Nitric Oxide Regulates Protein Methylation during Stress Responses in Plants.

Methylation and nitric oxide (NO)-based S-nitrosylation are highly conserved protein posttranslational modifications that regulate diverse biological processes. In higher eukaryotes, PRMT5 catalyzes Arg symmetric dimethylation, including key components of the spliceosome. The Arabidopsis prmt5 mutant shows severe developmental defects and impaired stress responses. However, little is known about the mechanisms regulating the PRMT5 activity. Here, we report that NO positively regulates the PRMT5 activity through S-nitrosylation at Cys-125 during stress responses. In prmt5-1 plants, a PRMT5C125S transgene, carrying a non-nitrosylatable mutation at Cys-125, fully rescues the developmental defects, but not the stress hypersensitive phenotype and the responsiveness to NO during stress responses. Moreover, the salt-induced Arg symmetric dimethylation is abolished in PRMT5C125S/prmt5-1 plants, correlated to aberrant splicing of pre-mRNA derived from a stress-related gene. These findings define a mechanism by which plants transduce stress-triggered NO signal to protein methylation machinery through S-nitrosylation of PRMT5 in response to environmental alterations.

Redox proteomics of PANC-1 cells reveals the significance of HIF-1 signaling protein oxidation in pancreatic ductal adenocarcinoma pathogenesis.

BACKGROUND: Protein cysteine oxidation is substantially involved in various biological and pathogenic processes, but its implications in pancreatic cancer development remains poorly understood. METHODS AND RESULTS: In this study, we performed a global characterization of protein oxidation targets in PDAC cells through iodoTMT-based quantitative proteomics, which identified over 4300 oxidized cysteine sites in more than 2100 proteins in HPDE6c7 and PANC-1 cells. Among them, 1715 cysteine residues were shown to be differentially oxidized between HPDE6c7 and PANC-1 cells. Also, charged amino acids including aspartate, glutamate and lysine were significantly overrepresented in flanking sequences of oxidized cysteines. Differentially oxidized proteins in PANC-1 cells were enriched in multiple cancer-related biological processes and signaling pathways. Specifically, the HIF-1 signaling proteins exhibited significant oxidation alterations in PANC-1 cells, and the reduced PHD2 oxidation in human PDAC tissues was correlated with lower survival time in pancreatic cancer patients. CONCLUSION: These investigations provided new insights into protein oxidation-regulated signaling and biological processes during PDAC pathogenesis, which might be further explored for pancreatic cancer diagnosis and treatment.

Volumetric compression develops noise-driven single-cell heterogeneity.

Recent studies have revealed that extensive heterogeneity of biological systems arises through various routes ranging from intracellular chromosome segregation to spatiotemporally varying biochemical stimulations. However, the contribution of physical microenvironments to single-cell heterogeneity remains largely unexplored. Here, we show that a homogeneous population of non-small-cell lung carcinoma develops into heterogeneous subpopulations upon application of a homogeneous physical compression, as shown by single-cell transcriptome profiling. The generated subpopulations stochastically gain the signature genes associated with epithelial-mesenchymal transition (EMT; VIM, CDH1, EPCAM, ZEB1, and ZEB2) and cancer stem cells (MKI67, BIRC5, and KLF4), respectively. Trajectory analysis revealed two bifurcated paths as cells evolving upon the physical compression, along each path the corresponding signature genes (epithelial or mesenchymal) gradually increase. Furthermore, we show that compression increases gene expression noise, which interplays with regulatory network architecture and thus generates differential cell-fate outcomes. The experimental observations of both single-cell sequencing and single-molecule fluorescent in situ hybridization agrees well with our computational modeling of regulatory network in the EMT process. These results demonstrate a paradigm of how mechanical stimulations impact cell-fate determination by altering transcription dynamics; moreover, we show a distinct path that the ecology and evolution of cancer interplay with their physical microenvironments from the view of mechanobiology and systems biology, with insight into the origin of single-cell heterogeneity.

Helical nanofiber yarn enabling highly stretchable engineered microtissue.

Development of microtissues that possess mechanical properties mimicking those of native stretchable tissues, such as muscle and tendon, is in high demand for tissue engineering and regenerative medicine. However, regardless of the significant advances in synthetic biomaterials, it remains challenging to fabricate living microtissue with high stretchability because application of large strains to microtissues can damage the cells by rupturing their structures. Inspired by the hierarchical helical structure of native fibrous tissues and its behavior of nonaffine deformation, we develop a highly stretchable and tough microtissue fiber made up of a hierarchical helix yarn scaffold, scaling from nanometers to millimeters, that can overcome this limitation. This microtissue can be stretched up to 15 times its initial length and has a toughness of 57 GJ m-3 More importantly, cells grown on this scaffold maintain high viability, even under severe cyclic strains (up to 600%) that can be attributed to the nonaffine deformation under large strains, mimicking native biopolymer scaffolds. Furthermore, as proof of principle, we demonstrate that the nanotopography of the helical nanofiber yarn is able to induce cytoskeletal alignment and nuclear elongation, which promote myogenic differentiation of mesenchymal stem cells by triggering nuclear translocation of transcriptional coactivator with PDZ-binding motif (TAZ). The highly stretchable microtissues we develop here will facilitate a variety of tissue engineering applications and the development of engineered living systems.

High stretchability, strength, and toughness of living cells enabled by hyperelastic vimentin intermediate filaments.

In many developmental and pathological processes, including cellular migration during normal development and invasion in cancer metastasis, cells are required to withstand severe deformations. The structural integrity of eukaryotic cells under small deformations has been known to depend on the cytoskeleton including actin filaments (F-actin), microtubules (MT), and intermediate filaments (IFs). However, it remains unclear how cells resist severe deformations since both F-actin and microtubules yield or disassemble under moderate strains. Using vimentin containing IFs (VIFs) as a model for studying the large family of IF proteins, we demonstrate that they dominate cytoplasmic mechanics and maintain cell viability at large deformations. Our results show that cytoskeletal VIFs form a stretchable, hyperelastic network in living cells. This network works synergistically with other cytoplasmic components, substantially enhancing the strength, stretchability, resilience, and toughness of cells. Moreover, we find the hyperelastic VIF network, together with other quickly recoverable cytoskeletal components, forms a mechanically robust structure which can mechanically recover after damage.

Quantitative proteomics reveals redox-based functional regulation of photosynthesis under fluctuating light in plants.

Photosynthesis involves a series of redox reactions and is the major source of reactive oxygen species in plant cells. Fluctuating light (FL) levels, which occur commonly in natural environments, affect photosynthesis; however, little is known about the specific effects of FL on the redox regulation of photosynthesis. Here, we performed global quantitative mapping of the Arabidopsis thaliana cysteine thiol redox proteome under constant light and FL conditions. We identified 8857 redox-switched thiols in 4350 proteins, and 1501 proteins that are differentially modified depending on light conditions. Notably, proteins related to photosynthesis, especially photosystem I (PSI), are operational thiol-switching hotspots. Exposure of wild-type A. thaliana to FL resulted in decreased PSI abundance, stability, and activity. Interestingly, in response to PSI photodamage, more of the PSI assembly factor PSA3 dynamically switches to the reduced state. Furthermore, the Cys199 and Cys200 sites in PSA3 are necessary for its full function. Moreover, thioredoxin m (Trx m) proteins play roles in redox switching of PSA3, and are required for PSI activity and photosynthesis. This study thus reveals a mechanism for redox-based regulation of PSI under FL, and provides insight into the dynamic acclimation of photosynthesis in a changing environment.

Brain SegNet: 3D local refinement network for brain lesion segmentation.

MR images (MRIs) accurate segmentation of brain lesions is important for improving cancer diagnosis, surgical planning, and prediction of outcome. However, manual and accurate segmentation of brain lesions from 3D MRIs is highly expensive, time-consuming, and prone to user biases. We present an efficient yet conceptually simple brain segmentation network (referred as Brain SegNet), which is a 3D residual framework for automatic voxel-wise segmentation of brain lesion. Our model is able to directly predict dense voxel segmentation of brain tumor or ischemic stroke regions in 3D brain MRIs. The proposed 3D segmentation network can run at about 0.5s per MRIs - about 50 times faster than previous approaches Med Image Anal 43: 98-111, 2018, Med Image Anal 36:61-78, 2017. Our model is evaluated on the BRATS 2015 benchmark for brain tumor segmentation, where it obtains state-of-the-art results, by surpassing recently published results reported in Med Image Anal 43: 98-111, 2018, Med Image Anal 36:61-78, 2017. We further applied the proposed Brain SegNet for ischemic stroke lesion outcome prediction, with impressive results achieved on the Ischemic Stroke Lesion Segmentation (ISLES) 2017 database.

Size- and speed-dependent mechanical behavior in living mammalian cytoplasm.

Active transport in the cytoplasm plays critical roles in living cell physiology. However, the mechanical resistance that intracellular compartments experience, which is governed by the cytoplasmic material property, remains elusive, especially its dependence on size and speed. Here we use optical tweezers to drag a bead in the cytoplasm and directly probe the mechanical resistance with varying size a and speed V We introduce a method, combining the direct measurement and a simple scaling analysis, to reveal different origins of the size- and speed-dependent resistance in living mammalian cytoplasm. We show that the cytoplasm exhibits size-independent viscoelasticity as long as the effective strain rate V/a is maintained in a relatively low range (0.1 s-1 < V/a < 2 s-1) and exhibits size-dependent poroelasticity at a high effective strain rate regime (5 s-1 < V/a < 80 s-1). Moreover, the cytoplasmic modulus is found to be positively correlated with only V/a in the viscoelastic regime but also increases with the bead size at a constant V/a in the poroelastic regime. Based on our measurements, we obtain a full-scale state diagram of the living mammalian cytoplasm, which shows that the cytoplasm changes from a viscous fluid to an elastic solid, as well as from compressible material to incompressible material, with increases in the values of two dimensionless parameters, respectively. This state diagram is useful to understand the underlying mechanical nature of the cytoplasm in a variety of cellular processes over a broad range of speed and size scales.

Increased concentration of serum periostin is associated with poor outcome of patients with aneurysmal subarachnoid hemorrhage.

OBJECTIVE: To explore the role of serum periostin in patients with aneurysmal subarachnoid hemorrhage (aSAH). METHOD: We conducted a retrospective study and 124 aSAH patients treated in Shenzhen People's hospital during March 1st 2015 to December 30th 2016 were included. Baseline information, neurological status and clinical outcome were recorded. Blood samples on admission were collected and enzyme linked immunosorbent assay (ELISA) kits were used to detect the serum level of periostin. Spearman's Correlation Analysis was used to analyze the correlation between periostin and clinical severity. Receiver operating characteristic (ROC) curve was performed to investigate variables' prognostic value in patients with aSAH. RESULTS: The average age of patients included was 57.23 years old. Preliminary analysis revealed that serum periostin was significantly correlated with clinical severity. Patients with poor outcome at 12 months had higher level of periostin than patients with good outcome. Multivariate logistic regression analysis showed elevated level of periostin was significantly associated with poor outcome and the AUC was 0.85 for periostin in predicting poor outcome of patient with aSAH. CONCLUSION: Elevated serum periostin concentrations are significantly associated with clinical severity and poor outcome of aSAH patients, which indicate serum periostin can be used as a prognostic biomarker in patients with aSAH.

Characterization of an Integrated Active Glu-1Ay Allele in Common Wheat from Wild Emmer and Its Potential Role in Flour Improvement.

Glu-1Ay, one of six genes encoding a high molecular weight glutenin subunit (HMW-GS), is frequently silenced in hexaploid common wheat. Here, an active allele of Glu-1Ay was integrated from wild emmer wheat (Triticum turgidum ssp. dicoccoides) accession D97 into the common wheat (Triticum aestivum) cultivar Chuannong 16 via the repeated self-fertilization of the pentaploid interspecific hybrid, culminating in the selection of a line TaAy7-40 shown to express the wild emmer Glu-1Ay allele. The open reading frame of this allele was a 1830 bp long sequence, demonstrated by its heterologous expression in Escherichia coli to encode a 608-residue polypeptide. Its nucleotide sequence was 99.2% identical to that of the sequence within the wild emmer parent. The TaAy7-40 introgression line containing the active Glu-1Ay allele showed higher protein content, higher sodium dodecyl sulfate (SDS) sedimentation value, higher content of wet gluten in the flour, higher grain weight, and bigger grain size than Chuannong 16. The end-use quality parameters of the TaAy7-40 were superior to those of the medium gluten common wheat cultivars Mianmai 37 and Neimai 9. Thus, the active Glu-1Ay allele might be of potential value in breeding programs designed to improve wheat flour quality.

S-nitrosylation positively regulates ascorbate peroxidase activity during plant stress responses.

Nitric oxide (NO) and reactive oxygen species (ROS) are two classes of key signaling molecules involved in various developmental processes and stress responses in plants. The burst of NO and ROS triggered by various stimuli activates downstream signaling pathways to cope with abiotic and biotic stresses. Emerging evidence suggests that the interplay of NO and ROS plays a critical role in regulating stress responses. However, the underpinning molecular mechanism remains poorly understood. Here, we show that NO positively regulates the activity of the Arabidopsis (Arabidopsis thaliana) cytosolic ascorbate peroxidase1 (APX1). We found that S-nitrosylation of APX1 at cysteine (Cys)-32 enhances its enzymatic activity of scavenging hydrogen peroxide, leading to the increased resistance to oxidative stress, whereas a substitution mutation at Cys-32 causes the reduction of ascorbate peroxidase activity and abolishes its responsiveness to the NO-enhanced enzymatic activity. Moreover, S-nitrosylation of APX1 at Cys-32 also plays an important role in regulating immune responses. These findings illustrate a unique mechanism by which NO regulates hydrogen peroxide homeostasis in plants, thereby establishing a molecular link between NO and ROS signaling pathways.

Site-specific nitrosoproteomic identification of endogenously S-nitrosylated proteins in Arabidopsis.

Nitric oxide (NO) regulates multiple developmental events and stress responses in plants. A major biologically active species of NO is S-nitrosoglutathione (GSNO), which is irreversibly degraded by GSNO reductase (GSNOR). The major physiological effect of NO is protein S-nitrosylation, a redox-based posttranslational modification mechanism by covalently linking an NO molecule to a cysteine thiol. However, little is known about the mechanisms of S-nitrosylation-regulated signaling, partly due to limited S-nitrosylated proteins being identified. In this study, we identified 1,195 endogenously S-nitrosylated peptides in 926 proteins from the Arabidopsis (Arabidopsis thaliana) by a site-specific nitrosoproteomic approach, which, to date, is the largest data set of S-nitrosylated proteins among all organisms. Consensus sequence analysis of these peptides identified several motifs that contain acidic, but not basic, amino acid residues flanking the S-nitrosylated cysteine residues. These S-nitrosylated proteins are involved in a wide range of biological processes and are significantly enriched in chlorophyll metabolism, photosynthesis, carbohydrate metabolism, and stress responses. Consistently, the gsnor1-3 mutant shows the decreased chlorophyll content and altered photosynthetic properties, suggesting that S-nitrosylation is an important regulatory mechanism in these processes. These results have provided valuable resources and new clues to the studies on S-nitrosylation-regulated signaling in plants.

A Simple Scoring System for Predicting the Risk of Delayed Hyponatremia After Endoscopic Transsphenoidal Surgery for Pituitary Adenomas.

BACKGROUND: To identify high-risk patients for delayed postoperative hyponatremia (DPH) early, we constructed a simple and effective scoring system. METHODS: We retrospectively analyzed 141 consecutive patients who underwent endoscopic transsphenoidal surgery from January 2019 to December 2022. Patients were divided into DPH group and nondelayed postoperative hyponatremia group based on whether hyponatremia occurred after the third postoperative day. Multivariable logistic regression analysis was conducted to determine the predictive factors of DPH, and a simple scoring system was constructed based on these predictors. RESULTS: Among 141 patients, 36 (25.5%) developed DPH. Multivariable logistic regression analysis showed that age ≥48 years (odds ratio [OR], 3.74; 95% confidence interval [CI], 1.14-12.21; P = 0.029), Knosp grade ≥3 (OR, 5.17; 95% CI, 1.20-22.27; P = 0.027), postoperative hypokalemia within three days (OR, 3.13; 95% CI, 1.05-9.33; P = 0.040), a difference in blood sodium levels between the first and second day after surgery ≥1 mEq/L (OR, 3.65; 95% CI, 1.05-12.77; P = 0.043), and postoperative diabetes insipidus (OR, 3.57; 95% CI, 1.16-10.96; P = 0.026) were independent predictors of DPH. CONCLUSIONS: This scoring system for predicting DPH has an area under the receiver operating characteristic curve of 0.856 (95% CI, 0.787-0.925), indicating moderate to good predictive value for DPH in our cohort, but further prospective external validation is needed.

Hederagenin reduces Aß-induced oxidative damage, decreases Aß deposition and promotes cell survival by the P13†K/Akt signaling pathway.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by memory loss and cognitive impairment. ß-amyloid (Aß) is one of the typical pathological features of AD, and its accumulation leads to neuronal death from oxidative stress. Here, we found that hederagenin (HG), a natural product, exhibits anti-tumor, anti-inflammatory, anti-depressant, anti-neurodegenerative biological activities. However, whether HG has anti-Aß activity remains unclear. Based on the characteristics of HG, it is hypothesized that HG has biological activity against Aß injury. Therefore, Aß-injured SH-SY5Y cells were constructed, and the protective effect of HG against Aß injury was further evaluated using C. elegans. The results showed that HG increased superoxide dismutase activity, effectively reduced Aß-induced oxidative damage, and reduced apoptosis via the PI3 K/Akt signaling pathway. HG inhibited Aß deposition and delayed senescence and paralysis in the C. elegans strain, CL4176. HG showed inhibitory effects on Aß; therefore, more natural active products are expected to be applied in AD therapy.

microRNA-130b May Induce Cerebral Vasospasm after Subarachnoid Hemorrhage via Modulating Kruppel-like Factor 4.

Recently, the diverse functions of microRNAs (miRNAs) in brain diseases have been demonstrated. We intended to uncover the functional role of microRNA-130b (miR-130b) in cerebral vasospasm (CVS) following subarachnoid hemorrhage (SAH). SAH was induced by injecting the autologous blood into the cisterna magna of Sprague Dawley rats. The cerebral vascular smooth muscle cells (cVSMCs) were extracted for in vitro experimentation. In vitro and in vivo assays were implemented with transfection of miR-130b mimic/inhibitor, sh-Kruppel-like factor 4 (KLF4), oe-KLF4 plasmids or p38/MAPK signaling pathway agonist (anisomycin), respectively, to elaborate the role of miR-130b in CVS following SAH. Elevated miR-130b and reduced KLF4 were found in SAH patients and rat models of SAH. KLF4 was the target gene of miR-130b. miR-130b promoted the proliferation and migration of cVSMCs through the Inhibition of KLF4. Besides, KLF4 inhibited the proliferation and migration of cVSMCs through blockage of the p38/MAPK pathway. Furthermore, in vivo assay confirmed the inhibitory effect of decreased miR-130b in CVS following SAH. In conclusion, miR-130b may activate the p38/MAPK signaling pathway through targeted inhibition of KLF4, thereby contributing to some extent to the development of cerebral vasospasm after SAH.

Angelica oil restores the intestinal barrier function by suppressing S100A8/A9 signalling in mice with ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) progression is driven by the activation of immune cells that release pro-inflammatory mediators to disrupt intestinal epithelial barrier integrity. This study aimed to investigate the potential protective effects of Angelica oil (AO) on the intestinal epithelial barrier in mice with UC and the underlying mechanisms. METHODS: Improvement of the disease state and protective effect of AO on the intestinal epithelial barrier were observed in mice with dextran sulphate sodium salt (DSS)-induced UC. Protein microarrays were used to screen AO-affected cytokine pools and their recruited immune cells for accumulation in the tissues. Furthermore, quantitative proteomics was applied to search for AO-acting molecules and to verify in vitro the functions of key molecules between inflammation and the intestinal mucosal barrier. RESULTS: AO significantly alleviated intestinal inflammation, reduced intestinal permeability, and retained barrier function in mice with UC. Furthermore, cytokines inhibited by AO mainly promoted monocyte and neutrophil activation or chemotaxis. Moreover, proteomic screening revealed that S100A8/A9 was a key molecule significantly regulated by AO, and its mediated TLR4/NF-κB pathway was also inhibited. Finally, we verified that AO inhibited the activation of the S100A8/A9/TLR4 signalling pathway and enhanced the expression of tight junctions (TJs) proteins using a cellular model of intestinal barrier damage induced by S100A8/A9 or macrophage-derived medium. And the enhancement of TJs in intestinal epithelial cells and the inhibition of inflammatory signalling by AO were significantly attenuated due to the application of S100A8/A9 monoclonal antibody. CONCLUSION: These results demonstrated that AO improves intestinal mucosal barrier damage in the inflammatory environment of mice with UC by inhibiting the expression of S100A8/A9 and the activation of its downstream TLR4/NF-κB signalling pathway.

Dual roles for CND1 in maintenance of nuclear and chloroplast genome stability in plants.

The coordination of chloroplast and nuclear genome status is critical for plant cell function. Here, we report that Arabidopsis CHLOROPLAST AND NUCLEUS DUAL-LOCALIZED PROTEIN 1 (CND1) maintains genome stability in the chloroplast and the nucleus. CND1 localizes to both compartments, and complete loss of CND1 results in embryo lethality. Partial loss of CND1 disturbs nuclear cell-cycle progression and photosynthetic activity. CND1 binds to nuclear pre-replication complexes and DNA replication origins and regulates nuclear genome stability. In chloroplasts, CND1 interacts with and facilitates binding of the regulator of chloroplast genome stability WHY1 to chloroplast DNA. The defects in nuclear cell-cycle progression and photosynthesis of cnd1 mutants are respectively rescued by compartment-restricted CND1 localization. Light promotes the association of CND1 with HSP90 and its import into chloroplasts. This study provides a paradigm of the convergence of genome status across organelles to coordinately regulate cell cycle to control plant growth and development.

Emergent phases of ecological diversity and dynamics mapped in microcosms.

From tropical forests to gut microbiomes, ecological communities host notably high numbers of coexisting species. Beyond high biodiversity, communities exhibit a range of complex dynamics that are difficult to explain under a unified framework. Using bacterial microcosms, we performed a direct test of theory predicting that simple community-level features dictate emergent behaviors of communities. As either the number of species or the strength of interactions increases, we show that microbial ecosystems transition between three distinct dynamical phases, from a stable equilibrium in which all species coexist to partial coexistence to emergence of persistent fluctuations in species abundances, in the order predicted by theory. Under fixed conditions, high biodiversity and fluctuations reinforce each other. Our results demonstrate predictable emergent patterns of diversity and dynamics in ecological communities.

miR-133a-5p Inhibits Glioma Cell Proliferation by Regulating IGFBP3.

Objective: This research aims to investigate the expression of miR-133a-5p in glioma tissues and its impact on glioma cell proliferation. Methods: Fluorescence-quantitative PCR was used to detect the expression of miR-133a-5p in 25 cases of glioma and adjuncent tissues. CCK-8 and colony formation analyses were used to evaluate the impact of transfection with miR-133a-5p inhibitors or mimics on glioma cell growth and colony formation. The IGFBP3 (insulin-like growth factor-binding protein-3) and miR-133a-5p binding sites were predicted using Starbase, and the miR-133a-5p binding capacity with 3'UTR of IGFBP3 gene was determined using a luciferase gene reporter system. Following transfection with miR-133a-5p mimics or inhibitors, the IGFBP3 protein expression in glioma cells was determined by western blotting. The colony formation assay was applied to evaluate the influence of IGFBP3 overexpression on the miR-133a-5p in glioma cell proliferation. For assessment of the IGFBP3 expression in glioma tissues and prognosis, TCGA database was employed. Results: The expression of miR-133a-5p was considerably reduced in glioma tissue compared to adjuncent control tissue. In addition, miR-133a-5p expression decreased with increasing glioma malignancy. Glioma cell growth and colony formation were reduced after miR-133a-5p mimics were transfected, while transfection of miR-133a-5p inhibitors had a reverse impact. The expression of IGFBP3 was affected by miR-133a-5p by binding to its 3'UTR region. Additional study demonstrated that the overall survival (OS) of subjects with increased IGFBP3 expression was considerably lower compared to patients with decreased IGFBP3 expression. The IGFBP3 overexpression effectively counteracts the glioma cell proliferation-inhibiting impact of miR-133a-5p. Conclusion: miR-133a-5p acts as a glioma tumor suppressor gene. It reduces glioma cell proliferation by modulating IGFBP3 and could be a target for glioma therapy.

A combined analysis of bulk and single-cell sequencing data reveals that depleted extracellular matrix and enhanced immune processes co-contribute to fluorouracil beneficial responses in gastric cancer.

Fluorouracil, also known as 5-FU, is one of the most commonly used chemotherapy drugs in the treatment of advanced gastric cancer (GC). Whereas, the presence of innate or acquired resistance largely limits its survival benefit in GC patients. Although accumulated studies have demonstrated the involvement of tumor microenvironments (TMEs) in chemo-resistance induction, so far little is known about the relevance of GC TMEs in 5-FU resistance. To this end, in this study, we investigated the relationship between TME features and 5-FU responses in GC patients using a combined analysis involving both bulk sequencing data from the TCGA database and single-cell RNA sequencing data from the GEO database. We found that depleted extracellular matrix (ECM) components such as capillary/stroma cells and enhanced immune processes such as increased number of M1 polarized macrophages/Memory T cells/Natural Killer T cells/B cells and decreased number of regulatory T cells are two important features relating to 5-FU beneficial responses in GC patients, especially in diffuse-type patients. We further validated these two features in the tumor tissues of 5-FU-benefit GC patients using immunofluorescence staining experiments. Based on this finding, we also established a Pro (63 genes) and Con (199 genes) gene cohort that could predict 5-FU responses in GC with an AUC (area under curve) score of 0.90 in diffuse-type GC patients, and further proved the partial applicability of this gene panel pan-cancer-wide. Moreover, we identified possible communications mediated by heparanase and galectin-1 which could regulate ECM remodeling and tumor immune microenvironment (TIME) reshaping. Altogether, these findings deciphered the relationship between GC TMEs and 5-FU resistance for the first time, as well as provided potential therapeutic targets and predicting rationale to overcome this chemo-resistance, which could shed some light on developing novel precision treatment strategies in clinical practice.