Comprehensive circulating microRNA profile as a supersensitive biomarker for early-stage lung cancer screening.

PURPOSE:  Less-invasive early diagnosis of lung cancer is essential for improving patient survival rates. The purpose of this study is to demonstrate that serum comprehensive miRNA profile is high sensitive biomarker to early-stage lung cancer in direct comparison to the conventional blood biomarker using next-generation sequencing (NGS) technology combined with automated machine learning (AutoML). METHODS: We first evaluated the reproducibility of our measurement system using Pearson's correlation coefficients between samples derived from a single pooled RNA sample. To generate comprehensive miRNA profile, we performed NGS analysis of miRNAs in 262 serum samples. Among the discovery set (57 patients with lung cancer and 57 healthy controls), 1123 miRNA-based diagnostic models for lung cancer detection were constructed and screened using AutoML technology. The diagnostic faculty of the best performance model was evaluated by inspecting the validation samples (74 patients with lung cancer and 74 healthy controls). RESULTS: The Pearson's correlation coefficients between samples derived from the pooled RNA sample ≥ 0.98. In the validation analysis, the best model showed a high AUC score (0.98) and a high sensitivity for early stage lung cancer (85.7%, n = 28). Furthermore, in comparison to carcinoembryonic antigen (CEA), a conventional blood biomarker for adenocarcinoma, the miRNA-based model showed higher sensitivity for early-stage lung adenocarcinoma (CEA, 27.8%, n = 18; miRNA-based model, 77.8%, n = 18). CONCLUSION: The miRNA-based diagnostic model showed a high sensitivity for lung cancer, including early-stage disease. Our study provides the experimental evidence that serum comprehensive miRNA profile can be a highly sensitive blood biomarker for early-stage lung cancer.

Clinical Significance of Circulating Tumor Cell Induced Epithelial-Mesenchymal Transition in Patients with Metastatic Colorectal Cancer by Single-Cell RNA-Sequencing.

BACKGROUND: Circulating tumor cells (CTCs) are a prognostic marker in patients with metastatic colorectal cancer (mCRC). However, little is known about the characterization of CTCs in mCRC at the single-cell level using RNA sequencing. The purpose of this study was to validate the capability to detect and isolate single CTCs for single-cell RNA sequencing (scRNA-seq) and to identify clinical significance at a single CTC level. METHODS: Single CTCs from 27 mCRC patients were collected by CTC-FIND, which is comprised of filter separation and immunomagnetic depletion to collect ultra-pure CTC samples. To address tumor heterogeneity, CTCs were collected without relying on any traditional CTC markers, such as epithelial and mesenchymal cell antigens, and were undertaken by scRNA-seq using SMART-Seq v4. RESULTS: We identified 59 single CTCs which were classified into four groups by epithelial, epithelial-mesenchymal transition (EMT) and stem cell-related gene expression. Patients receiving second or later-line treatment who had EMT gene expressing CTCs had a significantly shorter PFS and OS. CONCLUSIONS: Exploiting CTC-FIND with SMART-Seq v4 showed that scRNA-seq of CTCs may shed new insight into tumor heterogeneity of mCRC and that the presence of CTCs expressing EMT-related genes at the single-cell level could have prognostic value in mCRC patients.

Cancer marker-free enrichment and direct mutation detection in rare cancer cells by combining multi-property isolation and microfluidic concentration.

Genetic analysis, rather than simply counting the number of circulating tumor cells (CTCs), which are rare cancer cells in peripheral blood, has great potential for non-invasive biopsy or "liquid biopsy." However, a practical problem in conventional enrichment of CTCs is that the isolated target cells are mixed with numerous residual leukocytes, and are suspended in a large volume. Hence, further isolation (i.e., cytokeratin (CK)-positive cell picking) or DNA purification is required for downstream genetic analysis after isolation. Here, we propose a novel cancer marker-free method of CTC enrichment by size-based Filtration and Immunomagnetic Negative selection followed by Dielectrophoretic concentration (CTC-FIND) for direct detection of genetic mutations in rare cancer cells suspended in whole blood. A combination of two independent isolation methods based on physical (filtration) and biochemical properties (immunomagnetic negative selection) in CTC-FIND allowed highly efficient cancer marker-free purification (5.1-log depletion of leukocytes). The isolated cells were trapped and concentrated using a microfluidic step-channel device using dielectrophoresis for discrimination and downstream genetic analysis. The feasibility of cancer marker-free enrichment by CTC-FIND was successfully demonstrated by directly detecting mutations in various cancer cells with a very high sensitivity of 1 cell per mL, including EpCAM and CK-negative cells, which were used to spike 8 mL of whole blood. Thus, CTC-FIND can be used with liquid biopsy to detect genetic mutations in wide-ranging CTC subsets, independent of cancer cell-specific marker expression.

A novel detection strategy for living circulating tumor cells using 5-aminolevulinic acid.

Most circulating tumor cell (CTC) detection methods have technical limitations, allowing the detection of only cells expressing epithelial antigens, and they cannot identify if the CTCs are alive or dead. Herein, we constructed a novel CTC detection system comprised of filter separation and 5-aminolevulinic acid (5-ALA)-based labeling, termed "Fs-ALA". Blood specimens (7.5 mL) were subjected to this method. Cells enriched on the filter were incubated with 5-ALA and Hoechst 33342 as positive markers for CTCs. Images of the whole filter surface were obtained using a fluorescence microscope. No 5-ALA positive cells were detected in healthy blood specimens. The Fs-ALA method was capable of detecting not only EpCAM-positive, but also EpCAM-negative tumor cells. In the Fs-ALA method, one or more CTCs were detected in samples from 13 of 18 (72.2%) colorectal cancer patients. The Fs-ALA method had a significantly higher CTC detection rate than CellSearch™ in colorectal cancer patients (P <0.05), and only the former was capable of identifying live cells. This method is highly efficient for detecting CTC populations having undergone phenotypic changes, such as epithelial-mesenchymal transition.

Stimulatory Effects of CO(2) Laser, Er:YAG Laser and Ga-Al-As Laser on Exposed Dentinal Tubule Orifices.

We investigated the effects of lasers irradiation on the exposed dentinal tubule. Human tooth specimens with exposed dentinal tubule orifices were used. Three types of lasers (CO(2) laser, Er:YAG laser and Ga-Al-As laser) were employed. The parameters were 1.0 W in continuous-wave mode with an irradiation time of 30 s for the CO(2) laser, 30 mJ in continuous-wave mode with an irradiation time of 60 s for the Er:YAG laser, and 1.0 W in continuous-wave mode with an irradiation time of 60 s for the Ga-Al-As laser. A non-irradiated group was used as a control. After laser irradiation, the dentinal surface of each sample was observed using SEM. Afterwards, all samples were immersed in methylene blue dye solution in order to evaluate the penetration of the dye solution and observe the change in dentinal permeability after laser irradiation. SEM observation showed that the control group had numerous exposed dentinal tubule orifices, whereas these orifices were closed in the laser-irradiated groups. There was consistent dye penetration into the pulp chamber in the control group, whereas no dye penetration was evident in the laser-irradiated groups. Therefore, laser appears to be a promising treatment for reducing permeation through exposed dentinal tubules.

Dentin is dissolved by high concentrations of L-ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl-hydrogen phosphate] potassium salt with or without hydrogen peroxide.

L-Ascorbic acid 2-[3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl)-2H-1-benzopyran-6-yl-hydrogen phosphate] potassium salt (EPC-K(1)) is a conjugate of vitamin C and vitamin E that is water-soluble and stable at room temperature. EPC-K(1) has been developed as a hydroxyl radical (.OH) scavenger and antioxidant. In a previous tooth whitening experiment, it was accidentally found that tooth (dentin) blocks were dissolved by EPC-K(1) with H(2)O(2). In the current study, high concentrations of EPC-K(1) (2.5, 25 mM) with 3% H(2)O(2) dissolved and caused the collapse of dentin blocks. Similar concentrations of EPC-K(1) without 3% H(2)O(2), however, dissolved the dentin blocks without collapse over a 3-week period. In these cases, a.OH-like signal was detected using an ESR spin-trapping method. The volume of calcium in solution (including the dentin block) increased on the addition of EPC-K(1) in a concentration-dependent manner. In addition, the calcium : phosphorus ratio changed from 2 : 1 in sound dentin to 1 : 2 in the collapsed dentin block. High concentrations of EPC-K(1) are therefore considered to have calcium chelating and dentin dissolving activity. The dentin dissolving activity was enhanced when EPC-K(1) was used with H(2)O(2). EPC-K(1) had no protective effect when used in tooth whitening with H(2)O(2).