Comparison of cell type distribution between single-cell and single-nucleus RNA sequencing: enrichment of adherent cell types in single-nucleus RNA sequencing.

Single-cell ribonucleic acid (RNA) sequencing (scRNA-seq) is an effective technique for estimating the cellular composition and transcriptional profiles of individual cells from fresh tissue. Single-nucleus RNA sequencing (snRNA-seq) is necessary to perform this type of analysis in frozen or difficult-to-dissociate tissues, which cannot be subjected to scRNA-seq. This difference in the state of tissues leads to variation in cell-type distributions among each platform. To identify the characteristics of these methods and their differences, scRNA-seq and snRNA-seq were performed in parallel for colon and liver tissues. The two platforms revealed similar diversity but different proportions of cell types in matched tissues. The proportions of epithelial cells in the colon and hepatocytes in the liver were relatively high in snRNA-seq and that of immune cells was relatively high in scRNA-seq. This difference could be explained by variations in the expression scores of adhesion genes due to the disruption of the cytoplasmic contents during scRNA-seq. The enrichment of epithelial cells in the colon resulted in a discrepancy in the differentiation of epithelial cells. This enrichment was also well matched with the images of hematoxylin and eosin staining and the estimated distribution of cell types in bulk RNA sequencing. These results showed that snRNA-seq could be used to analyze tissues that cannot be subjected to scRNA-seq and provides more information in specific cell type analysis.

Spatiotemporal genomic architecture informs precision oncology in glioblastoma.

Precision medicine in cancer proposes that genomic characterization of tumors can inform personalized targeted therapies. However, this proposition is complicated by spatial and temporal heterogeneity. Here we study genomic and expression profiles across 127 multisector or longitudinal specimens from 52 individuals with glioblastoma (GBM). Using bulk and single-cell data, we find that samples from the same tumor mass share genomic and expression signatures, whereas geographically separated, multifocal tumors and/or long-term recurrent tumors are seeded from different clones. Chemical screening of patient-derived glioma cells (PDCs) shows that therapeutic response is associated with genetic similarity, and multifocal tumors that are enriched with PIK3CA mutations have a heterogeneous drug-response pattern. We show that targeting truncal events is more efficacious than targeting private events in reducing the tumor burden. In summary, this work demonstrates that evolutionary inference from integrated genomic analysis in multisector biopsies can inform targeted therapeutic interventions for patients with GBM.

A trachea-inspired bifurcated microfilter capturing viable circulating tumor cells via altered biophysical properties as measured by atomic force microscopy.

Circulating tumor cells (CTCs), though exceedingly rare in the blood, are nonetheless becoming increasingly important in cancer diagnostics. Despite this keen interest and the growing number of potential clinical applications, there has been limited success in developing a CTC isolation platform that simultaneously optimizes recovery rates, purity, and cell compatibility. Herein, a novel tracheal carina-inspired bifurcated (TRAB) microfilter system is reported, which uses an optimal filter gap size satisfying both 100% theoretical recovery rate and purity, as determined by biomechanical analysis and fluid-structure interaction (FSI) simulations. Biomechanical properties are also used to clearly discriminate between cancer cells and leukocytes, whereby cancer cells are selectively bound to melamine microbeads, which increase the size and stiffness of these cells. Nanoindentation experiments are conducted to measure the stiffness of leukocytes as compared to the microbead-conjugated cancer cells, with these parameters then being used in FSI analyses to optimize the filter gap size. The simulation results show that given a flow rate of 100 µL min(-1), an 8 µm filter gap optimizes the recovery rate and purity. MCF-7 breast cancer cells with solid microbeads are spiked into 3 mL of whole blood and, by using this flow rate along with the optimized microfilter dimensions, the cell mixture passes through the TRAB filter, which achieves a recovery rate of 93% and purity of 59%. Regarding cell compatibility, it is verified that the isolation procedure does not adversely affect cell viability, thus also confirming that the re-collected cancer cells can be cultured for up to 8 days. This work demonstrates a CTC isolation technology platform that optimizes high recovery rates and cell purity while also providing a framework for functional cell studies, potentially enabling even more sensitive and specific cancer diagnostics.

Highly efficient assay of circulating tumor cells by selective sedimentation with a density gradient medium and microfiltration from whole blood.

Isolation of circulating tumor cells (CTCs) by size exclusion can yield poor purity and low recovery rates, due to large variations in size of CTCs, which may overlap with leukocytes and render size-based filtration methods unreliable. This report presents a very sensitive, selective, fast, and novel method for isolation and detection of CTCs. Our assay platform consists of three steps: (i) capturing CTCs with anti-EpCAM conjugated microbeads, (ii) removal of unwanted hematologic cells (e.g., leukocytes, erythrocytes, etc.) by selective sedimentation of CTCs within a density gradient medium, and (iii) simple microfiltration to collect these cells. To demonstrate the efficacy of this assay, MCF-7 breast cancer cells (average diameter, 24 µm) and DMS-79 small cell lung cancer cells (average diameter, 10 µm) were used to model CTCs. We investigated the relative sedimentation rates for various cells and/or particles, such as CTCs conjugated with different types of microbeads, leukocytes, and erythrocytes, in order to maximize differences in the physical properties. We observed that greater than 99% of leukocytes in whole blood were effectively removed at an optimal centrifugal force, due to differences in their sedimentation rates, yielding a much purer sample compared to other filter-based methods. We also investigated not only the effect of filtration conditions on recovery rates and sample purity but also the sensitivity of our assay platform. Our results showed a near perfect recovery rate (~99%) for MCF-7 cells and very high recovery rate (~89%) for DMS-79 cells, with minimal amounts of leukocytes present.

KR33426, [2-(2,5-dichlorophenyl)-5-methyloxazol-4yl]carbonylguanidine, is a novel compound to be effective on mouse systemic lupus erythematosus.

B cell-activating factor (BAFF) is a key regulator of B lymphocyte development. Signals from BAFF are transmitted through binding to a specific BAFF receptor (BAFF-R). Here, we established screening method to find a specific inhibitor for the interference of BAFF-BAFF-R interactions. We screened oxazole-4-carbonylguanidine derivatives and selected KR33426, [2-(2,5-dichlorophenyl)-5-methyloxazol-4yl]carbonylguanidine, as a candidate to interfere BAFF-BAFF-R interactions. KR33426 inhibited BAFF-mediated anti-apoptotic effect on splenocytes as judged by hypodiploid cell formation. KR33426 also increased the degradation of procaspase-3 that was inhibited by BAFF protein. In addition, we examined whether KR33426 was effective on the treatment of systemic lupus erythematosus-like symptom in MRL(lpr/lpr) mouse. When 5 or 10mg/kg KR33426 was intraperitoneally administered to MRL(lpr/lpr) mice for 4 weeks, histopathological changes were ameliorated in the narrowed space between renal glomerulus and glomerulus capsule. KR33426 reduced B220(+) B cell population and B cell mitogen, lipopolysaccharide-stimulated lymphocyte proliferation in splenocytes. KR33426 attenuated an increase in CD43(-)IgM(+) immature pro-B and a decrease in CD21(+) IgM(+) T2-B and IgD(+) IgM(-)recirculating-B cells on B cell development. Data show that KR33426 inhibits BAFF-BAFF-R interactions and it is effective on the treatment of systemic lupus erythematosus-like symptom in MRL(lpr/lpr) mice. Thus, it suggests that KR33426 is a novel candidate to develop anti-autoimmune therapeutics by the interference of BAFF-BAFF-R interactions, specifically.

Actin-sequestering protein, thymosin beta-4, induces paclitaxel resistance through ROS/HIF-1alpha stabilization in HeLa human cervical tumor cells.

AIMS: We investigated whether actin-sequestering protein, thymosin beta-4 (TB4)-induced reactive oxygen species (ROS) affect the stabilization of hypoxia-inducible transcription factor (HIF)-1alpha and paclitaxel-resistance induction. MAIN METHODS: HeLa human cervical tumor cells were used. The percentage of cell survival was determined by MTT assay. ROS production, cell cycle and hypodiploid cell formation were assessed by flow cytometry analysis. HIF-1alpha stabilization and molecular changes were analyzed by western blotting or RT-PCR. NF-kappaB activation was assessed by EMSA and western blotting. KEY FINDINGS: TB4 protein (TB4P) significantly increased intracellular ROS level and HIF-1alpha. The increased level of HIF-1alpha by TB4P was reduced by the treatment with N-acetylcysteine (NAC), a well-known ROS scavenger. TB4P-induced ROS production was confirmed by the activation of nuclear factor kappa B. TB4P-induced Erk phosphorylation was attenuated by the treatment with NAC. In addition, tumor cell death was decreased by TB4 gene overexpression and TB4P treatment. NAC treatment attenuated tumor cell density increased by TB4P. Tumor cell death by paclitaxel was also increased by NAC treatment or the transfection with HIF-1alpha-siRNA. Paclitaxel-induced B16F10 mouse melanoma regression was physiologically inhibited in TB4-transgenic mice compared to wildtype mice. SIGNIFICANCE: These findings demonstrate that TB4-induced ROS and ROS-mediated HIF-1alpha stabilization could play a role in tumor cell resistance to anticancer agents like paclitaxel. It suggests that soluble TB4 could be a novel endogenous regulator to control intracellular ROS production in tumor cells.

Clitocybins, novel isoindolinone free radical scavengers, from mushroom Clitocybe aurantiaca inhibit apoptotic cell death and cellular senescence.

High concentration of intracellular reactive oxygen species (ROS) plays a role in damaging biological systems. We isolated clitocybin A from the culture broth of Clitocybe aurantiaca and then clitocybin B and C derivatives were synthesized from clitocybin A. IMR-90 lung fibroblast cells were pre-treated or post-treated with clitocybin A, B and C to the addition of 100 muM H(2)O(2). These compounds inhibited the level of intracellular reactive oxygen species (ROS) and H(2)O(2)-induced cell death as judged by hypodiploid cell formation. The inhibitory effect of clitocybins on H(2)O(2)-induced cell death was comparable to that with N-acetylcysteine (NAC), a well-known ROS scavenger. The inhibition of H(2)O(2)-induced cell death by clitocybins was mediated by the reduction of caspase 3 and 9 activation, cytochrome c release from mitochondria and the degradation of IkappaB-alpha and IkappaB-beta, which could be resulted in the prevention of cellular senescence. It suggests that clitocybins are novel compounds scavenging ROS and protect cells from apoptosis and cellular senescence.

Paclitaxel induces vascular endothelial growth factor expression through reactive oxygen species production.

The antineoplastic drug paclitaxel is known to block cells in the G2/M phase of the cell cycle through stabilization of microtubules. The development of paclitaxel resistance in tumors is one of the most significant obstacles to successful therapy. Vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1 (HIF-1) are important regulators of neovascularization. HIF-1 regulates VEGF expression at the transcriptional level. Here, we investigated whether paclitaxel treatment affects VEGF expression for the development of paclitaxel resistance. Paclitaxel treatment induced dose-dependent cell death and increased VEGF expression. Paclitaxel also induced nuclear factor-kappaB activation and stabilized HIF-1alpha, which stimulated luciferase activity of HIF-1alpha response element on VEGF gene. As paclitaxel treatment produced reactive oxygen species (ROS), VEGF expression was increased by H2O2 treatment and reduced by various ROS scavengers such as N-acetyl-L-cysteine, pyrrolidine dithiocarbamate and diphenylene iodonium. Paclitaxel-induced cell death was aggravated by incubation with those ROS scavengers. Collectively, this suggests that paclitaxel-induced VEGF expression could be mediated by paclitaxel-induced ROS production through nuclear factor-kappaB activation and HIF-1alpha stabilization, which could affect resistance induction to antitumor therapeutics during cancer treatment.

Hypoxia-inducible transcription factor (HIF)-1 alpha stabilization by actin-sequestering protein, thymosin beta-4 (TB4) in Hela cervical tumor cells.

Thymosin beta-4 (TB4) is an actin-sequestering protein to control cytoskeletal reorganization. Here, we investigated whether TB4 proteins (TB4P) affect tumor microenvironment by measuring hypoxia-inducible transcription factor (HIF)-1 alpha stabilization in cervical tumor cells, since TB4P reduced paclitaxel-induced cell death rate. TB4P increased HIF-1 alpha stabilization and transactivation, which is measured by the increase of hypoxia response element (HRE)-luciferase activity and target gene, vascular endothelial growth factor (VEGF) transcription. TB4P also elevated ERK phosphorylation. PD98059, ERK inhibitor reduced HIF-1 alpha increased by TB4P. Paclitaxel-induced cell death was inhibited by hypoxia conditioning that increased HIF-1 alpha stabilization and ERK phosphorylation. PD98059 reversed paclitaxel-induced cell death which was attenuated by hypoxia. Collectively, TB4P could lead tumor cell microenvironment to hypoxia condition, which might be resulted in antitumor drug-resistance induction. It suggests that soluble TB4P could be a novel target to control tumor cell death by regulating tumor cell microenvironment.