Metformin promotes anti-tumor immunity in STK11 mutant NSCLC through AXIN1-dependent upregulation of multiple nucleotide metabolites.

Background: Metformin has pleiotropic effects beyond glucose reduction, including tumor inhibition and immune regulation. It enhanced the anti-tumor effects of programmed cell death protein 1 (PD-1) inhibitors in serine/threonine kinase 11 (STK11) mutant non-small cell lung cancer (NSCLC) through an axis inhibition protein 1 (AXIN1)-dependent manner. However, the alterations of tumor metabolism and metabolites upon metformin administration remain unclear. Methods: We performed untargeted metabolomics using liquid chromatography (LC)-mass spectrometry (MS)/MS system and conducted cell experiments to verify the results of bioinformatics analysis. Results: According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, most metabolites were annotated into metabolism, including nucleotide metabolism. Next, the differentially expressed metabolites in H460 (refers to H460 cells), H460_met (refers to metformin-treated H460 cells), and H460_KO_met (refers to metformin-treated Axin1 -/- H460 cells) were distributed into six clusters based on expression patterns. The clusters with a reversed expression pattern upon metformin treatment were selected for further analysis. We screened out metabolic pathways through KEGG pathway enrichment analysis and found that multiple nucleotide metabolites enriched in this pathway were upregulated. Furthermore, these metabolites enhanced the cytotoxicity of activated T cells on H460 cells in vitro and can activate the stimulator of the interferon genes (STING) pathway independently of AXIN1. Conclusion: Relying on AXIN1, metformin upregulated multiple nucleotide metabolites which promoted STING signaling and the killing of activated T cells in STK11 mutant NSCLC, indicating a potential immunotherapeutic strategy for STK11 mutant NSCLC.

scRank infers drug-responsive cell types from untreated scRNA-seq data using a target-perturbed gene regulatory network.

Cells respond divergently to drugs due to the heterogeneity among cell populations. Thus, it is crucial to identify drug-responsive cell populations in order to accurately elucidate the mechanism of drug action, which is still a great challenge. Here, we address this problem with scRank, which employs a target-perturbed gene regulatory network to rank drug-responsive cell populations via in silico drug perturbations using untreated single-cell transcriptomic data. We benchmark scRank on simulated and real datasets, which shows the superior performance of scRank over existing methods. When applied to medulloblastoma and major depressive disorder datasets, scRank identifies drug-responsive cell types that are consistent with the literature. Moreover, scRank accurately uncovers the macrophage subpopulation responsive to tanshinone IIA and its potential targets in myocardial infarction, with experimental validation. In conclusion, scRank enables the inference of drug-responsive cell types using untreated single-cell data, thus providing insights into the cellular-level impacts of therapeutic interventions.

Revealing spatial multimodal heterogeneity in tissues with SpaTrio.

Capturing and depicting the multimodal tissue information of tissues at the spatial scale remains a significant challenge owing to technical limitations in single-cell multi-omics and spatial transcriptomics sequencing. Here, we developed a computational method called SpaTrio that can build spatial multi-omics data by integrating these two datasets through probabilistic alignment and enabling further analysis of gene regulation and cellular interactions. We benchmarked SpaTrio using simulation datasets and demonstrated its accuracy and robustness. Next, we evaluated SpaTrio on biological datasets and showed that it could detect topological patterns of cells and modalities. SpaTrio has also been applied to multiple sets of actual data to uncover spatially multimodal heterogeneity, understand the spatiotemporal regulation of gene expression, and resolve multimodal communication among cells. Our data demonstrated that SpaTrio could accurately map single cells and reconstruct the spatial distribution of various biomolecules, providing valuable multimodal insights into spatial biology.

Inhibiting the activation of MAPK (ERK1/2) in stressed Müller cells prevents photoreceptor degeneration.

Rationale: Müller cells play an essential role in maintaining the health of retinal photoreceptors. Dysfunction of stressed Müller cells often results in photoreceptor degeneration. However, how these cells communicate under stress and the signalling pathways involved remain unclear. In this study, we inhibited the MAPK (ERK1/2) signalling, mainly activated in Müller cells, evaluated the protective effects on the photoreceptors and further explored the signalling communication between stressed Müller cells and degenerating photoreceptors. Methods: We evaluated the changes of MAPK (ERK1/2) signalling and its downstream targets in human retinal explants treated with PD98059, a specific phosphorylated ERK1/2 inhibitor, by western blot and immunostaining. We further assessed photoreceptor degeneration by TUNEL staining and outer nuclear layer thickness. We also injected PD98059 into the eyes of mice exposed to photo-oxidative stress. We evaluated the protective effects on photoreceptor degeneration by optical coherence tomography (OCT) and electroretinography (ERG). The crosstalk between Müller cells and photoreceptors was further dissected based on the changes of transcription factors by RNA sequencing and protein profiles of multiple signalling pathways. Results: We found that MAPK (ERK1/2) signalling was mainly activated in Müller cells under stress, both ex vivo and in vivo. PD98059 inhibited the phosphorylation of ERK1/2, reduced expression of the gliotic marker glial fibrillary acidic protein (GFAP) in Müller cells and increased levels of the neuroprotective factor, interphotoreceptor retinoid-binding protein (IRBP) in photoreceptors. Inhibition of pERK1/2 also reduced retinal photo-oxidative damage in mice retinas assessed by OCT and ERG. We also identified that the JAK/STAT3 signalling pathway might mediate signalling transduction from Müller cells to photoreceptors. Conclusion: MAPK (ERK1/2) deactivation through chemical inhibition, mainly in stressed Müller cells, can alleviate gliosis in Müller cells and restore the expression of IRBP in photoreceptors, which appears to prevent retinal degeneration. Our findings suggested a new way to prevent photoreceptor degeneration by manipulating the stress response in Müller cells.

Transketolase in human Müller cells is critical to resist light stress through the pentose phosphate and NRF2 pathways.

The Pentose Phosphate Pathway (PPP), a metabolic offshoot of the glycolytic pathway, provides protective metabolites and molecules essential for cell redox balance and survival. Transketolase (TKT) is the critical enzyme that controls the extent of "traffic flow" through the PPP. Here, we explored the role of TKT in maintaining the health of the human retina. We found that Müller cells were the primary retinal cell type expressing TKT in the human retina. We further explored the role of TKT in human Müller cells by knocking down its expression in primary cultured Müller cells (huPMCs), isolated from the human retina (11 human donors in total), under light-induced oxidative stress. TKT knockdown and light stress reduced TKT enzymatic activities and the overall metabolic activities of huPMCs with no detectable cell death. TKT knockdown restrained the PPP traffic flow, reduced the expression of NAD(P)H Quinone Dehydrogenase 1 (NQO1), impaired the antioxidative response of NRF2 to light stress and aggravated the endoplasmic reticulum (ER) stress. TKT knockdown also inhibited overall glucose intake, reduced expression of Dihydrolipoamide dehydrogenase (DLD) and impaired the energy supply of the huPMCs. In summary, Müller cell-mediated TKT activity plays a critical protective role in the stressed retina. Knockdown of TKT disrupted the PPP and impaired overall glucose utilisation by huPMCs and rendered huPMCs more vulnerable to light stress by impairing energy supply and antioxidative NRF2 responses.

Single-Cell RNA Sequencing Reveals the Temporal Diversity and Dynamics of Cardiac Immunity after Myocardial Infarction.

Myocardial infarction (MI) is strongly associated with the temporal regulation of cardiac immunity. However, a variety of current clinical trials have failed because of the lack of post-MI immunomodulating/anti-inflammatory targets. Single-cell RNA sequencing analysis of the cardiac Cd45+ immune cell at 0, 3, 7, and 14 d after injury in a mouse left anterior descending coronary artery ligation model is performed. Major immune cell populations, distinct subsets, and dynamic changes are identified. Macrophages (Mø) are most abundant, peaking at 3 d after infarction. Mø-5 and Mø-6 are the predominant infiltrated subsets at this time point, with strong expression of inflammatory factors. Further analysis demonstrates that suppressing these sets attenuated pathological MI progression by preventing subsequent leukocyte extravasation and adverse remodeling. Abundant apoptotic neutrophils and a profibrotic macrophage subset on days 7 and 14, respectively, are also detected. These results provide a basis for developing cell type- and time-specific interventions in MI.

Danshen-Chuanxiongqin Injection attenuates cerebral ischemic stroke by inhibiting neuroinflammation via the TLR2/ TLR4-MyD88-NF-κB Pathway in tMCAO mice.

Danshen-Chuanxiongqin Injection (DCI) is a commonly used traditional Chinese medicine for the treatment of cerebral ischemic stroke in China. However, its underlying mechanisms remain completely understood. The current study was designed to explore the protective mechanisms of DCI against cerebral ischemic stroke through integrating whole-transcriptome sequencing coupled with network pharmacology analysis. First, using a mouse model of cerebral ischemic stroke by transient middle cerebral artery occlusion (tMCAO), we found that DCI (4.10 mL·kg-1) significantly alleviated cerebral ischemic infarction, neurological deficits, and the pathological injury of hippocampal and cortical neurons in mice. Next, the whole-transcriptome sequencing was performed on brain tissues. The cerebral ischemia disease (CID) network was constructed by integrating transcriptome sequencing data and cerebrovascular disease-related genes. The results showed CID network was imbalanced due to tMCAO, but a recovery regulation was observed after DCI treatment. Pathway analysis of the key genes with recovery efficiency showed that the neuroinflammation signaling pathway was highly enriched, while the TLR2/TLR4-MyD88-NF-κB pathway was predicted to be affected. Consistently, the in vivo validation experiments confirmed that DCI exhibited protective effects against cerebral ischemic stroke by inhibiting neuroinflammation via the TLR2/TLR4-MyD88-NF-κB pathway. More interestingly, DCI markedly suppressed the neutrophils infiltrated into the brain parenchyma via the choroid plexus route and showed anti-neuroinflammation effects. In conclusion, our results provide dependable evidence that inhibiting neuroinflammation via the TLR2/TLR4-MyD88-NF-κB pathway is the main mechanism of DCI against cerebral ischemic stroke in mice.