Liquid-liquid phase separation underpins the formation of replication factories in rotaviruses.

RNA viruses induce the formation of subcellular organelles that provide microenvironments conducive to their replication. Here we show that replication factories of rotaviruses represent protein-RNA condensates that are formed via liquid-liquid phase separation of the viroplasm-forming proteins NSP5 and rotavirus RNA chaperone NSP2. Upon mixing, these proteins readily form condensates at physiologically relevant low micromolar concentrations achieved in the cytoplasm of virus-infected cells. Early infection stage condensates could be reversibly dissolved by 1,6-hexanediol, as well as propylene glycol that released rotavirus transcripts from these condensates. During the early stages of infection, propylene glycol treatments reduced viral replication and phosphorylation of the condensate-forming protein NSP5. During late infection, these condensates exhibited altered material properties and became resistant to propylene glycol, coinciding with hyperphosphorylation of NSP5. Some aspects of the assembly of cytoplasmic rotavirus replication factories mirror the formation of other ribonucleoprotein granules. Such viral RNA-rich condensates that support replication of multi-segmented genomes represent an attractive target for developing novel therapeutic approaches.

Mesoporous Biodegradable Magnesium Phosphate-Citrate Nanocarriers Amplify Methotrexate Anticancer Activity in HeLa Cells.

We present the synthesis of amorphous, mesoporous, colloidal magnesium phosphate-citrate nanoparticles (MPCs) from biogenic precursors, resulting in a biocompatible and biodegradable nanocarrier that amplifies the action of the anticancer drug methotrexate (MTX). Synthesis conditions were gradually tuned to investigate the influence of the chelating agent citric acid on the colloidal stability and the mesoporosity of the obtained nanoparticles. With optimized synthesis conditions, a large BET surface area of 560 m2/g was achieved. We demonstrate the potential of these biocompatible and biodegradable mesoporous MPCs as a drug delivery system. Lipid-coated MPCs were used to load the fluorescent dye calcein and the chemotherapeutic agent MTX into the mesopores. In vitro experiments show very low premature release of the cargo but efficient stimuli-responsive release in an environment of pH 5.5, in which MPCs degrade. Lipid-coated MPCs are taken up by cancer cells and are nontoxic up to concentrations of 100 µg/mL. When loaded with MTX serving as a representative model drug for in vitro studies, MPCs induced efficient cell death with an IC50 value of 1.1 µg/mL. Compared to free MTX, its delivery with MPCs enhances its efficiency by an order of magnitude. In summary, we have developed a biodegradable nanomaterial synthesized from biocompatible precursors that are neither toxic by themselves nor in the form of nanoparticles. With these features, MPCs may be applied as drug delivery systems and have the potential to reduce the side effects of current chemotherapies.

Volumetric measurements of target lesions: does it improve inter-reader variability for oncological response assessment according to RECIST 1.1 guidelines compared to standard unidimensional measurements?

PURPOSE: Target lesion selection is known to be a major factor for inter-reader discordance in RECIST 1.1. The purpose of this study was to assess whether volumetric measurements of target lesions result in different response categorization, as opposed to standard unidimensional measurements, and to evaluate the impact on inter-reader agreement for response categorization when different readers select different sets of target lesions. MATERIAL AND METHODS: Fifty patients with measurable disease from solid tumours, in which 3 readers had blindly and independently selected different sets of target lesions and subsequently reached clinically significant discordant response categorizations (progressive disease [PD] vs. non-progressive disease [non-PD]) based on RECIST 1.1 analyses were included in this study. Additional volumetric measurements of all target lesions were performed by the same readers in a second read. Intra-reader agreement between standard unidimensional measurements (uRECIST) and volumetric measurements (vRECIST) was assessed using Cohen's k statistics. Fleiss k statistics was used to analyse the inter-reader agreement for uRECIST and vRECIST results. RESULTS: The 3 readers assigned the same response classifications based on uRECIST and vRECIST in 33/50 (66%), 42/50 patients (84%), and 44/50 patients (88%), respectively. Inter-reader agreement improved from 0% when using uRECIST to 36% when using vRECIST. CONCLUSIONS: Volumetric measurement of target lesions may improve inter-reader variability for response assessment as opposed to standard unidimensional measurements. However, in about two-thirds of patients, readers disagreed regardless of the measurement method, indicating that a limited set of target lesions may not be sufficiently representative of the whole-body tumour burden.

Supersensitive Multifluorophore RNA-FISH for Early Virus Detection and Flow-FISH by Using Click Chemistry.

The reliable detection of transcription events through the quantification of the corresponding mRNA is of paramount importance for the diagnostics of infections and diseases. The quantification and localization analysis of the transcripts of a particular gene allows disease states to be characterized more directly compared to an analysis on the transcriptome wide level. This is particularly needed for the early detection of virus infections as now required for emergent viral diseases, e. g. Covid-19. In situ mRNA analysis, however, is a formidable challenge and currently performed with sets of single-fluorophore-containing oligonucleotide probes that hybridize to the mRNA in question. Often a large number of probe strands (>30) are required to get a reliable signal. The more oligonucleotide probes are used, however, the higher the potential off-target binding effects that create background noise. Here, we used click chemistry and alkyne-modified DNA oligonucleotides to prepare multiple-fluorophore-containing probes. We found that these multiple-dye probes allow reliable detection and direct visualization of mRNA with only a very small number (5-10) of probe strands. The new method enabled the in situ detection of viral transcripts as early as 4 hours after infection.

A Chemiluminescent Metal-Organic Framework.

The synthesis and characterization of a chemiluminescent metal-organic framework with high porosity is reported. It consists of Zr6 O6 (OH)4 nodes connected by 4,4'-(anthracene-9,10-diyl)dibenzoate as the linker and luminophore. It shows the topology known for UiO-66 and is therefore denoted PAP-UiO. The MOF was not only obtained as bulk material but also as a thin film. Exposure of PAP-UiO as bulk or film to a mixture of bis-(2,4,6-trichlorophenyl) oxalate, hydrogen peroxide, and sodium salicylate in a mixture of dimethyl and dibutyl phthalate evoked strong and long lasting chemiluminescence of the PAP-UiO crystals. Time dependent fluorescence spectroscopy on bulk PAP-UiO and, for comparison, on dimethyl 4,4'-(anthracene-9,10-diyl)dibenzoate provided evidence that the chemiluminescence originates from luminophores being part of the PAP-UiO, including the luminophores inside the crystals.

Guiding 3D cell migration in deformed synthetic hydrogel microstructures.

The ability of cells to navigate through the extracellular matrix, a network of biopolymers, is controlled by an interplay of cellular activity and mechanical network properties. Synthetic hydrogels with highly tuneable compositions and elastic properties are convenient model systems for the investigation of cell migration in 3D polymer networks. To study the impact of macroscopic deformations on single cell migration, we present a novel method to introduce uniaxial strain in matrices by microstructuring photo-polymerizable hydrogel strips with embedded cells in a channel slide. We find that such confined swelling results in a strained matrix in which cells exhibit an anisotropic migration response parallel to the strain direction. Surprisingly, however, the anisotropy of migration reaches a maximum at intermediate strain levels and decreases strongly at higher strains. We account for this non-monotonic response in the migration anisotropy with a computational model, in which we describe a cell performing durotactic and proteolytic migration in a deformable elastic meshwork. Our simulations reveal that the macroscopically applied strain induces a local geometric anisotropic stiffening of the matrix. This local anisotropic stiffening acts as a guidance cue for directed cell migration, resulting in a non-monotonic dependence on strain, as observed in our experiments. Our findings provide a mechanism for mechanical guidance that connects network properties on the cellular scale to cell migration behaviour.

Dendrimer-Based Signal Amplification of Click-Labelled DNA in Situ.

The in vivo incorporation of alkyne-modified bases into the genome of cells is today the basis for the efficient detection of cell proliferation. Cells are grown in the presence of ethinyl-dU (EdU), fixed and permeabilised. The incorporated alkynes are then efficiently detected by using azide-containing fluorophores and the CuI -catalysed alkyne-azide click reaction. For a world in which constant improvement in the sensitivity of a given method is driving diagnostic advancement, we developed azide- and alkyne-modified dendrimers that allow the establishment of sandwich-type detection assays that show significantly improved signal intensities and signal-to-noise ratios far beyond that which is currently possible.

Rapid disorganization of mechanically interacting systems of mammary acini.

Cells and multicellular structures can mechanically align and concentrate fibers in their ECM environment and can sense and respond to mechanical cues by differentiating, branching, or disorganizing. Here we show that mammary acini with compromised structural integrity can interconnect by forming long collagen lines. These collagen lines then coordinate and accelerate transition to an invasive phenotype. Interacting acini begin to disorganize within 12.5 ± 4.7 h in a spatially coordinated manner, whereas acini that do not interact mechanically with other acini disorganize more slowly (in 21.8 ± 4.1 h) and to a lesser extent (P < 0.0001). When the directed mechanical connections between acini were cut with a laser, the acini reverted to a slowly disorganizing phenotype. When acini were fully mechanically isolated from other acini and also from the bulk gel by box-cuts with a side length <900 µm, transition to an invasive phenotype was blocked in 20 of 20 experiments, regardless of waiting time. Thus, pairs or groups of mammary acini can interact mechanically over long distances through the collagen matrix, and these directed mechanical interactions facilitate transition to an invasive phenotype.

Mutational scanning pinpoints distinct binding sites of key ATGL regulators in lipolysis.

ATGL is a key enzyme in intracellular lipolysis and plays an important role in metabolic and cardiovascular diseases. ATGL is tightly regulated by a known set of protein-protein interaction partners with activating or inhibiting functions in the control of lipolysis. Here, we use deep mutational protein interaction perturbation scanning and generate comprehensive profiles of single amino acid variants that affect the interactions of ATGL with its regulatory partners: CGI-58, G0S2, PLIN1, PLIN5 and CIDEC. Twenty-three ATGL amino acid variants yield a specific interaction perturbation pattern when validated in co-immunoprecipitation experiments in mammalian cells. We identify and characterize eleven highly selective ATGL switch mutations which affect the interaction of one of the five partners without affecting the others. Switch mutations thus provide distinct interaction determinants for ATGL's key regulatory proteins at an amino acid resolution. When we test triglyceride hydrolase activity in vitro and lipolysis in cells, the activity patterns of the ATGL switch variants trace to their protein interaction profile. In the context of structural data, the integration of variant binding and activity profiles provides insights into the regulation of lipolysis and the impact of mutations in human disease.

Directed invasion of cancer cell spheroids inside 3D collagen matrices oriented by microfluidic flow in experiment and simulation.

Invasion is strongly influenced by the mechanical properties of the extracellular matrix. Here, we use microfluidics to align fibers of a collagen matrix and study the influence of fiber orientation on invasion from a cancer cell spheroid. The microfluidic setup allows for highly oriented collagen fibers of tangential and radial orientation with respect to the spheroid, which can be described by finite element simulations. In invasion experiments, we observe a strong bias of invasion towards radial as compared to tangential fiber orientation. Simulations of the invasive behavior with a Brownian diffusion model suggest complete blockage of migration perpendicularly to fibers allowing for migration exclusively along fibers. This slows invasion toward areas with tangentially oriented fibers down, but does not prevent it.

Biomimetic Mineralization of Iron-Fumarate Nanoparticles for Protective Encapsulation and Intracellular Delivery of Proteins.

Biomimetic mineralization of proteins and nucleic acids into hybrid metal-organic nanoparticles allows for protection and cellular delivery of these sensitive and generally membrane-impermeable biomolecules. Although the concept is not necessarily restricted to zeolitic imidazolate frameworks (ZIFs), so far reports about intracellular delivery of functional proteins have focused on ZIF structures. Here, we present a green room-temperature synthesis of amorphous iron-fumarate nanoparticles under mildly acidic conditions in water to encapsulate bovine serum albumin (BSA), horseradish peroxidase (HRP), green fluorescent protein (GFP), and Cas9/sgRNA ribonucleoproteins (RNPs). The synthesis conditions preserve the activity of enzymatic model proteins and the resulting nanoparticles deliver functional HRP and Cas9 RNPs into cells. Incorporation into the iron-fumarate nanoparticles preserves and protects the activity of RNPs composed of the acid-sensitive Cas9 protein and hydrolytically labile RNA even during exposure to pH 3.5 and storage for 2 months at 4 °C, which are conditions that strongly impair the functionality of unprotected RNPs. Thus, the biomimetic mineralization into iron-fumarate nanoparticles presents a versatile platform for the delivery of biomolecules and protects them from degradation during storage under challenging conditions.

Colchicine-loaded lipid bilayer-coated 50 nm mesoporous nanoparticles efficiently induce microtubule depolymerization upon cell uptake.

We report on a one-step assembly route where supported lipid bilayers (SLB) are deposited on functionalized colloidal mesoporous silica (CMS) nanoparticles, resulting in a core-shell hybrid system (SLB@CMS). The supported membrane acts as an intact barrier against the escape of encapsulated dye molecules. These stable SLB@CMS particles loaded with the anticancer drug colchicine are readily taken up by cells and lead to the depolymerization of microtubules with remarkably enhanced efficiency as compared to the same dose of drug in solution.

Protocol for photoactivation of YAP in cancer cell spheroids embedded in collagen gels.

This protocol describes the necessary preparations and procedures to photo-activate Yes-associated protein (YAP) with optoYAP in cancer cell spheroids in 3D collagen matrices. We detail steps for immunofluorescent staining of the resulting YAP-activated HeLa spheroids. In addition, we describe handling of optoYAP on 2D substrates. While this protocol focuses on the use of optoYAP in 3D HeLa cell culture, it can be modified for other cell types. For complete details on the use and execution of this protocol, please refer to Illes et al. (2021).

CT-based whole-body tumor volumetry versus RECIST 1.1: Feasibility and implications for inter-reader variability.

PURPOSE: To investigate whether volumetric measurements of the whole-body tumor volume (WBTV) are feasible and whether they improve inter-reader variability in patients in whom conventional RECIST 1.1 assessment yielded discordant results. METHODS: 50 patients (29 male, 21 female, mean age 60.9 ±â€¯12.3 years) with metastases of solid tumors in whom three readers had selected different sets of target lesions and subsequently reached different results for response assessment (progressive vs. non-progressive disease) when using RECIST 1.1 were included. In a second read, all readers performed volumetric measurements of the WBTV on neck/chest/abdomen/pelvis CTs and measured the time needed for these measurements. Cohen's kappa and Fleiss kappa statistics were used to compare the intra- and inter-reader agreement for response assessment. RESULTS: In 8/50 patients (16 %), the WBTV was too extensive for volumetric measurements and these patients were therefore excluded. In the remaining 42 patients, WBTV measurements required a mean time of 18 min and 9 s. Readers assigned the same response categorizations based on unidimensional RECIST measurements and WBTV measurements in 15/42 patients (33 %), 24/42 patients (57 %) and 30/42 patients (71 %) for reader 1,2 and 3 respectively. When performing response assessment based on WBTV measurements, the three readers agreed in 40/42 patients (95 %) regarding the distinction progressive vs. non-progressive disease, resulting in a near-perfect agreement on a patient-based level (Fleiss' κ = 0.921, 0.95-CI:0.746-1.095). CONCLUSIONS: WBTV measurements yielded an almost perfect inter-reader agreement in a cohort of patients, in which three readers reached discordant response assessment results when following conventional RECIST 1.1 guidelines. This supports the hypothesis, that a limited subset of metastases may not be sufficient to accurately assess response-to-treatment.

Factors That Drive Heterogeneity of Response-to-Treatment of Different Metastatic Deposits Within the Same Patients as Measured by RECIST 1.1 Analyses.

RATIONALE AND OBJECTIVE: This study uses the rate of between-reader variability under Response Evaluation Criteria for Solid Tumors (RECIST) 1.1 as a metric to estimate the prevalence of biologic heterogeneity of individual metastases, and to determine whether this prevalence is modulated by the type of primary tumor, or type of treatment administered. MATERIALS AND METHODS: Three radiologists independently used dedicated oncologic response-assessment software (MintLesion) to prospectively determine RECIST1.1 treatment response in contrast-enhanced computed tomography studies of 355 patients with metastatic disease of different primaries between 07/2015 and 12/2017. In 200 patients, readers had chosen different sets of target lesions; these cases were used for further analysis. Clinically significant heterogeneity of response was considered to be present when RECIST1.1 results differed regarding the distinction of progressive versus non-progressive disease. Rates of response heterogeneity were compared for different types of primary cancers, and different types of systemic treatment. RESULTS: Heterogeneous treatment response was observed in 67 of 200 (34%) patients. Breast cancer was the only primary tumor associated with statistically significantly increased odds for heterogeneity of treatment response (Odds Ratio: 3.972, 0.95 Confidence Interval: 1.275-12.376, p = 0.017). No association was found between type of systemic treatment and rate of biologic heterogeneity. CONCLUSION: Clinically significant heterogeneity of response-to-treatment is a frequent phenomenon, observed in about one-third of patients undergoing contemporary systemic therapies. Patients with breast cancer are more likely to exhibit such heterogeneity. Type of systemic treatment did not modulate the likelihood of exhibiting metastases with diverging treatment response.

Spatio-selective activation of nuclear translocation of YAP with light directs invasion of cancer cell spheroids.

The mechanical properties of the extracellular matrix strongly influence tumor progression and invasion. Yes-associated protein (YAP) has been shown to be a key regulator of this process translating mechanical cues from the extracellular matrix into intracellular signals. Despite its apparent role in tumor progression and metastasis, it is not clear yet, whether YAP activation can actively trigger the onset of invasion. To address this question, we designed a photo-activatable YAP (optoYAP), which allows for spatiotemporal control of its activation. The activation mechanism of optoYAP is based on optically triggered nuclear translocation of the protein. Activation of optoYAP induces downstream signaling for several hours and leads to increased proliferation in two- and three-dimensional cultures. Applied to cancer spheroids, optoYAP activation induces invasion. Site-selective activation of optoYAP in cancer spheroids strikingly directs invasion into the activated direction. Thus, nuclear translocation of YAP may be enough to trigger the onset of invasion.

Mesoporous Silica Nanoparticles as pH-Responsive Carrier for the Immune-Activating Drug Resiquimod Enhance the Local Immune Response in Mice.

Nanoparticle-based delivery systems for cancer immunotherapies aim to improve the safety and efficacy of these treatments through local delivery to specialized antigen-presenting cells (APCs). Multifunctional mesoporous silica nanoparticles (MSNs), with their large surface areas, their tunable particle and pore sizes, and their spatially controlled functionalization, represent a safe and versatile carrier system. In this study, we demonstrate the potential of MSNs as a pH-responsive drug carrier system for the anticancer immune-stimulant R848 (resiquimod), a synthetic Toll-like receptor 7 and 8 agonist. Equipped with a biotin-avidin cap, the tailor-made nanoparticles showed efficient stimuli-responsive release of their R848 cargo in an environmental pH of 5.5 or below. We showed that the MSNs loaded with R848 were rapidly taken up by APCs into the acidic environment of the lysosome and that they potently activated the immune cells. Upon subcutaneous injection into mice, the particles accumulated in migratory dendritic cells (DCs) in the draining lymph nodes, where they strongly enhanced the activation of the DCs. Furthermore, simultaneous delivery of the model antigen OVA and the adjuvant R848 by MSNs resulted in an augmented antigen-specific T-cell response. The MSNs significantly improved the pharmacokinetic profile of R848 in mice, as the half-life of the drug was increased 6-fold, and at the same time, the systemic exposure was reduced. In summary, we demonstrate that MSNs represent a promising tool for targeted delivery of the immune modulator R848 to APCs and hold considerable potential as a carrier for cancer vaccines.

Probing GFP-actin diffusion in living cells using fluorescence correlation spectroscopy.

The cytoskeleton of eukaryotic cells is continuously remodeled by polymerization and depolymerization of actin. Consequently, the relative content of polymerized filamentous actin (F-actin) and monomeric globular actin (G-actin) is subject to temporal and spatial fluctuations. Since fluorescence correlation spectroscopy (FCS) can measure the diffusion of fluorescently labeled actin it seems likely that FCS allows us to determine the dynamics and hence indirectly the structural properties of the cytoskeleton components with high spatial resolution. To this end we investigate the FCS signal of GFP-actin in living Dictyostelium discoideum cells and explore the inherent spatial and temporal signatures of the actin cytoskeleton. Using the free green fluorescent protein (GFP) as a reference, we find that actin diffusion inside cells is dominated by G-actin and slower than diffusion in diluted cell extract. The FCS signal in the dense cortical F-actin network near the cell membrane is probed using the cytoskeleton protein LIM and is found to be slower than cytosolic G-actin diffusion. Furthermore, we show that polymerization of the cytoskeleton induced by Jasplakinolide leads to a substantial decrease of G-actin diffusion. Pronounced fluctuations in the distribution of the FCS correlation curves can be induced by latrunculin, which is known to induce actin waves. Our work suggests that the FCS signal of GFP-actin in combination with scanning or spatial correlation techniques yield valuable information about the local dynamics and concomitant cytoskeletal properties.

Particle-Size-Dependent Delivery of Antitumoral miRNA Using Targeted Mesoporous Silica Nanoparticles.

Multifunctional core-shell mesoporous silica nanoparticles (MSN) were tailored in size ranging from 60 to 160 nm as delivery agents for antitumoral microRNA (miRNA). The positively charged particle core with a pore diameter of about 5 nm and a stellate pore morphology allowed for an internal, protective adsorption of the fragile miRNA cargo. A negatively charged particle surface enabled the association of a deliberately designed block copolymer with the MSN shell by charge-matching, simultaneously acting as a capping as well as endosomal release agent. Furthermore, the copolymer was functionalized with the peptide ligand GE11 targeting the epidermal growth factor receptor, EGFR. These multifunctional nanoparticles showed an enhanced uptake into EGFR-overexpressing T24 bladder cancer cells through receptor-mediated cellular internalization. A luciferase gene knock-down of up to 65% and additional antitumoral effects such as a decreased cell migration as well as changes in cell cycle were observed. We demonstrate that nanoparticles with a diameter of 160 nm show the fastest cellular internalization after a very short incubation time of 45 min and produce the highest level of gene knock-down.

Metal-Organic Framework Nanoparticles Induce Pyroptosis in Cells Controlled by the Extracellular pH.

Ion homeostasis is essential for cellular survival, and elevated concentrations of specific ions are used to start distinct forms of programmed cell death. However, investigating the influence of certain ions on cells in a controlled way has been hampered due to the tight regulation of ion import by cells. Here, it is shown that lipid-coated iron-based metal-organic framework nanoparticles are able to deliver and release high amounts of iron ions into cells. While high concentrations of iron often trigger ferroptosis, here, the released iron induces pyroptosis, a form of cell death involving the immune system. The iron release occurs only in slightly acidic extracellular environments restricting cell death to cells in acidic microenvironments and allowing for external control. The release mechanism is based on endocytosis facilitated by the lipid-coating followed by degradation of the nanoparticle in the lysosome via cysteine-mediated reduction, which is enhanced in slightly acidic extracellular environment. Thus, a new functionality of hybrid nanoparticles is demonstrated, which uses their nanoarchitecture to facilitate controlled ion delivery into cells. Based on the selectivity for acidic microenvironments, the described nanoparticles may also be used for immunotherapy: the nanoparticles may directly affect the primary tumor and the induced pyroptosis activates the immune system.