Metabolomics of small extracellular vesicles derived from isocitrate dehydrogenase 1-mutant HCT116 cells collected by semi-automated size exclusion chromatography.

Cancer-derived small extracellular vesicles (sEVs) are multifunctional particles with a lipid bilayer structure that are involved in cancer progression, such as malignant proliferation, distant metastasis, and cancer immunity evasion. The separation protocol used to isolate sEVs is an important process and thus, several have been developed, including ultracentrifugation (UC), size exclusion chromatography (SEC), and affinity purification using antibodies against sEV surface antigens. However, the effects of different separation methods on sEV components have not been adequately examined.

Quantification and Imaging of Exosomes via Luciferase-Fused Exosome Marker Proteins: ExoLuc System.

Bioluminescence (BL) has been widely used to quantitatively monitor various biological phenomena. Here, we describe a protocol for preparing and using cells expressing exosomes labeled with luciferase. The BL of the culture medium of these cells is proportional to the number of secreted exosome particles obtained by well-established nanoparticle tracking analysis, allowing easy, rapid, and sensitive quantification of exosomes in vitro and in vivo.

Asteltoxin inhibits extracellular vesicle production through AMPK/mTOR-mediated activation of lysosome function.

Cancer cells secrete aberrantly large amounts of extracellular vesicles (EVs) including exosomes, which originate from multivesicular bodies (MVBs). Because EVs potentially contribute to tumor progression, EV inhibitors are of interest as novel therapeutics. We screened a fungal natural product library.

MEK/ERK-mediated oncogenic signals promote secretion of extracellular vesicles by controlling lysosome function.

Cancer cells secrete large amounts of extracellular vesicles (EVs) originating from multivesicular bodies (MVBs). Mature MVBs fuse either with the plasma membrane for release as EVs, often referred as to exosomes or with lysosomes for degradation. However, the mechanisms regulating MVB fate remain unknown.

A mass spectrometric method for in-depth profiling of phosphoinositide regioisomers and their disease-associated regulation.

Phosphoinositides are a family of membrane lipids essential for many biological and pathological processes. Due to the existence of multiple phosphoinositide regioisomers and their low intracellular concentrations, profiling these lipids and linking a specific acyl variant to a change in biological state have been difficult. To enable the comprehensive analysis of phosphoinositide phosphorylation status and acyl chain identity, we develop PRMC-MS (Phosphoinositide Regioisomer Measurement by Chiral column chromatography and Mass Spectrometry).

Exosomes secreted from cancer-associated fibroblasts elicit anti-pyrimidine drug resistance through modulation of its transporter in malignant lymphoma.

The tumor microenvironment is deeply involved in the process of tumor growth and development. In this study, we focused on cancer-associated fibroblasts (CAFs) and their derived exosomes on the lymphoma microenvironment to uncover their clinical significance. CAFs were established from primary lymphoma samples, and exosomes secreted from CAFs were obtained by standard procedures.

Filopodium-derived vesicles produced by MIM enhance the migration of recipient cells.

Extracellular vesicles (EVs) are classified as large EVs (l-EVs, or microvesicles) and small EVs (s-EVs, or exosomes). S-EVs are thought to be generated from endosomes through a process that mainly depends on the ESCRT protein complex, including ALG-2 interacting protein X (ALIX). However, the mechanisms of l-EV generation from the plasma membrane have not been identified.

In vivo imaging of long-term accumulation of cancer-derived exosomes using a BRET-based reporter.

Monitoring of exosome dynamics in living organisms is essential to demonstrate the real functions of cancer-derived exosomes. Currently, these have been elucidated in vitro or under non-physiological conditions in vivo in most cases. To overcome these limitations, we developed an imaging method using Antares2-mediated bioluminescence resonance energy transfer (BRET) for observing long-term accumulation of exosomes in vivo.

Src in endosomal membranes promotes exosome secretion and tumor progression.

c-Src is a membrane-associated tyrosine kinase that has key roles in the signaling transduction that controls cell growth, adhesion, and migration. In the early stage of carcinogenesis, c-Src is activated under the plasma membrane and transduces oncogenic signals. Here we show that c-Src localized to the endosomal membrane has unique functions in c-Src-transformed cells.

Sensitive and rapid quantification of exosomes by fusing luciferase to exosome marker proteins.

Exosomes have emerged as important mediators of intercellular communication. Although their modes of action have been elucidated, the molecular mechanisms underlying their secretion, sorting of molecules, uptake into recipient cells, and biological distribution in vivo remain elusive. Here, we present a novel system for quantifying secreted exosomes by introducing ectopic or CRISPR/Cas9-mediated knock-in of luciferase-fusion exosome markers such as CD63.

BK-UM in patients with recurrent ovarian cancer or peritoneal cancer: a first-in-human phase-I study.

Background: BK-UM (CRM197) is a mutant form of diphtheria toxin and a specific inhibitor of heparin-binding epidermal growth factor-like growth factor (HB-EGF). We assessed the safety, pharmacokinetics, recommended dose, and efficacy of BK-UM in patients with recurrent ovarian cancer (OC) or peritoneal cancer (PC), and measured HB-EGF levels in serum and abdominal fluid after BK-UM administration.

Methods: Eleven patients with advanced or recurrent OC or PC were enrolled and treated with BK-UM via the intraperitoneal route.

Role of Sialidase in Long-Term Potentiation at Mossy Fiber-CA3 Synapses and Hippocampus-Dependent Spatial Memory.

Sialic acid bound to glycans in glycolipids and glycoproteins is essential for synaptic plasticity and memory. Sialidase (EC 3.2.

Isolation and characterization of monoclonal antibodies specific for chondroitin sulfate E.

Chondroitin sulfate E (CSE) is a polysaccharide containing mainly disaccharide units of D-glucuronic acid (GlcA) and 4,6-O-disulfated N-acetyl-D-galactosamine (GalNAc) residues (E-unit) in the amount of ∼ 60%. CSE is involved in many biological and pathological processes. In this study, we established new monoclonal antibodies, termed E-12C and E-18H, by using CSE that contained more than 70% of E-units as an immunogen.

Purvalanol A, a CDK inhibitor, effectively suppresses Src-mediated transformation by inhibiting both CDKs and c-Src.

The nonreceptor tyrosine kinase c-Src is frequently over-expressed or hyperactivated in various human cancers and contributes to cancer progression in cooperation with up-regulated growth factor receptors. However, Src-selective anticancer drugs are still in clinical trials. To identify more effective inhibitors of c-Src-mediated cancer progression, we developed a new screening platform using Csk-deficient cells that can be transformed by c-Src.

The lipid raft-anchored adaptor protein Cbp controls the oncogenic potential of c-Src.

The tyrosine kinase c-Src is upregulated in various human cancers irrespective of its negative regulator Csk, but the regulatory mechanisms remain unclear. Here, we show that a lipid raft-anchored Csk adaptor, Cbp/PAG, is directly involved in controlling the oncogenicity of c-Src. Using Csk-deficient cells that can be transformed by c-Src overexpression, we found that Cbp expression is markedly downregulated by c-Src activation and re-expression of Cbp efficiently suppresses c-Src transformation as well as tumorigenesis.

Functional dissection of transformation by c-Src and v-Src.

The c-src proto-oncogene product, c-Src, is frequently over-expressed and activated in various human malignant cancers, implicating a role for c-Src in cancer progression. To verify the role of c-Src, we analyzed the transforming ability of c-Src in mouse embryonic fibroblasts that lack Csk, a negative regulator of Src family kinases. Although Csk deficiency is not sufficient for cell transformation, c-Src over-expression induced characteristic transformed phenotypes including anchorage-independent growth and tumorigenecity.

Proteomic identification of ZO-1/2 as a novel scaffold for Src/Csk regulatory circuit.

To elucidate the regulatory mechanism of cell transformation induced by c-Src tyrosine kinase, we performed a proteomic analysis of tyrosine phosphorylated proteins that interact with c-Src and/or its negative regulator Csk. The c-Src interacting proteins were affinity-purified from Src transformed cells using the Src SH2 domain as a ligand. LC-MS/MS analysis of the purified proteins identified general Src substrates, such as focal adhesion kinase and paxillin, and ZO-1/2 as a transformation-dependent Src target.

Junctional adhesion molecule-C promotes metastatic potential of HT1080 human fibrosarcoma.

The junctional adhesion molecule (JAM) family is a key molecule in a process called transendothelial migration or diapedesis. Here, we report implications of JAM-C in cancer metastasis. We first determined the mRNA expression of JAMs in 19 kinds of cancer cell lines.

Antiangiogenic photodynamic therapy (PDT) by using long-circulating liposomes modified with peptide specific to angiogenic vessels.

For the improvement of therapeutic efficacy in photodynamic therapy (PDT) by using a photosensitizer, benzoporphyrin derivative monoacid ring A (BPD-MA), we previously prepared polyethylene glycol (PEG)-modified liposomes encapsulating BPD-MA (PEG-Lip BPD-MA). PEGylation of liposomes enhanced the accumulation of BPD-MA in tumor tissue at 3 h after injection of it into Meth-A-sarcoma-bearing mice, but, unexpectedly, decreased the suitability of the drug for PDT when laser irradiation was performed at 3 h after the injection of the liposomal photosensitizer. To improve the bioavailability of PEG-Lip BPD-MA, we endowed the liposomes with active-targeting characteristics by using Ala-Pro-Arg-Pro-Gly (APRPG) pentapeptide, which had earlier been isolated as a peptide specific to angiogenic endothelial cells.

Antiangiogenic photodynamic therapy (PDT) using Visudyne causes effective suppression of tumor growth.

We previously observed that antiangiogenic photodynamic therapy (PDT), namely, laser irradiation at 15 min after administration of photosensitizer, by using stable liposomal benzoporphyrin derivative monoacid ring A (BPD-MA), in which the liposomes were composed of dipalmitoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine, cholesterol, and dipalmitoylphosphatidylglycerol (10:10:10:2.5 as a molar ratio), was quite effective for cancer treatment. On the other hand, Visudyne, a commercialized liposomal formulation of BPD-MA, is based on more fluid lipids, namely, dimyristoylphosphatidylcholine and egg yolk phosphatidylglycerol, and is thought to be less stable in the presence of serum.

PEGylation of liposome decreases the susceptibility of liposomal drug in cancer photodynamic therapy.

For the purpose of the avoidance of reticuloendothelial system (RES)-trapping, liposome entrapped benzoporphyrin derivative monoacid ring A (BPD-MA), which is used for cancer photodynamic therapy (PDT), was modified with polyethylene glycol (PEG-LipBPD-MA). Tumor accumulation of BPD-MA at 3 h after injection with PEG-LipBPD-MA in Meth A-sarcoma-bearing mice was significantly higher than that after injection with non-modified liposomal BPD-MA (Cont-LipBPD-MA) as expected. On the contrary, significant tumor growth suppression after PDT was observed only for Cont-LipBPD-MA but not for PEG-LipBPD-MA.