Sublethal necroptosis signaling promotes inflammation and liver cancer.

It is currently not well known how necroptosis and necroptosis responses manifest in vivo. Here, we uncovered a molecular switch facilitating reprogramming between two alternative modes of necroptosis signaling in hepatocytes, fundamentally affecting immune responses and hepatocarcinogenesis. Concomitant necrosome and NF-κB activation in hepatocytes, which physiologically express low concentrations of receptor-interacting kinase 3 (RIPK3), did not lead to immediate cell death but forced them into a prolonged "sublethal" state with leaky membranes, functioning as secretory cells that released specific chemokines including CCL20 and MCP-1. This triggered hepatic cell proliferation as well as activation of procarcinogenic monocyte-derived macrophage cell clusters, contributing to hepatocarcinogenesis. In contrast, necrosome activation in hepatocytes with inactive NF-κB-signaling caused an accelerated execution of necroptosis, limiting alarmin release, and thereby preventing inflammation and hepatocarcinogenesis. Consistently, intratumoral NF-κB-necroptosis signatures were associated with poor prognosis in human hepatocarcinogenesis. Therefore, pharmacological reprogramming between these distinct forms of necroptosis may represent a promising strategy against hepatocellular carcinoma.

Spatial multi-omic map of human myocardial infarction.

Myocardial infarction is a leading cause of death worldwide1. Although advances have been made in acute treatment, an incomplete understanding of remodelling processes has limited the effectiveness of therapies to reduce late-stage mortality2. Here we generate an integrative high-resolution map of human cardiac remodelling after myocardial infarction using single-cell gene expression, chromatin accessibility and spatial transcriptomic profiling of multiple physiological zones at distinct time points in myocardium from patients with myocardial infarction and controls. Multi-modal data integration enabled us to evaluate cardiac cell-type compositions at increased resolution, yielding insights into changes of the cardiac transcriptome and epigenome through the identification of distinct tissue structures of injury, repair and remodelling. We identified and validated disease-specific cardiac cell states of major cell types and analysed them in their spatial context, evaluating their dependency on other cell types. Our data elucidate the molecular principles of human myocardial tissue organization, recapitulating a gradual cardiomyocyte and myeloid continuum following ischaemic injury. In sum, our study provides an integrative molecular map of human myocardial infarction, represents an essential reference for the field and paves the way for advanced mechanistic and therapeutic studies of cardiac disease.

Detection of PatIent-Level distances from single cell genomics and pathomics data with Optimal Transport (PILOT).

Although clinical applications represent the next challenge in single-cell genomics and digital pathology, we still lack computational methods to analyze single-cell or pathomics data to find sample-level trajectories or clusters associated with diseases. This remains challenging as single-cell/pathomics data are multi-scale, i.e., a sample is represented by clusters of cells/structures, and samples cannot be easily compared with each other. Here we propose PatIent Level analysis with Optimal Transport (PILOT). PILOT uses optimal transport to compute the Wasserstein distance between two individual single-cell samples. This allows us to perform unsupervised analysis at the sample level and uncover trajectories or cellular clusters associated with disease progression. We evaluate PILOT and competing approaches in single-cell genomics or pathomics studies involving various human diseases with up to 600 samples/patients and millions of cells or tissue structures. Our results demonstrate that PILOT detects disease-associated samples from large and complex single-cell or pathomics data. Moreover, PILOT provides a statistical approach to find changes in cell populations, gene expression, and tissue structures related to the trajectories or clusters supporting interpretation of predictions.

MOJITOO: a fast and universal method for integration of multimodal single-cell data.

MOTIVATION: The advent of multi-modal single-cell sequencing techniques have shed new light on molecular mechanisms by simultaneously inspecting transcriptomes, epigenomes and proteomes of the same cell. However, to date, the existing computational approaches for integration of multimodal single-cell data are either computationally expensive, require the delineation of parameters or can only be applied to particular modalities. RESULTS: Here we present a single-cell multi-modal integration method, named Multi-mOdal Joint IntegraTion of cOmpOnents (MOJITOO). MOJITOO uses canonical correlation analysis for a fast and parameter free detection of a shared representation of cells from multimodal single-cell data. Moreover, estimated canonical components can be used for interpretation, i.e. association of modality-specific molecular features with the latent space. We evaluate MOJITOO using bi- and tri-modal single-cell datasets and show that MOJITOO outperforms existing methods regarding computational requirements, preservation of original latent spaces and clustering. AVAILABILITY AND IMPLEMENTATION: The software, code and data for benchmarking are available at https://github.com/CostaLab/MOJITOO and https://doi.org/10.5281/zenodo.6348128. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Reversible Shielding between Dual Ligands for Enhanced Tumor Accumulation of ZnPc-Loaded Micelles.

Herein, we report a ligand-reversible-shielding strategy based on the mutual shielding of dual ligands tethered to the surface of nanoparticles. To exemplify this concept, phenylboronic acid-functionalized poly(ethylene glycol)- b-poly(ε-caprolactone) (PBA-PEG-PCL) and galactose-functionalized diblock polymer (Gal-PEG-PCL) were mixed to form dual-ligand micelles (PBA/Gal). PBA and Gal residues could form a complex at pH 7.4 and mutually shield their targeting function. At pH 6.8, the binding affinity between PBA and Gal weakened, and PBA preferred to bind with the sialic acid residues on the tumor cell surface rather than to Gal on the micellar surface; furthermore, the unbound Gal recovered its targeting ability toward the asialoglycoprotein receptor. When the pH decreased from 7.4 to 6.8, enzyme-linked immunosorbent assays exhibited that the percentage of exposed Gal on the micellar surface increased 1.9-fold, and flow cytometry showed that HepG2 cellular uptake increased 4.3-fold. More importantly, this process was reversible, confirming the reversible shielding and deshielding of dual ligands. With the encapsulation of a photosensitizer, zinc phthalocyanine (ZnPc), the reversible-shielding micelles showed a 48% improvement in the half-life ( t1/2) in blood circulation, a 54% decrease in liver capture, a 40% increase in tumor accumulation, and a 10.3% improvement in the tumor inhibition rate compared to the Gal-coated irreversible micelles. This dual-ligand mutual-shielding strategy provides a new perspective on reversible tumor targeting.

A glutathione responsive nitric oxide release system based on charge-reversal chitosan nanoparticles for enhancing synergistic effect against multidrug resistance tumor.

The combination therapy of nitric oxide (NO) and anticancer drug was developed for reversing multidrug resistance (MDR). In order to avoid NO release during the blood circulation, and realize pinpointed release in the tumor cells, we designed a tumor-specific NO-release system based on 10-hydroxycamptothecin (HCPT)-loaded charge-reversal chitosan nanoparticles and covalently linked phenylsulfonyl furoxan (glutathione (GSH)-responsive NO donor) on the surface. The results showed that only 6.0% of NO was eventually released under physiology condition (pH 7.4 and 2 µM GSH) within 8 h. In contrast, 93.0% of NO was released within 4 h in the presence of 10 mM GSH. Western blot result displayed the P-glycoprotein expression was significantly decreased by 50.1%. Hence, this system performed remarkable cytotoxicity in vitro and the highest tumor inhibition rate (79.7%) comparing with free HCPT group (20.7%) in vivo. Such GSH-responsive NO-release system is a promising candidate with prominent therapeutic effect against MDR tumor.

Lineage tracing reveals transient phenotypic adaptation of tubular cells during acute kidney injury.

Tubular injury is the hallmark of acute kidney injury (AKI) with a tremendous impact on patients and health-care systems. During injury, any differentiated proximal tubular cell (PT) may transition into a specific injured phenotype, so-called "scattered tubular cell" (STC)-phenotype. To understand the fate of this specific phenotype, we generated transgenic mice allowing inducible, reversible, and irreversible tagging of these cells in a murine AKI model, the unilateral ischemia-reperfusion injury (IRI). For lineage tracing, we analyzed the kidneys using single-cell profiling during disease development at various time points. Labeled cells, which we defined by established endogenous markers, already appeared 8 h after injury and showed a distinct expression set of genes. We show that STCs re-differentiate back into fully differentiated PTs upon the resolution of the injury. In summary, we show the dynamics of the phenotypic transition of PTs during injury, revealing a reversible transcriptional program as an adaptive response during disease.

Next-Generation Morphometry for pathomics-data mining in histopathology.

Pathology diagnostics relies on the assessment of morphology by trained experts, which remains subjective and qualitative. Here we developed a framework for large-scale histomorphometry (FLASH) performing deep learning-based semantic segmentation and subsequent large-scale extraction of interpretable, quantitative, morphometric features in non-tumour kidney histology. We use two internal and three external, multi-centre cohorts to analyse over 1000 kidney biopsies and nephrectomies. By associating morphometric features with clinical parameters, we confirm previous concepts and reveal unexpected relations. We show that the extracted features are independent predictors of long-term clinical outcomes in IgA-nephropathy. We introduce single-structure morphometric analysis by applying techniques from single-cell transcriptomics, identifying distinct glomerular populations and morphometric phenotypes along a trajectory of disease progression. Our study provides a concept for Next-generation Morphometry (NGM), enabling comprehensive quantitative pathology data mining, i.e., pathomics.

Mapping cardiac remodeling in chronic kidney disease.

Patients with advanced chronic kidney disease (CKD) mostly die from sudden cardiac death and recurrent heart failure. The mechanisms of cardiac remodeling are largely unclear. To dissect molecular and cellular mechanisms of cardiac remodeling in CKD in an unbiased fashion, we performed left ventricular single-nuclear RNA sequencing in two mouse models of CKD. Our data showed a hypertrophic response trajectory of cardiomyocytes with stress signaling and metabolic changes driven by soluble uremia-related factors. We mapped fibroblast to myofibroblast differentiation in this process and identified notable changes in the cardiac vasculature, suggesting inflammation and dysfunction. An integrated analysis of cardiac cellular responses to uremic toxins pointed toward endothelin-1 and methylglyoxal being involved in capillary dysfunction and TNFα driving cardiomyocyte hypertrophy in CKD, which was validated in vitro and in vivo. TNFα inhibition in vivo ameliorated the cardiac phenotype in CKD. Thus, interventional approaches directed against uremic toxins, such as TNFα, hold promise to ameliorate cardiac remodeling in CKD.

Chromatin-accessibility estimation from single-cell ATAC-seq data with scOpen.

A major drawback of single-cell ATAC-seq (scATAC-seq) is its sparsity, i.e., open chromatin regions with no reads due to loss of DNA material during the scATAC-seq protocol. Here, we propose scOpen, a computational method based on regularized non-negative matrix factorization for imputing and quantifying the open chromatin status of regulatory regions from sparse scATAC-seq experiments. We show that scOpen improves crucial downstream analysis steps of scATAC-seq data as clustering, visualization, cis-regulatory DNA interactions, and delineation of regulatory features. We demonstrate the power of scOpen to dissect regulatory changes in the development of fibrosis in the kidney. This identifies a role of Runx1 and target genes by promoting fibroblast to myofibroblast differentiation driving kidney fibrosis.

One-pot synthesis of acid-induced in situ aggregating theranostic gold nanoparticles with enhanced retention in tumor cells.

In this work, we took advantage of a one-pot reaction to prepare tumor-targeting nanoparticles (Au@T), which could respond to the intracellular acidic environment and form aggregates to enhance the retention effect of nanoparticles in tumor cells. Au@T is composed of gold nanoparticles (Au NPs) modified with 4-mercaptobenzoic acid (MCBA), p-hydroxythiophenol (HTP), LA (lipoic acid)-PEG2K-OCH3 and LA-PEG2K-biotin. During blood circulation, Au@T remains well dispersed, making it inconspicuous. Then, with the help of active targeted transport, much more Au@T becomes internalized at the tumor site. After being internalized by tumor cells, Au@T aggregates under the condition of pH = 6.0, thereby improving the retention effect of Au@T, stymieing exocytosis and reducing the amount of nanoparticles returned to the blood stream. Furthermore, the in vivo experimental results showed that aggregated Au@T exhibits excellent photothermal effects, with a tumor inhibition rate of 86.40%. The computed tomography (CT) value was found to be 1.5 times higher than that of the control group (Au@Bio), as Au@Bio was unable to aggregate in tumor cells. In conclusion, this work provides a simple method for synthesizing a type of gold nanoparticles (Au@T) with promising potential for tumor diagnosis and treatment through enhancing the retention effect in tumor cells.

Multivalent nanoparticles for personalized theranostics based on tumor receptor distribution behavior.

It is acknowledged that the targeting ability of multivalent ligand-modified nanoparticles (MLNs) strongly depends on the ligand spatial presentation determined by ligand valency. However, the receptor overexpression level varies between different types or stages of tumors. Thus, it is essential to explore the influence of ligand valency on the targeting ability of MLNs to tumors with different levels of receptor overexpression. In this study, a dual-acting agent raltitrexed was used as a ligand to target the folate receptor (FR). Different copies of the raltitrexed-modified multivalent dendritic polyethyleneimine ligand cluster PRn (n = 2, 4, and 8) were conjugated onto magnetic nanoparticles to form multivalent magnetic NPs (MMNs) with different valences. The in vitro studies demonstrated that Fe-PR4 was the most effective valency in the treatment of high FR overexpressing KB cells with a decentralized receptor distribution, owing to the fact that Fe-PR2 was negative in statistical rebinding and Fe-PR8 could induce steric hindrance in the limited binding area. Instead, in moderate FR overexpressing HeLa cells with clustered receptor display, the extra ligands on Fe-PR8 would facilitate statistical rebinding more beneficially. Furthermore, in in vivo tumor inhibition and targeted magnetic resonance imaging (MRI) of KB tumors and another moderate FR expressing H22 tumor, similar results were obtained with the cell experiments. Overall, the optimizable treatment effect of Fe-PRn by modulating the ligand valency based on the overexpressing tumor receptor distribution behavior supports the potential of Fe-PRn as a nanomedicine for personalized theranostics.

Zwitterionic chitooligosaccharide-modified ink-blue titanium dioxide nanoparticles with inherent immune activation for enhanced photothermal therapy.

Photothermal therapy (PTT) can trigger massive apoptosis of cancer cells, and this sharply increasing local apoptotic rate may recruit plenty of tumor-associated macrophages (TAMs). Although TAMs are recognized to display an M2-like subtype, which encourages tumor ontogenesis, they can be re-educated to a tumoricidal M1-like subtype by immunomodulatory reagents. Chitooligosaccharides (COSs) are endowed with immunomodulatory ability, but the positive electrical property limits their application; besides, their re-educating ability on TAMs is uncertain. Therefore, we proposed whether the combination of zwitterionic COS with a photothermal material can impair the undesirable tumor promotion of TAMs, thus enhancing the PTT treatment outcome. Herein, zwitterionic COS was obtained via the carboxymethylate method and then, the obtained COS was modified on the surface of ink-blue titanium dioxide (BTiO2) with photothermal ability to synthesize BTC NPs. In vitro, the immunofluorescence staining and cell survival assays indicated that BTC NPs could re-educate 87% of the M2-like RAW264.7 macrophages stimulated by apoptotic tumor cell secretion and significantly inhibit the liver tumor cell proliferation. Notably, in a mouse H22 liver cancer model, compared with mono PTT with BTiO2, the PTT treatment of BTC could reverse the ratio of M2 : M1 from 3.3 : 1 to 0.5 : 1, thus leading to 20.7% increase in the tumor inhibition rate. In general, our study demonstrated that zwitterionic COS can act as a potent immune activator to re-educate TAMs to M1. Furthermore, equipping the photothermal material with zwitterionic COS can be a potential treatment paradigm to achieve more forceful PTT.

Single NIR Laser-Activated Multifunctional Nanoparticles for Cascaded Photothermal and Oxygen-Independent Photodynamic Therapy.

Inconvenient dual-laser irradiation and tumor hypoxic environment as well as limited judgment of treating region have impeded the development of combined photothermal and photodynamic therapies (PTT and PDT). Herein, Bi2Se3@AIPH nanoparticles (NPs) are facilely developed to overcome these problems. Through a one-step method, free radical generator (AIPH) and phase transition material (lauric acid, LA, 44-46 °C) are encapsulated in hollow bismuth selenide nanoparticles (Bi2Se3 NPs). Under a single 808-nm laser irradiation at the tumor area, hyperthermia produced by Bi2Se3 not only directly leads to cell death, but also promotes AIPH release by melting LA and triggers free radical generation, which could further eradicate tumor cells in hypoxic environments. Moreover, Bi2Se3 with high X-ray attenuation coefficient endows the NPs with high computed tomography (CT) imaging capability, which is important for treating area determination. The results exhibit that Bi2Se3@AIPH NPs possesses 31.2% photothermal conversion efficiency for enhanced PTT, ideal free radical generation for oxygen-independent PDT, and 37.77 HU mL mg-1 X-ray attenuation coefficient for CT imaging with high quality. Most importantly, the tumor growth inhibition rate by synergistic PTT, PDT, and following immunotherapy is 99.6%, and even one tumor disappears completely, which demonstrates excellent cascaded synergistic effect of Bi2Se3@AIPH NPs for the tumor therapy.

"Three-in-One" Multifunctional Gatekeeper Gated Mesoporous Silica Nanoparticles for Intracellular pH-Activated Targeted Cancer Therapy.

Mesoporous silica nanoparticle (MSN)-based stimuli-responsive capped drug delivery systems have attracted extensive attention. However, most studies about capping agents only focus on the capping function. Here, a multifunctional capping agent (MFCA) was developed to integrate pore-capping, drug-loading, and tumor-targeting abilities. It was composed of phenylboronic acid (PBA)-modified sodium alginate (ALG) and doxorubicin (DOX), which were loaded in the channels and conjugated ALG via hydrazone bond through a one-pot method. Consequently, the drug-loading content of obtained nanoparticles (DOX@MSN-ALG-APBA) reached up to 10.6%, which increased by about 58.2% compared with that of ALG-APBA capped MSNs (i-DOX@MSN-ALG-APBA). The in vitro drug release at pH 7.4 was less than 10% in 3 days, which was comparable to i-DOX@MSN-ALG-APBA. More importantly, the initial accumulative drug release of DOX@MSN-ALG-APBA was about 2-fold than that of i-DOX@MSN-ALG-APBA at pH 6.0. Also, the targeting efficiency of DOX@MSN-ALG-APBA was 2.85-fold than that of DOX@MSN-ALG in vitro after introducing phenylboronic acid. Compared with free DOX and i-DOX@MSN-ALG-APBA, DOX@MSN-ALG-APBA induced remarkable cell apoptosis and inhibition effects in vitro, owing to the rapid release of drug during the initial phase. Overall, it is expected that this integrated strategy is promising to fabricate an excellent MSN-based drug delivery system.