homeostasis could be a general strategy for improving the efficacy of hyperthermia.

Reduction in PA28αß activation in HD mouse brain correlates to increased mHTT aggregation in cell models.

Huntington's disease is an autosomal dominant heritable disorder caused by an expanded CAG trinucleotide repeat at the N-terminus of the Huntingtin (HTT) gene. Lowering the levels of soluble mutant HTT protein prior to aggregation through increased degradation by the proteasome would be a therapeutic strategy to prevent or delay the onset of disease. Native PAGE experiments in HdhQ150 mice and R6/2 mice showed that PA28αß disassembles from the 20S proteasome during disease progression in the affected cortex, striatum and hippocampus but not in cerebellum and brainstem. Modulating PA28αß activated proteasomes in various in vitro models showed that PA28αß improved polyQ degradation, but decreased the turnover of mutant HTT. Silencing of PA28αß in cells lead to an increase in mutant HTT aggregates, suggesting that PA28αß is critical for overall proteostasis, but only indirectly affects mutant HTT aggregation.

Evaluation of the Heat Shock Protein 90 Inhibitor Ganetespib as a Sensitizer to Hyperthermia-Based Cancer Treatments.

Hyperthermia is being used as a radio- and chemotherapy sensitizer for a growing range of tumor subtypes in the clinic. Its potential is limited, however, by the ability of cancer cells to activate a protective mechanism known as the heat stress response (HSR). The HSR is marked by the rapid overexpression of molecular chaperones, and recent advances in drug development make their inhibition an attractive option to improve the efficacy of hyperthermia-based therapies. Our previous in vitro work showed that a single, short co-treatment with a HSR (HSP90) inhibitor ganetespib prolongs and potentiates the effects of hyperthermia on DNA repair, enhances hyperthermic sensitization to radio- and chemotherapeutic agents, and reduces thermotolerance. In the current study, we first validated these results using an extended panel of cell lines and more robust methodology. Next, we examined the effects of hyperthermia and ganetespib on global proteome changes. Finally, we evaluated the potential of ganetespib to boost the efficacy of thermo-chemotherapy and thermo-radiotherapy in a xenograft murine model of cervix cancer. Our results revealed new insights into the effects of HSR inhibition on cellular responses to heat and show that ganetespib could be employed to increase the efficacy of hyperthermia when combined with radiation.

A simple microscopy setup for visualizing cellular responses to DNA damage at particle accelerator facilities.

Cellular responses to DNA double-strand breaks (DSBs) not only promote genomic integrity in healthy tissues, but also largely determine the efficacy of many DNA-damaging cancer treatments, including X-ray and particle therapies. A growing body of evidence suggests that activation of the mechanisms that detect, signal and repair DSBs may depend on the complexity of the initiating DNA lesions. Studies focusing on this, as well as on many other radiobiological questions, require reliable methods to induce DSBs of varying complexity, and to visualize the ensuing cellular responses. Accelerated particles of different energies and masses are exceptionally well suited for this task, due to the nature of their physical interactions with the intracellular environment, but visualizing cellular responses to particle-induced damage - especially in their early stages - at particle accelerator facilities, remains challenging. Here we describe a straightforward approach for real-time imaging of early response to particle-induced DNA damage. We rely on a transportable setup with an inverted fluorescence confocal microscope, tilted at a small angle relative to the particle beam, such that cells can be irradiated and imaged without any microscope or beamline modifications. Using this setup, we image and analyze the accumulation of fluorescently-tagged MDC1, RNF168 and 53BP1-key factors involved in DSB signalling-at DNA lesions induced by 254 MeV α-particles. Our results provide a demonstration of technical feasibility and reveal asynchronous initiation of accumulation of these proteins at different individual DSBs.

Ultra-soft X-ray system for imaging the early cellular responses to X-ray induced DNA damage.

The majority of the proteins involved in processing of DNA double-strand breaks (DSBs) accumulate at the damage sites. Real-time imaging and analysis of these processes, triggered by the so-called microirradiation using UV lasers or heavy particle beams, yielded valuable insights into the underlying DSB repair mechanisms. To study the temporal organization of DSB repair responses triggered by a more clinically-relevant DNA damaging agent, we developed a system coined X-ray multi-microbeam microscope (XM3), capable of simultaneous high dose-rate (micro)irradiation of large numbers of cells with ultra-soft X-rays and imaging of the ensuing cellular responses. Using this setup, we analyzed the changes in real-time kinetics of MRE11, MDC1, RNF8, RNF168 and 53BP1-proteins involved in the signaling axis of mammalian DSB repair-in response to X-ray and UV laser-induced DNA damage, in non-cancerous and cancer cells and in the presence or absence of a photosensitizer. Our results reveal, for the first time, the kinetics of DSB signaling triggered by X-ray microirradiation and establish XM3 as a powerful platform for real-time analysis of cellular DSB repair responses.

Nanoparticle-Aided Characterization of Arterial Endothelial Architecture during Atherosclerosis Progression and Metabolic Therapy.

Atherosclerosis is associated with a compromised endothelial barrier, facilitating the accumulation of immune cells and macromolecules in atherosclerotic lesions. In this study, we investigate endothelial barrier integrity and the enhanced permeability and retention (EPR) effect during atherosclerosis progression and therapy in Apoe-/- mice using hyaluronan nanoparticles (HA-NPs). Utilizing ultrastructural and en face plaque imaging, we uncover a significantly decreased junction continuity in the atherosclerotic plaque-covering endothelium compared to the normal vessel wall, indicative of disrupted endothelial barrier. Intriguingly, the plaque advancement had a positive effect on junction stabilization, which correlated with a 3-fold lower accumulation of in vivo administrated HA-NPs in advanced plaques compared to early counterparts. Furthermore, by using super-resolution and correlative light and electron microscopy, we trace nanoparticles in the plaque microenvironment. We find nanoparticle-enriched endothelial junctions, containing 75% of detected HA-NPs, and a high HA-NP accumulation in the endothelium-underlying extracellular matrix, which suggest an endothelial junctional traffic of HA-NPs to the plague. Finally, we probe the EPR effect by HA-NPs in the context of metabolic therapy with a glycolysis inhibitor, 3PO, proposed as a vascular normalizing strategy. The observed trend of attenuated HA-NP uptake in aortas of 3PO-treated mice coincides with the endothelial silencing activity of 3PO, demonstrated in vitro. Interestingly, the therapy also reduced the plaque inflammatory burden, while activating macrophage metabolism. Our findings shed light on natural limitations of nanoparticle accumulation in atherosclerotic plaques and provide mechanistic insight into nanoparticle trafficking across the atherosclerotic endothelium. Furthermore, our data contribute to the rising field of endothelial barrier modulation in atherosclerosis.

Hyaluronan Nanoparticles Selectively Target Plaque-Associated Macrophages and Improve Plaque Stability in Atherosclerosis.

Hyaluronan is a biologically active polymer, which can be formulated into nanoparticles. In our study, we aimed to probe atherosclerosis-associated inflammation by using hyaluronan nanoparticles and to determine whether they can ameliorate atherosclerosis. Hyaluronan nanoparticles (HA-NPs) were prepared by reacting amine-functionalized oligomeric hyaluronan (HA) with cholanic ester and labeled with a fluorescent or radioactive label. HA-NPs were characterized in vitro by several advanced microscopy methods. The targeting properties and biodistribution of HA-NPs were studied in apoe-/- mice, which received either fluorescent or radiolabeled HA-NPs and were examined ex vivo by flow cytometry or nuclear techniques. Furthermore, three atherosclerotic rabbits received 89Zr-HA-NPs and were imaged by PET/MRI. The therapeutic effects of HA-NPs were studied in apoe-/- mice, which received weekly doses of 50 mg/kg HA-NPs during a 12-week high-fat diet feeding period. Hydrated HA-NPs were ca. 90 nm in diameter and displayed very stable morphology under hydrolysis conditions. Flow cytometry revealed a 6- to 40-fold higher uptake of Cy7-HA-NPs by aortic macrophages compared to normal tissue macrophages. Interestingly, both local and systemic HA-NP-immune cell interactions significantly decreased over the disease progression. 89Zr-HA-NPs-induced radioactivity in atherosclerotic aortas was 30% higher than in wild-type controls. PET imaging of rabbits revealed 6-fold higher standardized uptake values compared to the muscle. The plaques of HA-NP-treated mice contained 30% fewer macrophages compared to control and free HA-treated group. In conclusion, we show favorable targeting properties of HA-NPs, which can be exploited for PET imaging of atherosclerosis-associated inflammation. Furthermore, we demonstrate the anti-inflammatory effects of HA-NPs in atherosclerosis.

Human salivary peptide histatin-1 stimulates epithelial and endothelial cell adhesion and barrier function.

Histatins are multifunctional histidine-rich peptides secreted by the salivary glands and exclusively present in the saliva of higher primates, where they play a fundamental role in the protection of the oral cavity. Our previously published results demonstrated that histatin-1 (Hst1) promotes cell-substrate adhesion in various cell types and hinted that it could also be involved in cell-cell adhesion, a process of fundamental importance to epithelial and endothelial barriers. Here we explore the effects of Hst1 on cellular barrier function. We show that Hst1 improved endothelial barrier integrity, decreased its permeability for large molecules, and prevented translocation of bacteria across epithelial cell layers. These effects are mediated by the adherens junction protein E-cadherin (E-cad) and by the tight junction protein zonula occludens 1, as Hst1 increases the levels of zonula occludens 1 and of active E-cad. Hst1 may also promote epithelial differentiation as Hst1 induced transcription of the epithelial cell differentiation marker apolipoprotein A-IV (a downstream E-cad target). In addition, Hst1 counteracted the effects of epithelial-mesenchymal transition inducers on the outgrowth of oral cancer cell spheroids, suggesting that Hst1 affects processes that are implicated in cancer progression.-Van Dijk, I. A., Ferrando, M. L., van der Wijk, A.-E., Hoebe, R. A., Nazmi, K., de Jonge, W. J., Krawczyk, P. M., Bolscher, J. G. M., Veerman, E. C. I., Stap, J. Human salivary peptide histatin-1 stimulates epithelial and endothelial cell adhesion and barrier function.

SPAD imagers for super resolution localization microscopy enable analysis of fast fluorophore blinking.

sCMOS imagers are currently utilized (replacing EMCCD imagers) to increase the acquisition speed in super resolution localization microscopy. Single-photon avalanche diode (SPAD) imagers feature frame rates per bit depth comparable to or higher than sCMOS imagers, while generating microsecond 1-bit-frames without readout noise, thus paving the way to in-depth time-resolved image analysis. High timing resolution can also be exploited to explore fluorescent dye blinking and other photophysical properties, which can be used for dye optimization. We present the methodology for the blinking analysis of fluorescent dyes on experimental data. Furthermore, the recent use of microlenses has enabled a substantial increase of SPAD imager overall sensitivity (12-fold in our case), reaching satisfactory values for sensitivity-critical applications. This has allowed us to record the first super resolution localization microscopy results obtained with a SPAD imager, with a localization uncertainty of 20 nm and a resolution of 80 nm.

Photon-Counting Arrays for Time-Resolved Imaging.

The paper presents a camera comprising 512 × 128 pixels capable of single-photon detection and gating with a maximum frame rate of 156 kfps. The photon capture is performed through a gated single-photon avalanche diode that generates a digital pulse upon photon detection and through a digital one-bit counter. Gray levels are obtained through multiple counting and accumulation, while time-resolved imaging is achieved through a 4-ns gating window controlled with subnanosecond accuracy by a field-programmable gate array. The sensor, which is equipped with microlenses to enhance its effective fill factor, was electro-optically characterized in terms of sensitivity and uniformity. Several examples of capture of fast events are shown to demonstrate the suitability of the approach.

Radioprotection of IDH1-Mutated Cancer Cells by the IDH1-Mutant Inhibitor AGI-5198.

Isocitrate dehydrogenase 1 (IDH1) is mutated in various types of human cancer to IDH1(R132H), a structural alteration that leads to catalysis of α-ketoglutarate to the oncometabolite D-2-hydroxyglutarate. In this study, we present evidence that small-molecule inhibitors of IDH1(R132H) that are being developed for cancer therapy may pose risks with coadministration of radiotherapy. Cancer cells heterozygous for the IDH1(R132H) mutation exhibited less IDH-mediated production of NADPH, such that after exposure to ionizing radiation (IR), there were higher levels of reactive oxygen species, DNA double-strand breaks, and cell death compared with IDH1 wild-type cells. These effects were reversed by the IDH1(R132H) inhibitor AGI-5198. Exposure of IDH1 wild-type cells to D-2-hydroxyglutarate was sufficient to reduce IDH-mediated NADPH production and increase IR sensitivity. Mechanistic investigations revealed that the radiosensitivity of heterozygous cells was independent of the well-described DNA hypermethylation phenotype in IDH1-mutated cancers. Thus, our results argue that altered oxidative stress responses are a plausible mechanism to understand the radiosensitivity of IDH1-mutated cancer cells. Further, they offer an explanation for the relatively longer survival of patients with IDH1-mutated tumors, and they imply that administration of IDH1(R132H) inhibitors in these patients may limit irradiation efficacy in this setting.

Re-scan confocal microscopy: scanning twice for better resolution.

We present a new super-resolution technique, Re-scan Confocal Microscopy (RCM), based on standard confocal microscopy extended with an optical (re-scanning) unit that projects the image directly on a CCD-camera. This new microscope has improved lateral resolution and strongly improved sensitivity while maintaining the sectioning capability of a standard confocal microscope. This simple technology is typically useful for biological applications where the combination high-resolution and high-sensitivity is required.

Increased plasticity of the nuclear envelope and hypermobility of telomeres due to the loss of A-type lamins.

BACKGROUND: The nuclear lamina provides structural support to the nucleus and has a central role in defining nuclear organization. Defects in its filamentous constituents, the lamins, lead to a class of diseases collectively referred to as laminopathies. On the cellular level, lamin mutations affect the physical integrity of nuclei and nucleo-cytoskeletal interactions, resulting in increased susceptibility to mechanical stress and altered gene expression. METHODS: In this study we quantitatively compared nuclear deformation and chromatin mobility in fibroblasts from a homozygous nonsense LMNA mutation patient and a Hutchinson-Gilford progeria syndrome patient with wild type dermal fibroblasts, based on the visualization of mCitrine labeled telomere-binding protein TRF2 with light-economical imaging techniques and cytometric analyses. RESULTS: Without application of external forces, we found that the absence of functional lamin A/C leads to increased nuclear plasticity on the hour and minute time scale but also to increased intranuclear mobility down to the second time scale. In contrast, progeria cells show overall reduced nuclear dynamics. Experimental manipulation (farnesyltransferase inhibition or lamin A/C silencing) confirmed that these changes in mobility are caused by abnormal or reduced lamin A/C expression. CONCLUSIONS: These observations demonstrate that A-type lamins affect both nuclear membrane and telomere dynamics. GENERAL SIGNIFICANCE: Because of the pivotal role of dynamics in nuclear function, these differences likely contribute to or represent novel mechanisms in laminopathy development.

Analysis of the mobility of DNA double-strand break-containing chromosome domains in living mammalian cells.

DNA double-strand breaks (DSBs) are among the most dangerous types of DNA damage. Unrepaired, DSBs may lead to cell death, and when misrejoined, they can result in potentially carcinogenic chromosome rearrangements. The induction of DSBs and their repair take place in a chromatin microenvironment. Therefore, understanding and describing the dynamics of DSB-containing chromatin is of crucial importance for understanding interactions among DSBs and their repair. Recent developments have made it possible to study ionizing radiation-induced foci of DSB repair proteins in vivo. In this chapter, we describe techniques that can be applied to visualize and analyze the spatio-temporal dynamics of DSB-containing chromatin domains in mammalian cell nuclei. Analogous procedures may also be applied to the analysis of mobility of other intranuclear structures in living cells.

Dynamics of DNA double-strand breaks revealed by clustering of damaged chromosome domains.

Interactions between ends from different DNA double-strand breaks (DSBs) can produce tumorigenic chromosome translocations. Two theories for the juxta-position of DSBs in translocations, the static "contact-first" and the dynamic "breakage-first" theory, differ fundamentally in their requirement for DSB mobility. To determine whether or not DSB-containing chromosome domains are mobile and can interact, we introduced linear tracks of DSBs in nuclei. We observed changes in track morphology within minutes after DSB induction, indicating movement of the domains. In a subpopulation of cells, the domains clustered. Juxtaposition of different DSB-containing chromosome domains through clustering, which was most extensive in G1 phase cells, suggests an adhesion process in which we implicate the Mre11 complex. Our results support the breakage-first theory to explain the origin of chromosomal translocations.