Fibroblast-Derived Lysyl Oxidase Increases Oxidative Phosphorylation and Stemness in Cholangiocarcinoma.

BACKGROUND & AIMS: Metabolic and transcriptional programs respond to extracellular matrix-derived cues in complex environments, such as the tumor microenvironment. Here, we demonstrate how lysyl oxidase (LOX), a known factor in collagen crosslinking, contributes to the development and progression of cholangiocarcinoma (CCA). METHODS: Transcriptomes of 209 human CCA tumors, 143 surrounding tissues, and single-cell data from 30 patients were analyzed. The recombinant protein and a small molecule inhibitor of the LOX activity were used on primary patient-derived CCA cultures to establish the role of LOX in migration, proliferation, colony formation, metabolic fitness, and the LOX interactome. The oncogenic role of LOX was further investigated by RNAscope and in vivo using the AKT/NICD genetically engineered murine CCA model. RESULTS: We traced LOX expression to hepatic stellate cells and specifically hepatic stellate cell-derived inflammatory cancer-associated fibroblasts and found that cancer-associated fibroblast-driven LOX increases oxidative phosphorylation and metabolic fitness of CCA, and regulates mitochondrial function through transcription factor A, mitochondrial. Inhibiting LOX activity in vivo impedes CCA development and progression. Our work highlights that LOX alters tumor microenvironment-directed transcriptional reprogramming of CCA cells by facilitating the expression of the oxidative phosphorylation pathway and by increasing stemness and mobility. CONCLUSIONS: Increased LOX is driven by stromal inflammatory cancer-associated fibroblasts and correlates with diminished survival of patients with CCA. Modulating the LOX activity can serve as a novel tumor microenvironment-directed therapeutic strategy in bile duct pathologies.

Discovery of NSD2-Degraders from Novel and Selective DEL Hits.

NSD2 is a histone methyltransferase predominantly catalyzing di-methylation of histone H3 on lysine K36. Increased NSD2 activity due to mutations or fusion-events affecting the gene encoding NSD2 is considered an oncogenic event and a driver in various cancers, including multiple myelomas carrying t(4;14) chromosomal translocations and acute lymphoblastic leukemia's expressing the hyperactive NSD2 mutant E1099 K. Using DNA-encoded libraries, we have identified small molecule ligands that selectively and potently bind to the PWWP1 domain of NSD2, inhibit NSD2 binding to H3K36me2-bearing nucleosomes, but do not inhibit the methyltransferase activity. The ligands were subsequently converted to selective VHL1-recruiting NSD2 degraders and by using one of the most efficacious degraders in cell lines, we show that it leads to NSD2 degradation, decrease in K3 K36me2 levels and inhibition of cell proliferation.

Mesenchymal-epithelial crosstalk shapes intestinal regionalisation via Wnt and Shh signalling.

Organs are anatomically compartmentalised to cater for specialised functions. In the small intestine (SI), regionalisation enables sequential processing of food and nutrient absorption. While several studies indicate the critical importance of non-epithelial cells during development and homeostasis, the extent to which these cells contribute to regionalisation during morphogenesis remains unexplored. Here, we identify a mesenchymal-epithelial crosstalk that shapes the developing SI during late morphogenesis. We find that subepithelial mesenchymal cells are characterised by gradients of factors supporting Wnt signalling and stimulate epithelial growth in vitro. Such a gradient impacts epithelial gene expression and regional villus formation along the anterior-posterior axis of the SI. Notably, we further provide evidence that Wnt signalling directly regulates epithelial expression of Sonic Hedgehog (SHH), which, in turn, acts on mesenchymal cells to drive villi formation. Taken together our results uncover a mechanistic link between Wnt and Hedgehog signalling across different cellular compartments that is central for anterior-posterior regionalisation and correct formation of the SI.

Tracing the cellular dynamics of sebaceous gland development in normal and perturbed states.

The sebaceous gland (SG) is an essential component of the skin, and SG dysfunction is debilitating1,2. Yet, the cellular bases for its origin, development and subsequent maintenance remain poorly understood. Here, we apply large-scale quantitative fate mapping to define the patterns of cell fate behaviour during SG development and maintenance. We show that the SG develops from a defined number of lineage-restricted progenitors that undergo a programme of independent and stochastic cell fate decisions. Following an expansion phase, equipotent progenitors transition into a phase of homeostatic turnover, which is correlated with changes in the mechanical properties of the stroma and spatial restrictions on gland size. Expression of the oncogene KrasG12D results in a release from these constraints and unbridled gland expansion. Quantitative clonal fate analysis reveals that, during this phase, the primary effect of the Kras oncogene is to drive a constant fate bias with little effect on cell division rates. These findings provide insight into the developmental programme of the SG, as well as the mechanisms that drive tumour progression and gland dysfunction.

Spatial distribution and temporal evolution of DRONPA-fused SNAP25 clusters in adrenal chromaffin cells.

Sub-diffraction imaging of plasma membrane localized proteins, such as the SNARE (Soluble NSF Attachment Protein Receptor) proteins involved in exocytosis, in fixed cells have resulted in images with high spatial resolution, at the expense of dynamical information. Here, we have imaged localized fluorescence bursts of DRONPA-fused SNAP-25 molecules in live chromaffin cells by Total Internal Reflection Fluorescence (TIRF) imaging. We find that this method allows tracking protein cluster dynamics over relatively long times (∼20 min.), partly due to the diffusion into the TIRF field of fresh molecules, making possible the simultaneous identification of cluster size, location and temporal evolution. The results indicate that the DRONPA-fused SNAP-25 clusters display rich dynamics, going from staying constant to disappearing and reappearing in specific cluster domains within minutes.

Transfection of living HeLa cells with fluorescent poly-cytosine encapsulated Ag nanoclusters.

The fluorescence of silver clusters encapsulated by single stranded oligo-DNA (24 cytosine base pairs, C(24):Ag(n)) was used to monitor the transfection of this new silver/DNA fluorophore inside living HeLa cells. For this, the C(24):Ag(n) molecules were complexed with a commercially available transfection reagent Lipofectamine and the internalization of C(24):Ag(n) was followed with confocal fluorescence microscopy. Bright near-infrared fluorescence was observed from inside the transfected HeLa cells, when exciting with 633 nm excitation, opening up the possibility for the use of these C(24):Ag(n) clusters for biological labelling and imaging of living cells and for monitoring the transfection process with limited harm to the living cells.

Live cell surface labeling with fluorescent Ag nanocluster conjugates.

DNA-encapsulated silver clusters are readily conjugated to proteins and serve as alternatives to organic dyes and semiconductor quantum dots. Stable and bright on the bulk and single molecule levels, Ag nanocluster fluorescence is readily observed when staining live cell surfaces. Being significantly brighter and more photostable than organics and much smaller than quantum dots with a single point of attachment, these nanomaterials offer promising new approaches for bulk and single molecule biolabeling.

Oligonucleotide-stabilized Ag nanocluster fluorophores.

Single-stranded oligonucleotides stabilize highly fluorescent Ag nanoclusters, with emission colors tunable via DNA sequence. We utilized DNA microarrays to optimize these scaffold sequences for creating nearly spectrally pure Ag nanocluster fluorophores that are highly photostable and exhibit great buffer stability. Five different nanocluster emitters have been created with tunable emission from the blue to the near-IR and excellent photophysical properties. Ensemble and single molecule fluorescence studies show that oligonucleotide encapsulated Ag nanoclusters exhibit significantly greater photostability and higher emission rates than commonly used cyanine dyes.

Strongly emissive individual DNA-encapsulated Ag nanoclusters as single-molecule fluorophores.

The water-soluble, near-IR-emitting DNA-encapsulated silver nanocluster presented herein exhibits extremely bright and photostable emission on the single-molecule and bulk levels. The photophysics have been elucidated by intensity-dependent correlation analysis and suggest a heavy atom effect of silver that rapidly depopulates an excited dark level before quenching by oxygen, thereby conferring great photostability, very high single-molecule emission rates, and essentially no blinking on experimentally relevant time scales (0.1 to >1,000 ms). Strong antibunching is observed from these biocompatible species, which emit >10(9) photons before photobleaching. The significant dark-state quantum yield even enables bunching from the emissive state to be observed as a dip in the autocorrelation curve with only a single detector as the dark state precludes emission from the emissive level. These species represent significant improvements over existing dyes, and the nonpower law blinking kinetics suggest that these very small species may be alternatives to much larger and strongly intermittent semiconductor quantum dots.

Ag Nanocluster Formation Using a Cytosine Oligonucleotide Template.

The reduction of silver cations bound to the oligonucleotide dC(12) was used to form silver nanoclusters. Mass spectra show that the oligonucleotides have 2-7 silver atoms that form multiple species, as evident from the number of transitions in the fluorescence and absorption spectra. The variations in the concentrations of the nanoclusters with time are attributed to the changing reducing capacity of the solution, and the formation of oxidized nanoclusters is proposed. Via mass spectrometry and circular dichroism spectroscopy, double-stranded structures with Ag(+)-mediated interactions between the bases are observed, but these structures are not maintained with the reduced nanoclusters. Through variations in the pH, the nanoclusters are shown to bind with the N3 of cytosine.

Quantum mechanical single-gold-nanocluster electroluminescent light source at room temperature.

Electrically contacted gold-nanocluster arrays formed within electromigration-induced break junctions exhibit bright, field-dependent electroluminescence in the near infrared (650-800 nm). Intensity autocorrelation of spatially isolated individual nanocluster emission driven at high electrical frequency (f(ac)= approximately 200 MHz) reveals antibunched electroluminescence at room temperature. These results demonstrate the single quantum nature of several-atom gold molecules and suggest their use as room-temperature electrically driven single-photon sources.