Single-Cell Transcriptome Analysis in Tumor Tissues

The tumor microenvironment is comprised of cancer cells and their surroundings, including various normal cells and non-cellular components, and each tumor tissue has a distinctive microenvironment. Cancer progression is affected by different microenvironmental states, such as the heterogeneity of infiltrating immune cells. Therefore, it is necessary to understand the complex cell-to-cell interactions associated with tumor developmental stages in different tissues. Recent revolution of single-cell RNA sequencing technology can uncover the tumor microenvironment diversity. We have developed a novel strategy of single-cell transcriptome analysis: next generation 1-cell sequencing (Nx1-seq) technology, and it allows for profiling of thousands of single cells from tumor tissue. Our microwell with cell bar-code beads device can detect genes with high sensitivity, and it is easily transported anywhere without any other dedicated devices. Further, the developmental cost is relatively cheaper than other single-cell RNA sequencing methods. In this study, we introduce representative application of the single-cell RNA sequencing technique in gynecological cancers, and we show the result of Nx1-seq application in human endometrioid adenocarcinoma tissue

Neuropeptide signaling through neurokinin-1 and neurokinin-2 receptors augments antigen presentation by human dendritic cells

Background: Neurotransmitters, including substance P (SP) and neurokinin A (NKA), are widely distributed in both the central and peripheral nervous system and their receptors, neurokinin-1 receptor (NK1R) and neurokinin-2 receptor (NK2R), are expressed on immune cells. However, the role of the NKA-NK2R axis in immune responses relative to the SP-NK1R signaling cascade has not been elucidated. Objective: We sought to examine the effect of neuropeptide signaling through NK1Rand NK2R on antigen presentation by dendritic cells (DCs) and the subsequent activation of effector Th cells. Methods: Expression levels of NK1R, NK2R, HLA-class II and costimulatory molecules of human MoDCs and cytokine production by birch pollen antigen-specific CD4+ T cells cocultured with MoDCs in the presence of NK1R and NK2R antagonists were evaluated by quantitative RT-PCR, flow cytometry or ELISA. NK1R and NK2R expression in the lung of patients with asthma and hypersensitivity pneumonitis was evaluated by immunohistochemistry. Results: Human MoDCs significantly upregulated NK2R and NK1R expression in response to poly I:C stimulation in a STAT1-dependent manner. Both NK2R and NK1R were expressed on alveolar macrophages and lung DCs from patients with asthma and pneumonitis hypersensitivity. Surface expression levels of HLA-class II and costimulatory molecules on DCs were modulated by NK1R or NK2R antagonists. Activation of birch pollen-derived antigen-specific CD4+ T cells and their production of cytokines including IL-4 and IFN-γ as well as IL-12 production by MoDCs, were suppressed by blocking NK1R or NK2R after in vitro antigen stimulation. Conclusions: NK1R- and NK2R-mediated neuropeptide signaling promotes both innate and acquired immune responses through activation of human DCs

Inducible astrocytic glucose transporter-3 contributes to the enhanced storage of intracellular glycogen during reperfusion after ischemia

Glucose is a necessary source of energy to sustain cell activities and homeostasis in the brain, and enhanced glucose transporter (GLUT) activities are protective of cells during energy depletion including brain ischemia. Here we investigated whether and if so how the astrocytic expression of GLUTs crucial for the uptake of glucose changes in ischemic conditions. Under physiological conditions, cultured astrocytes primarily expressed GLUT1, and GLUT3 was only detected at extremely low levels. However, exposure to ischemic stress increased the expression of not only GLUT1 but also GLUT3. During ischemia, cultured astrocytes significantly increased production of the transcription factor nuclear factor-κB (NF-κB), leading to an increase in GLUT3 expression. Moreover, astrocytic GLUT3 was responsible for the enhanced storage of intracellular glucose during reperfusion, resulting in increased resistance to lethal ischemic stress. These results suggested that astrocytes promptly increase GLUT3 production in situations such as ischemia, and much glucose is quickly taken up, possibly contributing to the protection of astrocytes from ischemic damage

Functional significance of the negative-feedback regulation of ATP release via pannexin-1 hemichannels under ischemic stress in astrocytes

The opening of pannexin-1 (Px1) hemichannels is regulated by the activity of P2X7 receptors (P2X7Rs). At present, however, little is known about how extracellular ATP-sensitive P2X7Rs regulates the opening and closure of Px1 hemichannels. Several lines of evidence suggest that P2X7Rs are activated under pathological conditions such as ischemia, resulting in the opening of Px1 hemichannels responsible for the massive influx of Ca^[2+] from the extracellular space and the release of ATP from the cytoplasm, leading to cell death. Here we show in cultured astrocytes that the suppression of the activity of P2X7Rs during simulated ischemia (oxygen/glucose deprivation, OGD) resulted in the opening of Px1 hemichannels, leading to the enhanced release of ATP. In addition, the suppression of the activity of P2X7Rs during OGD resulted in a significant increase in astrocytic damage. Both the P2X7Rs suppression-induced enhancement of the release of ATP and cell damage were reversed by co-treatment with blockers of Px1 hemichannels, suggesting that suppression of the activity of PX7Rs resulted in the opening of Px1 hemichannels. All these findings suggested the existence of a negative-feedback loop regulating the release of ATP via Px1 hemichannels; ATP-induced suppression of ATP release. The present study indicates that ATP, released through Px1 hemichannels, activates P2X7Rs, resulting in the closure of Px1 hemichannels during ischemia. This negative-feedback mechanism, suppressing the loss of cellular ATP and Ca^[2+] influx, might contribute to the survival of astrocytes under ischemic stress

Possible involvement of nitric oxide in the modulation of photolytic flash-induced intercellular calcium waves in cultured astrocytes

Waves of elevated intracellular free calcium that propagate between neighboring astrocytes are important for the intercellular communication between astrocytes as well as between neurons and astrocytes. In this study, intercellular calcium waves were evoked by focal photolysis of a caged calcium inophore in cultured astrocytes. The focal photolysis of the caged compound resulted in an increase of intracellular calcium in the flashed cells, and this increase then propagated to neighboring astrocytes. The evoked calcium increase was inhibited by incubating cells with an inhibitor of nitric oxide synthase as well as with a scavenger of nitric oxide (NO). In addition, treatment of cultures with an NO donor resulted in the marked enhancement of the photolytic flash-induced calcium rise in astrocytes. This enhancement was reversed by treatment with inhibitors of soluble guanylyl cyclase as well as of protein kinase G

Photolytic flash-induced intercellular calcium waves using caged calcium ionophore in cultured astrocytes from newborn rats

Waves of elevated intracellular free calcium that propagate between neighboring astrocytes are important for the intercellular communication between astrocytes as well as between neurons and astrocytes. However, the mechanisms responsible for the initiation and propagation of astrocytic calcium waves remain unclear. In this study, intercellular calcium waves were evoked by focal photolysis of a caged calcium ionophore (DMNPE-caged Br A23187) in cultured astrocytes from newborn rats. The focal photolysis of the caged compound resulted in the increase in intracellular calcium in a single astrocyte, and this increase then propagated to neighboring astrocytes. We also analyzed the spatiotemporal characteristics of the intercellular calcium waves, and estimated the propagation pathways for them. The method using a caged calcium ionophore described in this study provides a new in vitro model for the analysis of intercellular calcium waves

Negative-feedback regulation of ATP release : ATP release from cardiomyocytes is strictly regulated during ischemia

Extracellular ATP acts as a potent agonist on cardiomyocytes, inducing a broad range of physiological responses via P2 purinoceptors. Its concentration in the interstitial space within the heart is elevated during ischemia or hypoxia due to its release from a number of cell types, including cardiomyocytes. However, the exact mechanism responsible for the release of ATP from cardiomyocytes during ischemia is not known. In this study, we investigated whether and how the release of ATP was strictly regulated during ischemia in cultured neonatal rat cardiomyocytes. lschemia was mimicked by oxygen-glucose deprivation (OGD). Exposure of cardiomyocytes to OGD resulted in an increase in the concentration of extracellular ATP shortly after the onset of OGD (15 min), and the increase was reversed by treatment with blockers of maxi-anion channels. Unexpectedly, at 1 and 2 hours after the onset of OGD, the blocking of maxi-anion channels increased the concentration of extracellular ATP, and the increase was significantly suppressed by co-treatment with blockers of hemichannels, suggesting that ATP release via maxi-anion channels was involved in the suppression of ATP release via hemichannels during persistent OGD. Here we show the possibility that the release of ATP from cardiomyocytes was strictly regulated during ischemia by negative-feedback mechanisms; that is, maxi-anion channel-derived ATP-induced suppression of ATP release via hemichannels in cardiomyocytes