Molecular subtypes of lung adenocarcinoma present distinct immune tumor microenvironments.

Overcoming resistance to immune checkpoint inhibitors is an important issue in patients with non-small-cell lung cancer (NSCLC). Transcriptome analysis shows that adenocarcinoma can be divided into three molecular subtypes: terminal respiratory unit (TRU), proximal proliferative (PP), and proximal inflammatory (PI), and squamous cell carcinoma (LUSQ) into four. However, the immunological characteristics of these subtypes are not fully understood. In this study, we investigated the immune landscape of NSCLC tissues in molecular subtypes using a multi-omics dataset, including tumor-infiltrating leukocytes (TILs) analyzed using flow cytometry, RNA sequences, whole exome sequences, metabolomic analysis, and clinicopathologic findings. In the PI subtype, the number of TILs increased and the immune response in the tumor microenvironment (TME) was activated, as indicated by high levels of tertiary lymphoid structures, and high cytotoxic marker levels. Patient prognosis was worse in the PP subtype than in other adenocarcinoma subtypes. Glucose transporter 1 (GLUT1) expression levels were upregulated and lactate accumulated in the TME of the PP subtype. This could lead to the formation of an immunosuppressive TME, including the inactivation of antigen-presenting cells. The TRU subtype had low biological malignancy and "cold" tumor-immune phenotypes. Squamous cell carcinoma (LUSQ) did not show distinct immunological characteristics in its respective subtypes. Elucidation of the immune characteristics of molecular subtypes could lead to the development of personalized immune therapy for lung cancer. Immune checkpoint inhibitors could be an effective treatment for the PI subtype. Glycolysis is a potential target for converting an immunosuppressive TME into an antitumorigenic TME in the PP subtype.

Tumor-infiltrating Leukocyte Profiling Defines Three Immune Subtypes of NSCLC with Distinct Signaling Pathways and Genetic Alterations.

Resistance to immune checkpoint blockade remains challenging in patients with non-small cell lung cancer (NSCLC). Tumor-infiltrating leukocyte (TIL) quantity, composition, and activation status profoundly influence responsiveness to cancer immunotherapy. This study examined the immune landscape in the NSCLC tumor microenvironment by analyzing TIL profiles of 281 fresh resected NSCLC tissues. Unsupervised clustering based on numbers and percentages of 30 TIL types classified adenocarcinoma (LUAD) and squamous cell carcinoma (LUSQ) into the cold, myeloid cell-dominant, and CD8+ T cell-dominant subtypes. These were significantly correlated with patient prognosis; the myeloid cell subtype had worse outcomes than the others. Integrated genomic and transcriptomic analyses, including RNA sequencing, whole-exome sequencing, T-cell receptor repertoire, and metabolomics of tumor tissue, revealed that immune reaction-related signaling pathways were inactivated, while the glycolysis and K-ras signaling pathways activated in LUAD and LUSQ myeloid cell subtypes. Cases with ALK and ROS1 fusion genes were enriched in the LUAD myeloid subtype, and the frequency of TERT copy-number variations was higher in LUSQ myeloid subtype than in the others. These classifications of NSCLC based on TIL status may be useful for developing personalized immune therapies for NSCLC. Significance: The precise TIL profiling classified NSCLC into novel three immune subtypes that correlates with patient outcome, identifying subtype-specific molecular pathways and genomic alterations that should play important roles in constructing subtype-specific immune tumor microenvironments. These classifications of NSCLC based on TIL status are useful for developing personalized immune therapies for NSCLC.

Identification of somatic mutations in postmortem human brains by whole genome sequencing and their implications for psychiatric disorders.

AIM: Somatic mutations in the human brain are hypothesized to contribute to the functional diversity of brain cells as well as the pathophysiology of neuropsychiatric diseases. However, there are still few reports on somatic mutations in non-neoplastic human brain tissues. This study attempted to unveil the landscape of somatic mutations in the human brain. METHODS: We explored the landscape of somatic mutations in human brain tissues derived from three individuals with no neuropsychiatric diseases by whole-genome deep sequencing at a depth of around 100. The candidate mutations underwent multi-layered filtering, and were validated by ultra-deep target amplicon sequencing at a depth of around 200 000. RESULTS: Thirty-one somatic mutations were identified in the human brain, demonstrating the utility of whole-genome sequencing of bulk brain tissue. The mutations were enriched in neuron-expressed genes, and two-thirds of the identified somatic single nucleotide variants in the brain tissues were cytosine-to-thymine transitions, half of which were in CpG dinucleotides. CONCLUSION: Our developed filtering and validation approaches will be useful to identify somatic mutations in the human brain. The vulnerability of neuron-expressed genes to mutational events suggests their potential relevance to neuropsychiatric diseases.

Combining onartuzumab with erlotinib inhibits growth of non-small cell lung cancer with activating EGFR mutations and HGF overexpression.

Erlotinib, a tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR-TKI), benefits survival of patients with non-small cell lung cancer (NSCLC) who harbor activating EGFR mutations. However, elevated expression of hepatocyte growth factor (HGF), a ligand of the MET receptor tyrosine kinase, causes erlotinib resistance. Because onartuzumab, a monovalent antibody to MET, blocks HGF-induced MET activation, the addition of onartuzumab to erlotinib may improve therapeutic efficacy. We engineered the human NSCLC cell line PC-9 (MET-positive cells harboring an exon 19 deletion of EGFR) to overexpress hHGF and evaluated the effects of an onartuzumab and erlotinib combination in vitro and in vivo in xenograft models. A stable clone of PC-9/hHGF was less sensitive to erlotinib than the parental PC-9, and the addition of onartuzumab to erlotinib suppressed the proliferation of these cells in vitro. In PC-9/hHGF xenograft tumors, onartuzumab or erlotinib alone minimally inhibited tumor growth; however, combining onartuzumab and erlotinib markedly suppressed tumor growth. The total MET protein level was decreased in PC-9/hHGF cells, because MET is constitutively phosphorylated by autocrine HGF, leading to its ubiquitination and degradation. Onartuzumab reduced phospho-MET levels, inhibited MET ubiquitination, and consequently restored MET protein levels. Moreover, in NSCLC clinical specimens harboring activating EGFR mutations, more than 30% of patients expressed high levels of HGF. Our findings raised the possibility that patients with NSCLC with EGFR mutations who express high levels of HGF may benefit from onartuzumab and erlotinib combination therapy, and that HGF can be a novel biomarker for selecting such patients.

The enzyme Cyp26b1 mediates inhibition of mast cell activation by fibroblasts to maintain skin-barrier homeostasis.

Mast cells (MCs) mature locally, thus possessing tissue-dependent phenotypes for their critical roles in both protective immunity against pathogens and the development of allergy or inflammation. We previously reported that MCs highly express P2X7, a receptor for extracellular ATP, in the colon but not in the skin. The ATP-P2X7 pathway induces MC activation and consequently exacerbates the inflammation. Here, we identified the mechanisms by which P2X7 expression on MCs is reduced by fibroblasts in the skin, but not in the other tissues. The retinoic-acid-degrading enzyme Cyp26b1 is highly expressed in skin fibroblasts, and its inhibition resulted in the upregulation of P2X7 on MCs. We also noted the increased expression of P2X7 on skin MCs and consequent P2X7- and MC-dependent dermatitis (so-called retinoid dermatitis) in the presence of excessive amounts of retinoic acid. These results demonstrate a unique skin-barrier homeostatic network operating through Cyp26b1-mediated inhibition of ATP-dependent MC activation by fibroblasts.

Stem cell therapy: a new approach to the treatment of refractory depression.

To better understand the relationship of repeated exposure to adversity during early development as a risk factor for refractory depression, we exposed pregnant female rats to ethanol and the resulting pups to corticosterone during adolescence. A stressful forced swim test was then used to induce depression-like behavior. The adolescent rat brains were examined for the possible therapeutic benefit of a combination of sertraline, an antidepressant, and neural stem cells (NSCs) complexed with atelocollagen in relation to the level of GABAergic interneuron and synaptic protein density in different brain regions. The combined exposures of prenatal and adolescent stress resulted in a reduction in parvalbumin (PV)-positive phenotype of GABAergic interneurons and reduced postsynaptic density protein 95 (PSD-95) levels in the anterior cingulate cortex, amygdala, and hippocampus. Treatments with sertraline and NSCs reversed the reductions in PV-positive cells and PSD-95 levels. Furthermore, the combined treatment of sertraline and NSCs resulted in reduced depressive-like behaviors. These experiments underscore a potentially important role for synaptic remodeling and GABAergic interneuron genesis in the treatment of refractory depression and highlight the therapeutic potential of stem cell and pharmacological combination treatments for refractory depression.

Effects of atelocollagen on neural stem cell function and its migrating capacity into brain in psychiatric disease model.

Stem cell therapy is well proposed as a potential method for the improvement of neurodegenerative damage in the brain. Among several different procedures to reach the cells into the injured lesion, the intravenous (IV) injection has benefit as a minimally invasive approach. However, for the brain disease, prompt development of the effective treatment way of cellular biodistribution of stem cells into the brain after IV injection is needed. Atelocollagen has been used as an adjunctive material in a gene, drug and cell delivery system because of its extremely low antigenicity and bioabsorbability to protect these transplants from intrabody environment. However, there is little work about the direct effect of atelocollagen on stem cells, we examined the functional change of survival, proliferation, migration and differentiation of cultured neural stem cells (NSCs) induced by atelocollagen in vitro. By 72-h treatment 0.01-0.05% atelocollagen showed no significant effects on survival, proliferation and migration of NSCs, while 0.03-0.05% atelocollagen induced significant reduction of neuronal differentiation and increase of astrocytic differentiation. Furthermore, IV treated NSCs complexed with atelocollagen (0.02%) could effectively migrate into the brain rather than NSC treated alone using chronic alcohol binge model rat. These experiments suggested that high dose of atelocollagen exerts direct influence on NSC function but under 0.03% of atelocollagen induces beneficial effect on regenerative approach of IV administration of NSCs for CNS disease.

LGR5-positive colon cancer stem cells interconvert with drug-resistant LGR5-negative cells and are capable of tumor reconstitution.

The cancer stem cell (CSC) concept has been proposed as an attractive theory to explain cancer development, and CSCs themselves have been considered as targets for the development of diagnostics and therapeutics. However, many unanswered questions concerning the existence of slow cycling/quiescent, drug-resistant CSCs remain. Here we report the establishment of colon cancer CSC lines, interconversion of the CSCs between a proliferating and a drug-resistant state, and reconstitution of tumor hierarchy from the CSCs. Stable cell lines having CSC properties were established from human colon cancer after serial passages in NOD/Shi-scid, IL-2Rγ(null) (NOG) mice and subsequent adherent cell culture of these tumors. By generating specific antibodies against LGR5, we demonstrated that these cells expressed LGR5 and underwent self-renewal using symmetrical divisions. Upon exposure to irinotecan, the LGR5(+) cells transitioned into an LGR5(-) drug-resistant state. The LGR5(-) cells converted to an LGR5(+) state in the absence of the drug. DNA microarray analysis and immunohistochemistry demonstrated that HLA-DMA was specifically expressed in drug-resistant LGR5(-) cells, and epiregulin was expressed in both LGR5(+) and drug-resistant LGR5(-) cells. Both cells sustained tumor initiating activity in NOG mice, giving rise to a tumor tissue hierarchy. In addition, anti-epiregulin antibody was found to be efficacious in a metastatic model. Both LGR5(+) and LGR5(-) cells were detected in the tumor tissues of colon cancer patients. The results provide new biological insights into drug resistance of CSCs and new therapeutic options for cancer treatment.

Heat stress activates ER stress signals which suppress the heat shock response, an effect occurring preferentially in the cortex in rats.

Although heat stress induces a variety of illnesses, there have been few studies designed to uncover the molecular mechanisms underlining the illnesses. We here demonstrate that heat activates ER stress, which inhibits heat shock responses (HSR) via translational block. In heat-stressed rats, ER stress responses, as represented by eIF2α phosphorylation and XBP1 splicing, occurred mainly in the cortex, where the HSR was substantially inhibited. Heat exposure also activated ER stress signals in primary cortical neurons. Since HSF1 knockdown enhanced heat-induced ER stress and subsequent cell death, HSR inhibition in turn augments ER stress, implying a vicious spiral of both stresses. Taken together, heat-induced ER stress impairs the HSR and enhances cell damage, thereby manifesting its unique effect on heat stress.

[Promising therapy of neural stem cell transplantation for FASD model--neural network reconstruction and behavior recovery].

OBJECTIVES: It has been elucidated that psychiatric disorders are associated with impairment of the brain neural network. Reduction in brain size and hypoplasia of the basal ganglia and corpus callosum have been reported in Fetal Alcohol Spectrum Disorder (FASD). It is believed that the formation of the neural network is influenced by alcohol exposure during the fetal period. Additionally, it is well known that the functional expression of CNS consequences of prenatal alcohol exposure includes cognitive and attentional processes, as well as social behavioral problems. It has also been reported that abnormal 5-HT neuron development can be reversed by treatment with a 5-HT1A agonist in a prenatal alcohol exposure model. However, these treatments are prophylactic. Without early intervention, the consequences of FASD are permanent. Recently, emerging evidence suggest that many clinical symptoms observed in psychiatric disease are likely related to neural network disruptions including neurogenesis dysfunction. Neural stem cell (NSC) transplantation has been investigated in areas such as brain injury, stroke and neurodegenerative diseases and may be a way to reverse neurogenesis dysfunction. In the present work, we evaluated the usefulness of intravenous transplantation of NSCs in the FASD model rat focusing on the possibility of regenerative therapy, particularly regarding behavioral abnormalities, for FASD rats. RESULTS: Abnormal behaviors FASD model rats suggest that reduced social activity , and cognitive dysfunction are major symptoms in FASD patients. Intravenous NSC transplantation appeared to partially correct these behavioral abnormalities in FASD model rats. In the Amygdala areas intravenous NSC transplantation appears to have partially regaenerates expression of PSD95 in FASD model rats. CONCLUSIONS: The results suggest that intravenous NSC transplantation may be an advanced approach to recover neural network damage and CNS dysfunction in FASD and possibly other psychiatric disorders.

The role of neural stem cells for in vitro models of schizophrenia: neuroprotection via Akt/ERK signal regulation.

Recent neuroimaging studies have revealed progressive morphological brain changes during the course of schizophrenia, and the neurotrophic and neurogenetic effects of atypical antipsychotics are believed to prevent or retard these brain volume reductions. In addition to drug-induced neural stem cell (NSC) activation, transplantation of exogenous NSCs has been proposed as a possible approach to repair the damaged brain in psychiatric disease. NSC transplantation embraces not only neuron replacement but also enhanced neuroprotection of existing neurons with the goal of restoring the impaired brain. However, little is known about the cell-cell interactions of exogenous NSCs with existing neurons, or about their neuroprotective actions especially in psychiatric diseases. In the present study, we used cortical neuron cultures to examine the neurotrophism and neuroprotection of exogenous NSCs against the neuronal damage induced by exposure to the NMDA receptor antagonist, MK-801. We also investigated their role in serum/nutrient deprivation stress. The exogenous NSCs exerted neuroprotective effects against both types of apoptotic injuries considered as in vitro schizophrenic disease models. Exogenous NSCs also altered cellular survival signaling in injured neurons by indirect cell-cell contact in an injury-dependent manner. In MK-801 exposure, NSCs increased phosphorylated Akt (p-Akt) and ERK (p-ERK), both of which were reduced by this stress. While, in serum/nutrient deprivation, NSCs increased p-Akt, but decreased p-ERK which was increased by this damage. Our results demonstrate that exogenous NSCs have anti-apoptotic activities and can rescue cortical neurons by directing cellular survival signaling of neurons into the proper direction, without cell contact.

The common aspects of pathophysiolgy of alcoholism and depression.

Recent biological studies suggest the existence of the common pathophysiological aspects in alcoholism and depression. Postmortem studies have revealed the impairment of cAMP signaling in the patients with alcoholism. The similar alteration of cAMP signaling was also reported in postmortem brains of depressed patients. In this study, we supported the notion that neurogenesis would be essential in pathophysiology of both alcoholism and depression. Alcohol affected the function of neural stem cells (NSCs) and decreased neurogenesis at doses which did not affects cell survival, and treatment of antidepressant or moodstabilizer rescued the alcohol-induced suppression of neurogenesis. As the key mechanism of NSC differentiation change by ethanol and psychotropics, we focused on the transcriptional repressor, NRSF/REST activity change. Our in vitro studies demonstrated the NRSF/REST activation by ethanol and suppressive effect of antidepressants and lithium against its activation by ethanol. We further described the ERK reduction and ER stress in the cellular mechanism of NRSF/REST activation. All these findings suggested that cAMP-CREB cascade reduction and NRSF/REST activation may be common underlying mechanisms in the pathophysiology of alcoholism and depression.

[Neural network abnormalities caused by alcohol: approach for repair using neural stem cells].

Recent clinical neuroimaging studies have revealed the possible relation between morphological brain changes, and memory, cognitive impairment in the course of alcoholism and depression. In the previous studies, we have been analyzing the mechanism of neural network disruption by ethanol using postmortem human brain and cultured cells, and identified the sensitive effect of ethanol on the neural stem cell (NSC) differentiation rather than the influence on neuronal cell survival. Furthermore, to develop a novel method for reconstruction of the neural network damaged by ethanol, we tried to analyze the usefulness of intravenous NSC transplantation in fetal alcohol syndrome spectrum disorder (FASD) model rats. In the in vitro studies, we have found the suppressive effect of ethanol on NSC differentiation to neurons, through alteration of transcription factor, CREB and NRSF/REST activities, by the cellular signaling cascade changes including trophic factors and endoplasmic reticulum (ER) function. In the in vivo studies, we have shown the effective migration of labeled NSCs into the brain of FASD model rats, and revealed the therapeutic potential of this transplantation for the treatment of anxiety/cognitive dysfunction and behavioral abnormalities in alcohol-induced brain neural network damage. We are going to the next step for analysis of transplanted NSC dynamics in the brain, which must play a pivotal role in the effective induction of behavioral recoveries.

[The analysis of impairment and repair of neural network by ethanol: in vitro and in vivo studies using neurons and neural stem cells].

Recent clinical neuroimaging studies have suggested the morphological brain changes occur and progress in the course of alcoholism and depression. The abnormality of neurogenesis has emerged as a potential epidemiological mechanism of these diseases. Previously, we have indicated the low dose of ethanol that could not influence on the survival of neurons and neural stem cell (NSC) suppress differentiation to neurons but glias through activation of neuron-restrictive silencing factor/repressor element-1 silencing transcription factor (NRSF/REST). We revealed the endoplasmic reticulum function and trophic factor signaling change implicated in this mechanism of ethanol action on NSC differentiation change. The analysis of potentials of psychotropic drugs on the ethanol-induced NSC function change may reveal the possible biological way of neural network impairment and its repair. Furthermore, the approach of using stem cells such as intravenous NSC transplantation can be a useful method to clarify the neural network reconstruction damaged by ethanol. The importance of interactive analysis of in vitro to in vivo should be documented for the pathophysiological understanding and new therapy development against alcohol-induced brain damage.

Ca2+ influx-mediated histamine synthesis and IL-6 release in mast cells activated by monomeric IgE.

We previously demonstrated that histamine synthesis is drastically induced upon sensitization with an anti-DNP IgE clone, SPE-7, in IL-3-dependent mouse bone marrow derived mast cells (BMMC). We found that Ca2+ mobilization induced by SPE-7 exhibited a similar profile to the capacitative Ca2+ entry evoked by thapsigargin. Potentials for activation of mast cells were found to vary between different IgE clones, and a monovalent hapten, DNP-lysine, suppressed the activation induced by SPE-7. Ca2+ mobilization induced by SPE-7 was suppressed potently by the specific store-operated Ca2+ channel inhibitor, SK&F 96365, but not at all by Ca2+ channel inhibitors with more broad spectrum, La3+ and Gd3+, whereas the Ca2+ mobilization induced by Ag stimulation was suppressed by these inhibitors. Ca2+ mobilization was also induced by SPE-7 in in vitro differentiated mast cells, although the increases in histamine synthesis and IL-6 release were smaller than those in BMMC. These results suggest that Ca2+ influx operated by a distinct mechanism from that in Ag stimulation is essential for increased histamine synthesis and IL-6 release in mast cells.

Augmentation of ethanol-induced cell damage and activation of nuclear factor-kappa B by annexin IV in cultured cells.

BACKGROUND: We previously reported that the amount of annexin IV expression was increased in culture cells by exposure to ethanol. Here, to investigate the physiologic role of annexin IV, we analyzed ethanol-induced cytotoxicity and nuclear factor (NF)-kappa B activity by using annexin IV-overexpressed cells. METHODS: Annexin IV overexpression was performed by transfection of expression vector, in which annexin IV complementary DNA was ligated, to culture cells (rat glioma C6 cells and human neuroblastoma SH-SY5Y cells). Ethanol-induced cytotoxicity was assayed by measuring the mitochondrial enzyme (dehydrogenase) activity or trypan blue exclusion. NF-kappa B activity was measured by electrophoretic mobility shift assay with a kappa B-oligonucleotide probe. RESULTS: Ethanol-induced cytotoxicity was increased by overexpression of annexin IV in both C6 cells and SH-SY5Y cells. Annexin IV overexpression augmented ethanol-induced NF-kappa B activation. CONCLUSIONS: Ethanol-induced increase in annexin IV expression might amplify ethanol-induced cytotoxicity via NF-kappa B activation.

Ethanol-induced augmentation of annexin IV in cultured cells and the enhancement of cytotoxicity by overexpression of annexin IV by ethanol.

The annexins are a family of highly homologous Ca(2+) and phospholipid binding proteins. The expressive amounts of several annexins have been shown to alter in certain pathological states such as brain ischemia and Alzheimer's disease. It has been demonstrated that ethanol induces cytotoxicity, which results in brain damage. In this study, we examined the relationship between ethanol-induced cytotoxicity and the intrinsic amount of annexins using cell lines (rat glioma C6 cells and human adenocarcinoma A549 cells). A decrease in the mitochondrial enzyme (dehydrogenase) activity, which is widely used to measure cytotoxicity, was observed with a high concentration of ethanol (200 mM or more) after a 24-h exposure in both C6 and A549 cells. Western blot analysis revealed that the amount of annexin IV was augmented in both cells by ethanol, whereas levels of annexins I and V were unchanged. The amount of annexin IV was augmented with increasing concentration of ethanol. The overexpression of annexin IV in C6 cells by transfection with annexin IV-DNA enhanced ethanol-induced cell lesion and was accompanied by NFkappaB activation. Thus, it might be indicated that the amount of annexin IV is selectively augmented and this augmentation facilitates the development of cell lesion by ethanol.

Ethanol-induced augmentation of annexin IV expression in rat C6 glioma and human A549 adenocarcinoma cells.

BACKGROUND: We previously reported that the amount of annexin IV significantly increased in the postmortem brains of alcoholics compared with controls. To investigate further whether ethanol directly affects cellular expression of annexins in a cell, we used cell lines and compared the amounts of annexins between ethanol-exposed and control cells. METHODS: Two kinds of cells (rat glioma C6 cells and human adenocarcinoma A549 cells) were used in the present study. Western blot analysis was performed to quantify expressed amounts of annexins using anti-annexin I, IV, and V antibodies. The mitochondrial enzyme (dehydrogenase) and caspase 3 activities were measured to assess cell damage (apoptosis) by ethanol. RESULTS: Expressive augmentation of annexin IV was shown in both C6 and A549 cells after a 5-hr exposure to 200 mM of ethanol, whereas amounts of annexins I and V were not changed. Both the mitochondrial dehydrogenase and caspase 3 activities were altered under the same conditions in C6 cells, indicating the induction of cell damage (apoptosis), whereas both of these enzyme activities were unchanged in A549 cells under the same conditions. CONCLUSIONS: The amount of annexin IV seemed to increase before the induction of cell damage by ethanol. Annexin IV might be one of the specific markers for the effect of ethanol.