Scalable spatial single-cell transcriptomics and translatomics in 3D thick tissue blocks.

Characterizing the transcriptional and translational gene expression patterns at the single-cell level within their three-dimensional (3D) tissue context is essential for revealing how genes shape tissue structure and function in health and disease. However, most existing spatial profiling techniques are limited to 5-20 µm thin tissue sections. Here, we developed Deep-STARmap and Deep-RIBOmap, which enable 3D in situ quantification of thousands of gene transcripts and their corresponding translation activities, respectively, within 200-µm thick tissue blocks. This is achieved through scalable probe synthesis, hydrogel embedding with efficient probe anchoring, and robust cDNA crosslinking. We first utilized Deep-STARmap in combination with multicolor fluorescent protein imaging for simultaneous molecular cell typing and 3D neuron morphology tracing in the mouse brain. We also demonstrate that 3D spatial profiling facilitates comprehensive and quantitative analysis of tumor-immune interactions in human skin cancer.

Triggering of endoplasmic reticulum stress via ATF4-SPHK1 signaling promotes glioblastoma invasion and chemoresistance.

Despite advances in therapies, glioblastoma (GBM) recurrence is almost inevitable due to the aggressive growth behavior of GBM cells and drug resistance. Temozolomide (TMZ) is the preferred drug for GBM chemotherapy, however, development of TMZ resistance is over 50% cases in GBM patients. To investigate the mechanism of TMZ resistance and invasive characteristics of GBM, analysis of combined RNA-seq and ChIP-seq was performed in GBM cells in response to TMZ treatment. We found that the PERK/eIF2α/ATF4 signaling was significantly upregulated in the GBM cells with TMZ treatment, while blockage of ATF4 effectively inhibited cell migration and invasion. SPHK1 expression was transcriptionally upregulated by ATF4 in GBM cells in response to TMZ treatment. Blockage of ATF4-SPHK1 signaling attenuated the cellular and molecular events in terms of invasive characteristics and TMZ resistance. In conclusion, GBM cells acquired chemoresistance in response to TMZ treatment via constant ER stress. ATF4 transcriptionally upregulated SPHK1 expression to promote GBM cell aggression and TMZ resistance. The ATF4-SPHK1 signaling in the regulation of the transcription factors of EMT-related genes could be the underlying mechanism contributing to the invasion ability of GBM cells and TMZ resistance. ATF4-SPHK1-targeted therapy could be a potential strategy against TMZ resistance in GBM patients.

Inhibition of human peptide deformylase by actinonin sensitizes glioblastoma cells to temozolomide chemotherapy.

Glioblastoma multiforme (GBM) is a common intracranial primary tumor of the central nervous system with high malignancy, poor prognosis, and short survival. Studies have shown that mitochondrial energy metabolism plays an important role in GBM chemotherapy resistance, suggesting that interrupting mitochondrial oxidative phosphorylation (OXPHOS) may improve GBM treatment. Human peptide deformylase (HsPDF) is a mitochondrial deformylase that removes the formylated methionine from the N-terminus of proteins encoded by mitochondrial DNA (mtDNA), thereby contributing to correct protein folding and participating in the assembly of the electron respiratory chain complex. In this study, we found that the expression of mtDNA-encoded proteins was significantly downregulated after treatment of GBM cells U87MG and LN229 with the HsPDF inhibitor, actinonin. In combination with temozolomide, a preferred chemotherapeutic medicine for GBM, the OXPHOS level decreased, mitochondrial protein homeostasis was unbalanced, mitochondrial fission increased, and the integrated stress response was activated to promote mitochondrial apoptosis. These findings suggest that HsPDF inhibition is an important strategy for overcoming chemoresistance of GBM cells.

Adaptive Changes Allow Targeting of Ferroptosis for Glioma Treatment.

Ferroptosis is a type of regulated cell death that plays an essential role in various brain diseases, including cranial trauma, neuronal diseases, and brain tumors. It has been reported that cancer cells rely on their robust antioxidant capacity to escape ferroptosis. Therefore, ferroptosis exploitation could be an effective strategy to prevent tumor proliferation and invasion. Glioma is a common malignant craniocerebral tumor exhibiting complicated drug resistance and survival mechanisms, resulting in a high mortality rate and short survival time. Recent studies have determined that metabolic alterations in glioma offer exploitable therapeutic targets. These metabolic alterations allow targeted therapy to achieve some initial efficacy but have failed to inhibit glioma growth, invasion, and drug resistance effectively. It has been proposed that the reason for the high malignancy and drug resistance observed with glioma is that these tumors can effectively evade ferroptosis. Ferroptosis-inducing drugs were found to exert a positive effect by targeting this particular characteristic of glioma cells. Moreover, gliomas develop enhanced drug resistance through anti-ferroptosis mechanisms. In this study, we provided an overview of the mechanisms by which glioma aggressiveness and drug resistance are mediated by the evasion of ferroptosis. This information might provide new targets for glioma therapy as well as new insights and ideas for future research.

Interfering with mitochondrial dynamics sensitizes glioblastoma multiforme to temozolomide chemotherapy.

Glioblastoma multiforme (GBM) is a primary tumour of the central nervous system (CNS) that exhibits the highest degree of malignancy. Radiotherapy and chemotherapy are essential to prolong the survival time of patients. However, clinical work has demonstrated that sensitivity of GBM to chemotherapy decreases with time. The phenomenon of multi-drug resistance (MDR) reminds us that there may exist some fundamental mechanisms in the process of chemo-resistance. We tried to explore the mechanism of GBM chemo-resistance from the perspective of energy metabolism. First, we found that the oxidative phosphorylation (OXPHOS) level of SHG44 and U87 cells increased under TMZ treatment. In further studies, it was found that the expression of PINK1 and mitophagy flux downstream was downregulated in GBM cells, which were secondary to the upregulation of TP53 in tumour cells under TMZ treatment. At the same time, we examined the mitochondrial morphology in tumour cells and found that the size of mitochondria in tumour cells increased under the treatment of TMZ, which originated from the regulation of AMPK on the subcellular localization of Drp1 under the condition of unbalanced energy supply and demand in tumour cells. The accumulation of mitochondrial mass and the optimization of mitochondrial quality accounted for the increased oxidative phosphorylation, and interruption of the mitochondrial fusion process downregulated the efficiency of oxidative phosphorylation and sensitized GBM cells to TMZ, which was also confirmed in the in vivo experiment. What is more, interfering with this process is an innovative strategy to overcome the chemo-resistance of GBM cells.

Coniferyl aldehyde alleviates LPS-induced WI-38 cell apoptosis and inflammation injury via JAK2-STAT1 pathway in acute pneumonia.

Pneumonia is a kind of inflammatory disease characterized by pathogen infection of lower respiratory track. Lipopolysaccharide (LPS) is the main bioactive component of Gram-negative bacteria responsible for inflammatory response. Recently, coniferyl aldehyde (CA) has been reported to play a crucial role because of its anti-inflammatory activity. However, the effect and mechanisms of CA in ameliorating symptoms of acute pneumonia remain unknown. Evaluating and identifying the value and exploring the mechanisms of CA on LPS-mediated WI-38 apoptosis and inflammation were the aims of this study. Here, CCK-8 cell viability assay was applied on WI-38 after treatment with or without LPS at different doses of CA to verify that CA can increase LPS-induced cell viability. Then, quantitative polymerase chain reaction (qPCR) and enzyme-linked-immunosorbent serologic assays (ELISA) suggested that LPS treatment dramatically decreased the expression level of IL-10 (anti-inflammatory factor) while strikingly increasing the expression levels of IL-1ß, IL-6, and TNF-α (tumor necrosis factor-α; proinflammatory factor) whereas CA treatment attenuates LPS-induced inflammation of WI-38. Further, flow cytometry and Western blot assay verified that LPS treatment dramatically promoted apoptosis of WI-38 cells, while administration of CA notably inhibited apoptosis of WI-38 cells. Moreover, the Western blot assay hinted that CA could inactivate LPS-induced JAK2-STAT1 signaling pathway. These findings indicated that CA could alleviate LPS-mediated WI-38 apoptosis and inflammation injury through JAK2-STAT1 pathway in acute pneumonia.

The MAMs Structure and Its Role in Cell Death.

The maintenance of cellular homeostasis involves the participation of multiple organelles. These organelles are associated in space and time, and either cooperate or antagonize each other with regards to cell function. Crosstalk between organelles has become a significant topic in research over recent decades. We believe that signal transduction between organelles, especially the endoplasmic reticulum (ER) and mitochondria, is a factor that can influence the cell fate. As the cellular center for protein folding and modification, the endoplasmic reticulum can influence a range of physiological processes by regulating the quantity and quality of proteins. Mitochondria, as the cellular "energy factory," are also involved in cell death processes. Some researchers regard the ER as the sensor of cellular stress and the mitochondria as an important actuator of the stress response. The scientific community now believe that bidirectional communication between the ER and the mitochondria can influence cell death. Recent studies revealed that the death signals can shuttle between the two organelles. Mitochondria-associated membranes (MAMs) play a vital role in the complex crosstalk between the ER and mitochondria. MAMs are known to play an important role in lipid synthesis, the regulation of Ca2+ homeostasis, the coordination of ER-mitochondrial function, and the transduction of death signals between the ER and the mitochondria. Clarifying the structure and function of MAMs will provide new concepts for studying the pathological mechanisms associated with neurodegenerative diseases, aging, and cancers. Here, we review the recent studies of the structure and function of MAMs and its roles involved in cell death, especially in apoptosis.

Inhibition of pyruvate dehydrogenase kinase­1 by dicoumarol enhances the sensitivity of hepatocellular carcinoma cells to oxaliplatin via metabolic reprogramming.

The Warburg effect is a unique metabolic feature of the majority of tumor cells and is closely related to chemotherapeutic resistance. Pyruvate dehydrogenase kinase 1 (PDK1) is considered a 'switch' that controls the fate of pyruvate in glucose metabolism. However, to date, to the best of our knowledge, there are only a few studies to available which had studied the reduction of chemotherapeutic resistance via the metabolic reprogramming of tumor cells with PDK1 as a target. In the present study, it was found dicoumarol (DIC) reduced the phosphorylation of pyruvate dehydrogenase (PDH) by inhibiting the activity of PDK1, which converted the metabolism of human hepatocellular carcinoma (HCC) cells to oxidative phosphorylation, leading to an increase in mitochondrial reactive oxygen species ROS (mtROS) and a decrease in mitochondrial membrane potential (MMP), thereby increasing the apoptosis induced by oxaliplatin (OXA). Furthermore, the present study elucidated that the targeting of PDK1 may be a potential strategy for targeting metabolism in the chemotherapy of HCC. In addition, DIC as an 'old drug' exhibits novel efficacy, bringing new hope for antitumor therapy.

Targeting off-target effects: endoplasmic reticulum stress and autophagy as effective strategies to enhance temozolomide treatment.

Glioblastoma multiforme (GBM) is the most common and aggressive adult primary central nervous system tumor. Unfortunately, GBM is resistant to the classic chemotherapy drug, temozolomide (TMZ). As well as its classic DNA-targeting effects, the off-target effects of TMZ can have pro-survival or pro-death roles and regulate GBM chemoradiation sensitivity. Endoplasmic reticulum (ER) stress is one of the most common off-target effects. ER stress and its downstream induction of autophagy, apoptosis, and other events have important roles in regulating TMZ sensitivity. Autophagy is an evolutionarily conserved cellular homeostasis mechanism that is closely associated with ER stress-induced apoptosis. Under ER stress, autophagy cannot only remove misfolded/unfolded proteins and damaged organelles and degrade and inhibit apoptosis-related caspase activation to reduce cell damage, but may also promote apoptosis dependent on ER stress intensity. Although some protein interactions between autophagy and apoptosis and common upstream signaling pathways have been found, the underlying regulatory mechanisms are still not fully understood. This review summarizes the possible mechanisms underlying the current known off-target roles of ER stress and downstream autophagy in the regulation of cell fate and evaluates their role in TMZ treatment and their potential as therapeutic targets.

Deficiency in IL-33/ST2 Axis Reshapes Mitochondrial Metabolism in Lipopolysaccharide-Stimulated Macrophages.

The polarization and function of macrophages play essential roles in controlling immune responses. Interleukin (IL)-33 is a member of the IL-1 family that has been shown to influence macrophage activation and polarization, but the underlying mechanisms are not fully understood. Mitochondrial metabolism has been reported to be a central player in shaping macrophage polarization; previous studies have shown that both aerobic glycolysis and oxidative phosphorylation uniquely regulate the functions of M1 and M2 macrophages. Whether IL-33 polarizes macrophages by reshaping mitochondrial metabolism requires further investigation. In this work, we examined the mitochondrial metabolism of bone marrow-derived macrophages (BMDMs) from either wild type (WT), Il33-overexpressing, or IL-33 receptor knockout (St2-/-) mice challenged with lipopolysaccharide (LPS). We found that after LPS stimulation, compared with WT BMDMs, St2-/- BMDMs had reduced cytokine production and increased numbers and activity of mitochondria via the metabolism regulator peroxisome proliferator-activated receptor-C coactivator-1 α (PGC1α). This was demonstrated by increased mitochondrial DNA copy number, mitochondria counts, mitochondria fission- and fusion-related gene expression, oxygen consumption rates, and ATP production, and decreased glucose uptake, lactate production, and extracellular acidification rates. For Il33-overexpressing BMDMs, the metabolic reprogramming upon LPS stimulation was similar to WT BMDMs, and was accompanied by increased M1 macrophage activity. Our findings suggested that the pleiotropic IL-33/ST2 pathway may influence the polarization and function of macrophages by regulating mitochondrial metabolism.

Regulation of Integrated Stress Response Sensitizes U87MG Glioblastoma Cells to Temozolomide Through the Mitochondrial Apoptosis Pathway.

Glioblastomas are the most frequently diagnosed and worst primary malignancy of the central nervous system, with very poor prognosis. The first-line antiglioma drug temozolomide shows decreasing therapeutic efficacy as treatment progresses. As the integrated stress response (ISR) may be a resistance factor and severe stress might transform the protective effect of the ISR into a damage effect, pharmacological regulation of ISR may be an effective way to sensitize glioma to temozolomide. The aim of the present study was to investigate the mechanisms of the ISR in regulating the therapeutic effect of temozolomide in the human glioblastoma multiforme cell line U87MG. Cultured U87MG cells were treated with temozolomide and PCR array was used to screen key factors in the response to treatment. Cells were co-treated with temozolomide and the eIF2α phosphatase inhibitor salubrinal, and cell apoptosis was measured. Combination treatment with temozolomide and salubrinal had a synergistic effect on cell viability. Salubrinal could upregulate the expression of ATF4, a key factor in the ISR, and enhance temozolomide-induced apoptosis. ATF4 transcriptionally regulated expression of the BH3-ONLY protein NOXA, thus inducing mitochondrial apoptosis. These findings suggest that ISR and ATF4 are involved in the death crosstalk between the endoplasmic reticulum and mitochondria and might be a potential target to enhance the therapeutic effect of temozolomide in patients with glioblastoma multiforme. Anat Rec, 2018. © 2018 Wiley Periodicals, Inc.

Genetic Variations of Kinase Inserts Domain Receptor (KDR) Gene Are Associated with the Risk of Astrocytomas.

Astrocytomas is one of the most common central nervous system (CNS) tumors with high mortality rate. Kinase insert domain receptor (KDR) is involved in the regulation of tumor angiogenesis, migration, and vascular permeability. The aim of the study was to explore the relationship between KDR polymorphisms and risk of astrocytomas. Blood samples were collected from 157 astrocytomas patients and 160 healthy controls. Three tag-SNPs (rs2071559C/T, rs2305948T/C, and rs1870377A/T) were identified from the International HapMap Project Databases and genotyped using the method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). We evaluated the astrocytomas risk caused by individual SNPs and haplotype using odds ratios (ORs) and their 95 % confidence intervals (CIs). In the overall individual SNP analysis, the C allele of rs2071559 was correlated with an increased risk of astrocytomas. However, individuals with mutant allele A and genotype TA + AA of rs1870377 showed a protective effect against astrocytomas. Subgroup analysis based on WHO tumor grade revealed that the C allele of rs2071559 had more influence with the risk of astrocytomas in the grade III-IV (OR = 1.91) subgroup than the grade I-II (OR = 1.47) group. Genotype TT of rs2305948 was found to be significantly associated with susceptibility of astrocytomas only in the grade III-IV subgroup. The protective effect of rs1870377 did not reveal significant difference between the grade III-IV and grade I-II subgroups. Meanwhile, stratified analysis demonstrated that mutation of rs2071559 and rs2305948 could elevate the risk of astrocytomas more significantly in the subgroup of smokers than the nonsmokers. Interestingly, the protective effect of rs1870377 was more obvious in the nonsmokers than the smokers. Additionally, haplotype-specific analysis showed that haplotype CCT and CTT were related with an increased risk of astrocytomas. We found that individual with variants of rs2071559*C and rs2305948*T might significantly elevate the risk of astrocytomas, while mutants of rs1870377*A was associated with the decreased risk of astrocytomas. Further studies about ethnically diverse populations with larger sample size should be performed to confirm the correlation between KDR gene polymorphisms and risk of astrocytomas.

Identification of New Candidate Genes and Chemicals Related to Esophageal Cancer Using a Hybrid Interaction Network of Chemicals and Proteins.

Cancer is a serious disease responsible for many deaths every year in both developed and developing countries. One reason is that the mechanisms underlying most types of cancer are still mysterious, creating a great block for the design of effective treatments. In this study, we attempted to clarify the mechanism underlying esophageal cancer by searching for novel genes and chemicals. To this end, we constructed a hybrid network containing both proteins and chemicals, and generalized an existing computational method previously used to identify disease genes to identify new candidate genes and chemicals simultaneously. Based on jackknife test, our generalized method outperforms or at least performs at the same level as those obtained by a widely used method--the Random Walk with Restart (RWR). The analysis results of the final obtained genes and chemicals demonstrated that they highly shared gene ontology (GO) terms and KEGG pathways with direct and indirect associations with esophageal cancer. In addition, we also discussed the likelihood of selected candidate genes and chemicals being novel genes and chemicals related to esophageal cancer.

ACTN4 and the pathways associated with cell motility and adhesion contribute to the process of lung cancer metastasis to the brain.

BACKGROUND: The aim of this study was to identify critical gene pathways that are associated with lung cancer metastasis to the brain. METHODS: The RNA-Seq approach was used to establish the expression profiles of a primary lung cancer, adjacent benign tissue, and metastatic brain tumor from a single patient. The expression profiles of these three types of tissues were compared to define differentially expressed genes, followed by serial-cluster analysis, gene ontology analysis, pathway analysis, and knowledge-driven network analysis. Reverse transcription-polymerase chain reaction (RT-PCR) was used to validate the expression of essential candidate genes in tissues from ten additional patients. RESULTS: Differential gene expression among these three types of tissues was classified into multiple clusters according to the patterns of their alterations. Further bioinformatic analysis of these expression profile data showed that the network of the signal transduction pathways related to actin cytoskeleton reorganization, cell migration, and adhesion was associated with lung cancer metastasis to the brain. The expression of ACTN4 (actinin, alpha 4), a cytoskeleton protein gene essential for cytoskeleton organization and cell motility, was significantly elevated in the metastatic brain tumor but not in the primary lung cancer tissue. CONCLUSIONS: The signaling pathways involved in the regulation of cytoskeleton reorganization, cell motility, and focal adhesion play a role in the process of lung cancer metastasis to the brain. The contribution of ACTN4 to the process of lung cancer metastasis to the brain could be mainly through regulation of actin cytoskeleton reorganization, cell motility, and focal adhesion.

Identification of novel thyroid cancer-related genes and chemicals using shortest path algorithm.

Thyroid cancer is a typical endocrine malignancy. In the past three decades, the continued growth of its incidence has made it urgent to design effective treatments to treat this disease. To this end, it is necessary to uncover the mechanism underlying this disease. Identification of thyroid cancer-related genes and chemicals is helpful to understand the mechanism of thyroid cancer. In this study, we generalized some previous methods to discover both disease genes and chemicals. The method was based on shortest path algorithm and applied to discover novel thyroid cancer-related genes and chemicals. The analysis of the final obtained genes and chemicals suggests that some of them are crucial to the formation and development of thyroid cancer. It is indicated that the proposed method is effective for the discovery of novel disease genes and chemicals.

A graphic method for identification of novel glioma related genes.

Glioma, as the most common and lethal intracranial tumor, is a serious disease that causes many deaths every year. Good comprehension of the mechanism underlying this disease is very helpful to design effective treatments. However, up to now, the knowledge of this disease is still limited. It is an important step to understand the mechanism underlying this disease by uncovering its related genes. In this study, a graphic method was proposed to identify novel glioma related genes based on known glioma related genes. A weighted graph was constructed according to the protein-protein interaction information retrieved from STRING and the well-known shortest path algorithm was employed to discover novel genes. The following analysis suggests that some of them are related to the biological process of glioma, proving that our method was effective in identifying novel glioma related genes. We hope that the proposed method would be applied to study other diseases and provide useful information to medical workers, thereby designing effective treatments of different diseases.