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1.
Biomaterials ; 296: 122087, 2023 05.
Article in English | MEDLINE | ID: mdl-36924663

ABSTRACT

The development of organoid culture technologies has triggered industrial interest in ex vivo drug test-guided clinical response prediction for precision cancer therapy. The three-dimensional culture encapsulated with basement membrane (BM) components is extremely important in establishing ex vivo organoids and drug sensitivity tests because the BM components confer essential structures resembling tumor histopathology. Although numerous studies have demonstrated three-dimensional culture-based drug screening methods, establishing a large-scale drug-screening platform with matrix-encapsulated tumor cells is challenging because the arrangement of microspots of a matrix-cell droplet onto each well of a microwell plate is inconsistent and difficult to standardize. In addition, relatively low scales and lack of reproducibility discourage the application of three-dimensional organoid-based drug screening data for precision treatment or drug discovery. To overcome these limitations, we manufactured an automated organospotter-integrated high-throughput organo-on-pillar (high-TOP) drug-screening platform. Our system is compatible with various extracellular matrices, including BM extract, Matrigel, collagen, and hydrogel. In addition, it can be readily utilized for high-content analyses by simply exchanging the bottom plates without disrupting the domes. Our system demonstrated considerable robustness, consistency, reproducibility, and biological relevancy in three-dimensional drug sensitivity analyses using Matrigel-encapsulated ovarian cancer cell lines. We also demonstrated proof-of-concept cases representing the clinical feasibility of high-TOP-assisted ex vivo drug tests linked to clinical chemo-response in ovarian cancer patients. In conclusion, our platform provides an automated and standardized method for ex vivo drug-sensitivity-guided clinical response prediction, suggesting effective chemotherapy regimens for patients with cancer.


Subject(s)
Cell Culture Techniques , Ovarian Neoplasms , Female , Humans , Cell Culture Techniques/methods , Reproducibility of Results , Drug Evaluation, Preclinical/methods , Drug Discovery , Organoids , Ovarian Neoplasms/pathology , High-Throughput Screening Assays/methods
2.
Sci Rep ; 11(1): 1644, 2021 01 18.
Article in English | MEDLINE | ID: mdl-33462311

ABSTRACT

Early recurrence in pancreatic ductal adenocarcinoma (PDAC) is a decisive factor in determining a patient's prognosis. We determined in our current study whether circulating tumour cells (CTCs) exist in the blood of PDAC patients and can be used as a predictor of recurrence patterns (i.e. time and site) after surgical resection. Between December 2017 and November 2018, the mononuclear cell layer was obtained from the peripheral blood of 36 patients diagnosed with PDAC. CTCs were then isolated using the CD-PRIME™ platform and detected via immunostaining. The patient records were analyzed to correlate these data with survival and recurrence patterns. Twelve patients were CTC-positive (33.3%) and showed a significantly frequent rate of systemic recurrence (distant metastases and peritoneal dissemination) (p = 0.025). On multi-variable logistic regression analysis, CTC positivity was an independent risk factor for early recurrence (p = 0.027) and for systemic recurrence (p = 0.033). In summary, the presence or absence of CTC in the blood of the patients with PDAC could help predict the recurrence pattern after surgery. PDAC patients with CTC positivity at tumour diagnosis should therefore undergo a comprehensive strategy for systemic therapy and active monitoring to detect possible early recurrence.


Subject(s)
Carcinoma, Pancreatic Ductal/blood , Neoplasm Recurrence, Local/blood , Neoplastic Cells, Circulating/pathology , Aged , Biomarkers, Tumor/blood , Biomarkers, Tumor/genetics , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/surgery , Humans , Male , Middle Aged , Neoplasm Recurrence, Local/genetics , Neoplasm Recurrence, Local/pathology , Neoplasm Recurrence, Local/surgery , Prognosis , Survival Rate
3.
Cancers (Basel) ; 12(9)2020 Aug 19.
Article in English | MEDLINE | ID: mdl-32825137

ABSTRACT

This study was conducted to identify the composition and diversity of the microbiome in tissues of pancreatic cancer and to determine its role. First, extracellular vesicles (EVs) were obtained from the paired tumor and normal tissues, and 16s rRNA gene sequencing was performed. We identified the microbiomes, compared the diversity between groups, and found that Tepidimonas was more abundant in tumors. Second, larger tumors resulted in lower levels of Leuconostoc and Sutterella, and increased lymph node metastasis resulted in higher levels of Comamonas and Turicibacter in tumor tissues. Moreover, in the case of tumor recurrence, the levels of Streptococcus and Akkermansia were decreased in tumor tissues. Finally, with the supernatant of Tepidimonasfonticaldi, proliferation and migration of cells increased, and epithelial-mesenchymal transition and the Tricarboxylic Acid (TCA) cycle-related metabolites were enhanced. The composition and diversity of EV-derived microbiomes are important for providing novel insights into theragnostic approaches in pancreatic cancer.

4.
Transl Oncol ; 11(3): 800-807, 2018 Jun.
Article in English | MEDLINE | ID: mdl-29705629

ABSTRACT

Nucleic acid sequencing is frequently used to determine the molecular basis of diseases. Therefore, proper storage of biological specimens is essential to inhibit nucleic acid degradation. RNA isolated from the human pancreas is generally of poor quality because of its high concentration of endogenous RNase. In this study, we optimized the method for extracting high quality RNA from paired tumor and normal pancreatic tissues obtained from eight pancreatic cancer patients post-surgery. RNA integrity number (RIN) was checked to evaluate the integrity of RNA, we tried to extract the RNA with an RIN value of 8 or higher that allows for the latest genetic analysis. The effect of several parameters, including the method used for tissue lysis, RNAlater treatment, tissue weight at storage, and the time to storage after surgical resection, on the quantity and quality of RNA extracted was examined. Data showed that the highest quantity of RNA was isolated using a combination of manual and mechanical methods of tissue lysis. Additionally, sectioning the tissues into small pieces (<100 mg) and treating them with RNAlater solution prior to storage increased RNA stability. Following these guidelines, high quality RNA was obtained from 100% (8/8) of tumor tissues and 75% (6/8) of normal tissues. High-quality RNA was still stable under repeated freezing and thawing. The application of these results during sample handling and storage in clinical settings will facilitate the genetic diagnosis of diseases and their subsequent treatment.

5.
Mol Biotechnol ; 57(6): 506-12, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25632893

ABSTRACT

Conventional procedures to assay RNA degradation by a protein with ribonuclease (RNase) activity require a step to isolate intact RNA molecules, which are used as a substrate. Here, we established a novel "In-cell RNA hydrolysis assay" in which RNAs within cells are used as a substrate for the RNA-hydrolyzing protein, thereby avoiding the need to prepare intact RNA molecules. In this method, the degree of RNA degradation is indicated by the fluorescence intensity of RNA molecules released from fixed and permeabilized cells following treatment with the potential RNase. A catalytic 3D8 antibody capable of degrading RNAs and pancreatic RNase A were used as model RNases. Our data demonstrate that the novel In-cell RNA hydrolysis assay is a reliable and sensitive method to analyze the activities of potential RNA-hydrolyzing proteins such as catalytic antibodies.


Subject(s)
RNA/metabolism , Ribonucleases/metabolism , Hydrolysis , Microscopy, Confocal , Spectrophotometry, Ultraviolet
6.
J Biol Chem ; 288(50): 35877-85, 2013 Dec 13.
Article in English | MEDLINE | ID: mdl-24155236

ABSTRACT

Many murine monoclonal anti-DNA antibodies (Abs) derived from mice models for systemic lupus erythematosus have additional cell-penetration and/or nucleic acid-hydrolysis properties. Here, we examined the influence of deactivating each complementarity-determining region (CDR) within a multifunctional anti-nucleic acid antibody (Ab) that possesses these activities, the catalytic 3D8 single chain variable fragment (scFv). CDR-deactivated 3D8 scFv variants were generated by replacing all of the amino acids within each CDR with Gly/Ser residues. The structure of 3D8 scFv accommodated single complete CDR deactivations. Different functional activities of 3D8 scFv were affected differently depending on which CDR was deactivated. The only exception was CDR1, located within the light chain (LCDR1); deactivation of LCDR1 abolished all of the functional activities of 3D8 scFv. A hybrid Ab, HW6/3D8L1, in which the LCDR1 from an unrelated Ab (HW6) was replaced with the LCDR1 from 3D8, acquired all activities associated with the 3D8 scFv. These results suggest that the activity of a multifunctional 3D8 scFv Ab can be modulated by single complete CDR deactivation and that the LCDR1 plays a crucial role in maintaining Ab properties. This study presents a new approach for determining the role of individual CDRs in multifunctional Abs with important implications for the future of Ab engineering.


Subject(s)
Complementarity Determining Regions/immunology , DNA/immunology , RNA/immunology , Single-Chain Antibodies/immunology , Amino Acid Sequence , Amino Acid Substitution , Complementarity Determining Regions/chemistry , Complementarity Determining Regions/genetics , Complementarity Determining Regions/metabolism , HeLa Cells , Heparin/metabolism , Humans , Hydrolysis , Immunoglobulin Light Chains/genetics , Immunoglobulin Light Chains/immunology , Molecular Sequence Data , Protein Structure, Secondary , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/genetics , Single-Chain Antibodies/metabolism
7.
Biochem Biophys Res Commun ; 395(4): 484-9, 2010 May 14.
Article in English | MEDLINE | ID: mdl-20382124

ABSTRACT

Some proteins with ribonuclease (RNase) activity have been shown to suppress viral replication. A well-characterized recombinant antibody, 3D8 single-chain variable fragment (3D8 scFv), has RNA-hydrolyzing and cell-penetrating activities. Here, we investigated antiviral activity of 3D8 scFv against classical swine fever virus (CSFV). In a cell line expressing 3D8 scFv (C26), intracellular RNA-hydrolysis activity was higher compared to control PK-15 cells and viral replication was strongly suppressed at the viral RNA level, with the evidence of independency of IFN-alpha/beta induction. Exogenous treatment of 3D8 scFv, prior to or post-CSFV infection, was also shown to suppress CSFV replication at the viral RNA level. These observations suggest that antiviral activity of 3D8 scFv may be due to the intrinsic RNase activity of 3D8 scFv, which is capable of targeting viral RNA genomes or transcripts.


Subject(s)
Antibodies, Antinuclear/pharmacology , Antibodies, Monoclonal/pharmacology , Antiviral Agents/pharmacology , Classical Swine Fever Virus/drug effects , Classical Swine Fever/virology , Recombinant Proteins/pharmacology , Single-Chain Antibodies/pharmacology , Animals , Antibodies, Antinuclear/metabolism , Antibodies, Monoclonal/metabolism , Antiviral Agents/metabolism , Cell Line , Classical Swine Fever/immunology , Down-Regulation , Hydrolysis , Interferon Type I/immunology , RNA, Viral/immunology , RNA, Viral/metabolism , Recombinant Proteins/metabolism , Ribonucleases/metabolism , Ribonucleases/pharmacology , Single-Chain Antibodies/immunology , Swine , Virus Replication/drug effects
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