Assessing Cell-to-Cell DNA Methylation Variability on Individual Long Reads

Understanding cell-to-cell variability in cytosine methylation is essential for understanding cellular perturbation and its molecular machinery. However, conventional methylation studies have focused on the differences in the average levels between cell types while overlooking methylation heterogeneity within cell types. Little information has been uncovered using recent single-cell methods because of either technical limitations or the great labor required to process many single cells. Here, we report the highly efficient detection of cell-to-cell DNA methylation variability in liver tissue, based on comparing the methylation status of adjacent CpG sites on long sequencing reads. This method provides abundant methylation linkage information and enables genome-wide estimation of cell-to-cell variability. We observed repressed methylation variability in hypomethylated regions compared with the variability in hypomethylated regions across the genome, which we confirmed using public human sperm data. A gradual change in methylation status at the boundaries of hypomethylated regions was observed for the first time. This approach allows the concise, comprehensive assessment of cell-to-cell DNA methylation variability

Lack of interleukin-6 in the tumor microenvironment augments type-1 immunity and increases the efficacy of cancer immunotherapy

Conquering immunosuppression in tumor microenvironments is crucial for effective cancer immunotherapy. It is well known that interleukin (IL)-6, a pleiotropic cytokine, is produced in the tumor-bearing state. In the present study, we investigated the precise effects of IL-6 on antitumor immunity and the subsequent tumorigenesis in tumor-bearing hosts. CT26 cells, a murine colon cancer cell line, were intradermally injected into wild-type and IL-6-deficient mice. As a result, we found that tumor growth was decreased significantly in IL-6-deficient mice compared with wild-type mice and the reduction was abrogated by depletion of CD8+ T cells. We further evaluated the immune status of tumor microenvironments and confirmed that mature dendritic cells, helper T cells and cytotoxic T cells were highly accumulated in tumor sites under the IL-6-deficient condition. In addition, higher numbers of interferon (IFN)-γ-producing T cells were present in the tumor tissues of IL-6-deficient mice compared with wild-type mice. Surface expression levels of programmed death-ligand 1 (PD-L1) and MHC class I on CT26 cells were enhanced under the IL-6-deficient condition in vivo and by IFN-γ stimulation in vitro. Finally, we confirmed that in vivo injection of an anti-PD-L1 antibody or a Toll-like receptor 3 ligand, polyinosinic-polycytidylic acid, effectively inhibited tumorigenesis under the IL-6-deficient condition. Based on these findings, we speculate that a lack of IL-6 produced in tumor-bearing host augments induction of antitumor effector T cells and inhibits tumorigenesis in vivo, suggesting that IL-6 signaling may be a promising target for the development of effective cancer immunotherapies

Differentiation capacities of PS-clusters, adult pituitary stem/progenitor cell clusters located in the parenchymal-niche, of the rat anterior lobe

<div><p>Pituitary endocrine cells are supplied by <i>Sox2</i>-expressing stem/progenitor cells in the anterior lobe of the adult pituitary. In relation to their microenvironment (“niche”), SOX2-positive cells exist in two types of niches; the marginal cell layer-niche and the parenchymal-niche. Recently, we isolated dense stem/progenitor cell clusters from the parenchymal-niche as parenchymal stem/progenitor cell (PS)-clusters. We classified these PS-clusters into three subtypes based on differences in <i>S100β</i>-expression (S100β-positive, -negative, and -mixed type), and reported that S100β-positive PS-clusters exhibited the capacity for differentiation into endocrine cells under 3-dimensional cultivation system. In the present study, we further characterized S100β-positive PS-clusters using an <i>in vitro</i> 2-dimensional cultivation system. The results demonstrated that S100β-positive PS-clusters in the 2-dimensional cultivation system proliferated more actively than S100β-negative clusters. Moreover, in 2-dimensional cultivation conditions, S100β-positive PS-clusters showed differentiation capacity into non-endocrine cells (Myogenin-, αSMA-, NG2-, or SOX17-positive cells) but not into endocrine cells, whereas S100β-negative PS-clusters did not. Collectively, PS-clusters were heterogeneous, exhibiting different proliferation and differentiation properties based on the difference in <i>S100β</i>-expression. Specifically, a part of SOX2-positive cells in the parenchymal-niche had capacities for differentiation into non-endocrine cells, and S100β-positive PS-clusters may be in more progressive stages toward differentiation than S100β-negative clusters.</p></div

Analysis of the proliferative activity of S100β-positive and -negative PS-clusters upon 2D-cultivation using Matrigel-coated glass slides.

<p>(A, B): PS-clusters were isolated from the anterior lobe of adult S100β/GFP-TG rats after enzymatic treatment. Time-lapse images of GFP- (A) and null-GFP-clusters (B) during 2D-cultivation in growth and differentiation-medium (GD-medium) on Matrigel-coated glass slides for 5 days are shown. Phase-contrast (PC) (upper panels) and fluorescence images (lower panels) of each PS-cluster during cultivation. Images were obtained at 0, 1, 3, and 5 days after seeding. Bars: 50 μm. (C, D): Immunostaining for BrdU on GFP- and null-GFP-clusters after 2D-cultivation. Each GFP- (C) and null-GFP-cluster (D) was treated with BrdU for 24 h after days 3 and 6, followed by immunostaining for BrdU. BrdU visualized with Cy3 (<i>red</i>) and merged image with nuclear staining by DAPI (<i>blue</i>) are shown in upper and lower panels, respectively. Bars: 50 μm. (E): The proportion of BrdU-positive cells in the cells derived from each GFP- and null-GFP-cluster after treatment of BrdU for 24 h from 3 and 6 days-cultivation. White and black bars indicate GFP- and null-GFP-clusters, respectively. The data are presented as the mean ± SE (n = 3) in three independent experiments with triplicate wells. The statistical significance between the groups of GFP- and null-GFP-clusters was determined by Student’s <i>t</i>-test. *<i>P</i> < 0.01.</p

Immunocytochemistry for the pituitary cell lineage markers on GFP-clusters after 2D-cultivation.

<p>Immunostaining for PROP1 (A) and pituitary hormones (B) on GFP-clusters after 2D-cultivation in GD-medium for 7 days was performed. SOX2 visualized with Cy5 (<i>green</i>), and PROP1 (A) or pituitary hormones with Cy3 (<i>red</i>: B), and merged image with nuclear staining by DAPI (<i>blue</i>) are shown. Merged images with DAPI and PROP1 (A') or pituitary hormones (B') in the cells before cultivation are also shown. Arrowheads indicate SOX2-negative cells. Bars: 20 μm.</p